Secondary literature sources for the CYCc domain

For each of the hand selected primary papers associated with the CYCc domain, 100 neighbouring papers are retrieved from PubMed. If one of these neighbouring papers is referenced by more than two primary papers, it is shown in the table below.

CO(2)/HCO(3)(-)-responsive soluble adenylyl cyclase as a putative metabolic sensor.

Zippin JH, Levin LR, Buck J
Trends Endocrinol Metab. 2001; 12: 366-70

Cyclic AMP (cAMP) is an evolutionarily conserved regulator of metabolism. Recently, we identified a novel mammalian source of cAMP - soluble adenylyl cyclase (sAC) - that is regulated directly by bicarbonate ions (HCO(3)(-)). As the concentration of HCO(3)(-) reflects cellular levels of carbon dioxide (CO(2)), energy-generating metabolic processes (which increase intracellular CO(2)) are poised to activate bicarbonate-responsive sAC. This direct link between metabolic activity, sAC and cAMP could represent an evolutionarily conserved mechanism of metabolic feedback regulation.

Mutations uncover a role for two magnesium ions in the catalytic mechanism of adenylyl cyclase.

Zimmermann G, Zhou D, Taussig R
J Biol Chem. 1998; 273: 19650-5

The recent determination of the crystal structure of adenylyl cyclase has elucidated many structural features that determine the regulatory properties of the enzyme. In addition, the characterization of adenylyl cyclase by mutagenic techniques and the identification of the binding site for P-site inhibitors have led to modeling studies that describe the ATP-binding site. Despite these advances, the catalytic mechanism of adenylyl cyclase remains uncertain, especially with respect to the role that magnesium ions may play in this process. We have identified four mutant mammalian adenylyl cyclases defective in their metal dependence, allowing us to further characterize the function of metal ions in the catalytic mechanism of this enzyme. The wild-type adenylyl cyclase shows a biphasic Mg2+ dose-response curve in which the high-affinity component displays cooperativity (Hill coefficient of 1.4). Two mutations (C441R and Y442H) reduce the affinity of the adenylyl cyclase for Mg2+ dramatically without affecting the binding of MgATP, suggesting that there is a metal requirement in addition to the ATP-bound Mg2+. The results of this study thus demonstrate multiple metal requirements of adenylyl cyclase and support the existence of a Mg2+ ion essential for catalysis and distinct from the ATP-bound ion. We propose that adenylyl cyclase employs a catalytic mechanism analogous to that of DNA polymerase, in which two key magnesium ions facilitate the nucleophilic attack of the 3'-hydroxyl group and the subsequent elimination of pyrophosphate.

Activating mutation of adenylyl cyclase reverses its inhibition by G proteins.

Zimmermann G, Zhou D, Taussig R
Mol Pharmacol. 1999; 56: 895-901

We have implemented a yeast genetic selection developed previously by our laboratory to identify mutant mammalian type V adenylyl cyclases insensitive to inhibition by G(ialpha.) One mutation isolated was localized to the first cytoplasmic domain at a Phe residue (position 400), which is conserved in all nine isoforms of membrane-bound mammalian adenylyl cyclase. Biochemical characterization of the F400Y mutant revealed a dramatic conversion of the G(ialpha) response from inhibitory to stimulatory. This mutation results in additional activating effects. The mutant exhibits an enhanced sensitivity toward activation by either G(salpha) or forskolin. Synergism between G(salpha) and forskolin is not observed for the F400Y mutant, presumably because the mutant already is in the sensitized state. Additionally, an enhancement of the basal unstimulated activity was observed. This mutation, which is the first demonstration of an activating point in a mammalian adenylyl cyclase, mimics a sensitized conformation of the wild-type enzyme that underlies the synergism between stimulatory inputs, and additionally, removes the inhibitory regulatory input provided by G(ialpha). Because sensitizing adenylyl cyclase toward its stimulators can have profound biological implications, this raises the possibility that naturally occurring mutations resembling those at the Phe400 residue may be associated with human disease states.

Genetic selection of mammalian adenylyl cyclases insensitive to stimulation by Gsalpha.

Zimmermann G, Zhou D, Taussig R
J Biol Chem. 1998; 273: 6968-75

We describe the development of a genetic system allowing for the isolation of mutant mammalian adenylyl cyclases defective in their responses to G protein subunits, thus allowing for the identification of structural elements within the cyclase that are responsible for the recognition of these regulators. Expression of mammalian type V adenylyl cyclase in a cyclase-deleted yeast strain can conditionally complement the lethal phenotype of this strain. Type V adenylyl cyclase-expressing yeast grow only when the cyclase is activated by coexpression of Gsalpha or addition of forskolin to the medium; however, growth arrest is observed in the presence of both activators or under basal conditions. Utilizing this genetic system, we have isolated 25 adenylyl cyclase mutants defective in their response to Gsalpha. Sequence analysis and biochemical characterization of these mutants have identified residues in both cytoplasmic domains of the cyclase that are involved in the specific binding of and regulation by Gsalpha.

Diltiazem prevents the depression of adenylyl cyclase activity induced by the calcium paradox in rat.

Ziegelhoeffer A, Will-Shahab L, Ravingerova T, Kuettner I
Cardioscience. 1993; 4: 151-5

In isolated rat hearts the calcium paradox, induced by perfusion for 3 minutes in the absence of calcium followed by perfusion for 10 minutes in the presence of calcium, depressed the activation of adenylyl cyclase by l-isoproterenol, NaF and forskolin. The characteristics of the beta-adrenoceptors and the activation of adenylyl cyclase by guanylyl imidodiphosphate were not changed. The findings suggest an uncoupling of beta-adrenoceptors from the catalytic site of the adenylate cyclase complex. Diltiazem, at 0.4 microM in the perfusion medium, greatly reduced the diminution of the activation of adenylate cyclase by isoproterenol and forskolin, and completely prevented the depression of the activation of adenylate cyclase by NaF. These effects may be due to interference by diltiazem with the mechanisms that promote an excessive influx of calcium into the heart during the calcium paradox.

Characterization and crystallization of a minimal catalytic core domain from mammalian type II adenylyl cyclase.

Zhang G, Liu Y, Qin J, Vo B, Tang WJ, Ruoho AE, Hurley JH
Protein Sci. 1997; 6: 903-8

Adenylyl cyclases play a pivotal role in signal transduction by carrying out the regulated synthesis of cyclic AMP. The nine cloned mammalian adenylyl cyclases all share two conserved regions of sequence, C1 and C2, which are homologous to each other and are together responsible for catalytic activity. Recombinant C1 and C2 domains catalyze the synthesis of cyclic AMP when they are mixed and activated by forskolin, and C2 domains alone also manifest reduced levels of forskolin-stimulated enzyme activity. Using limited proteolysis and mass spectrometry, we have mapped the boundaries of a minimal stable and active C2 catalytic domain to residues 871-1090 of type II adenylyl cyclase. We report the properties and crystallization of this trimmed domain, termed IIC2-delta 4. Crystals belong to space group P4n2(1)2, where n = 1 or 3; a = b = 81.3, and c = 180.5 A; and there are two molecules per asymmetric unit related by an approximate body centering operation. Flash-frozen crystals diffract anisotropically to 2.2 A along the c* direction and to 2.8 A along the a* and b* directions using synchrotron radiation.

Two separate functions are encoded by the carboxyl-terminal domains of the yeast cyclase-associated protein and its mammalian homologs. Dimerization and actin binding.

Zelicof A, Protopopov V, David D, Lin XY, Lustgarten V, Gerst JE
J Biol Chem. 1996; 271: 18243-52

The yeast adenylyl cyclase-associated protein, CAP, was identified as a component of the RAS-activated cyclase complex. CAP consists of two functional domains separated by a proline-rich region. One domain, which localizes to the amino terminus, mediates RAS signaling through adenylyl cyclase, while a domain at the carboxyl terminus is involved in the regulation of cell growth and morphogenesis. Recently, the carboxyl terminus of yeast CAP was shown to sequester actin, but whether this function has been conserved, and is the sole function of this domain, is unclear. Here, we demonstrate that the carboxyl-terminal domains of CAP and CAP homologs have two separate functions. We show that carboxyl-terminals of both yeast CAP and a mammalian CAP homolog, MCH1, bind to actin. We also show that this domain contains a signal for dimerization, allowing both CAP and MCH1 to form homodimers and heterodimers. The properties of actin binding and dimerization are mediated by separate regions on the carboxyl terminus; the last 27 amino acids of CAP being critical for actin binding. Finally, we present evidence that links a segment of the proline-rich region of CAP to its localization in yeast. Together, these results suggest that all three domains of CAP proteins are functional.

Guanylyl cyclase expression in specific sensory neurons: a new family of chemosensory receptors.

Yu S, Avery L, Baude E, Garbers DL
Proc Natl Acad Sci U S A. 1997; 94: 3384-7

A guanylyl cyclase (GC-D) was recently shown to be expressed in a subclass of neurons within the neuroepithelim of the rat, but given that only a single cyclase was discovered, whether it represents an odorant/pheromone receptor as has been suggested for the large family of seven-transmembrane receptors remains unclear. Through cloning and expression of cDNA we now demonstrate that at least 29 genomic or cDNA sequences found in Caenorhabditis elegans represent guanylyl cyclases. Many of the membrane forms retain cysteine residues conserved within the extracellular, ligand-binding domain of known cyclase receptors. Of eight orphan cyclase receptor::GFP (green fluroescence protein) fusion constructs for which signals were obtained, all were expressed in specific sensory neurons. Furthermore, a cyclase/GFP fusion protein (GCY-10/GFP) was principally expressed in the sensory cilium, suggesting these cyclases function as primary chemosensory receptors. For the first time, we also found that chemosensory neurons (ASE), known to be bilaterally symmetric, demonstrate absolute right or left sidedness with respect to the expression of three different cyclases. Thus, the guanylyl cyclases represent an unexpectedly large and new family of sensory neuron receptors that may complement the 7-transmembrane family of odorant/pheromone receptors.

Inhibition of retinal guanylyl cyclase by the RGS9-1 N-terminus.

Yu H, Bondarenko VA, Yamazaki A
Biochem Biophys Res Commun. 2001; 286: 12-9

Cyclic GMP plays a key role in retinal phototransduction and its photoreceptor concentration is precisely controlled by the cooperative action of cGMP phosphodiesterase (PDE) and retinal guanylyl cyclase (retGC). However, studies of the relationship between these two systems have focused only on a Ca(2+)-mediated, indirect connection. Using a retinal "regulator of G-protein signaling" (RGS9-1) and its fragments, we show that the N-terminus of RGS9-1 inhibits retGC activity. We also indicate that the GGL domain and/or the RGS domain function as an internal suppressor against the N-terminus, suggesting that proteins bound to these domains regulate the inhibitory activity of the N-terminus. Direct interaction of retGC with RGS9-1 and its N-terminus is also proved by immunoprecipitation and an overlay technique. Since RGS9-1 also controls the lifetime of transducin-activated PDE through regulating GTPase activity of transducin, this study strongly suggests that RGS9-1 mediates the direct interaction between PDE and retGC systems, and that this ingenious mechanism plays an important role in tuning of cGMP concentration in photoreceptors.

Dominant negative mutants of nitric oxide-sensitive guanylyl cyclase.

Yuen PS, Doolittle LK, Garbers DL
J Biol Chem. 1994; 269: 791-3

Since a nitric oxide-sensitive form of guanylyl cyclase exists as a heterodimer, mutations disrupting catalysis but not heterodimer formation could serve as dominant negative mutations. Two mutations within the catalytic region of the alpha subunit (alpha 1D513A, alpha 1D529A) caused complete losses of basal and sodium nitroprusside-stimulated guanylyl cyclase activity; however, the mutant alpha subunits continued to form heterodimers with wild-type beta-subunit. Rat insulinoma cells, which contain the alpha 1 beta 1 form of guanylyl cyclase, were stably transfected with alpha 1D513A or alpha 1D529A. The response to sodium nitroprusside, which exceeded 200-fold in the presence of wild-type alpha 1, was markedly reduced by the expression of either mutant subunit. In contrast, the mutant subunits failed to inhibit heat-stable enterotoxin-induced cGMP elevations; the bacterial peptide elevated insulinoma cell cGMP approximately 100-fold. The two point mutations, therefore, result in dominant negative proteins that can effectively and specifically block the NO/cGMP signaling pathway. These are also the first studies to show that, although both the alpha and beta subunits contain regions homologous to putative cyclase catalytic regions, a point mutation in just one of the subunits can completely inhibit cyclase activity.

Dominant negative mutants of guanylyl cyclase: probes for global functions and intramolecular mechanisms.

Yuen PS
Methods. 1999; 19: 532-44

Dominant negative mutants are unique tools to define functions of a protein, not only within complex cellular and organismal contexts, but also mechanistically within a protein. Guanylyl cyclases are amenable to studies with dominant negative mutants, with their own sets of opportunities for insight and pitfalls to overcome. Membrane and soluble forms of guanylyl cyclase represent self-contained signal transduction modules that recognize, transduce, and amplify an external signal to give a carefully controlled response. Beginning with recognition of peptide hormones versus nitric oxide, membrane and soluble guanylyl cyclases are considerably different, except that their catalytic domains are closely related. Studies on these catalytic domains and their counterparts in adenylyl cyclases have raised an integral question of whether one or two domains form a catalytic site, which remains unresolved. Regardless of which model is correct, guanylyl cyclases appear to require an oligomeric state to function properly. The inferred relationship between protein-protein interaction and function is the basis for developing dominant negative mutants, which can be designed without prior structural information. Soluble guanylyl cyclases exist in a heterodimeric state, whereas membrane guanylyl cyclases are homodimeric, or possibly higher-order oligomers. These properties dictate that dominant negative mutants of membrane and soluble guanylyl cyclases be approached in fundamentally different ways, with regard to their design, their functional consequences, and their limitations. Using dominant negative mutants as specific inhibitors in complex systems, such as transgenic animals, represents a significant advance, and continuing improvements are just an inkling of the extraordinary potential of this approach. For example, the function of a protein can be obscured because it is expressed in multiple cell types; by restricting its pattern of expression, a cell-specific promoter, coupled to a dominant negative mutant, can pinpoint this function. As more sophisticated methods are developed, dominant negative mutants will provide additional opportunities to unveil new regulatory mechanisms, new signaling pathways, or even new therapeutic approaches.

Cloning and expression of a Ca(2+)-inhibitable adenylyl cyclase from NCB-20 cells.

Yoshimura M, Cooper DM
Proc Natl Acad Sci U S A. 1992; 89: 6716-20

A cDNA that encodes an adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] has been cloned from NCB-20 cells, in which adenylyl cyclase activity is inhibited by Ca2+ at physiological concentrations. The cDNA clone (5.8 kilobases) was isolated by polymerase chain reaction (PCR) using degenerate primers designed by comparison of three adenylyl cyclase sequences (types I, II, and III) and subsequent library screening. Northern analysis revealed expression of mRNA (6.1 kilobases) corresponding to this cDNA in cardiac tissue, which is a prominent source of Ca(2+)-inhibitable adenylyl cyclase. The clone encodes a protein of 1165 amino acids, whose hydrophilicity profile was very similar to those of other mammalian adenylyl cyclases that have recently been cloned. A noticeable difference between this protein and other adenylyl cyclases was a lengthy aminoterminal region before the first transmembrane span. Transient expression of this cDNA in the human embryonic kidney cell line 293 revealed a 3-fold increase in cAMP production in response to forskolin compared with control transfected cells. In purified plasma membranes from transfected cells, increased adenylyl cyclase activity was also detected, which was susceptible to inhibition by submicromolar Ca2+. Thus, this adenylyl cyclase seems to represent the Ca(2+)-inhibitable form that is encountered in NCB-20 cells, cardiac tissue, and elsewhere. Its identification should permit a determination of the structural features that determine the mode of regulation of adenylyl cyclase by Ca2+.

Isolation of a Drosophila gene encoding a head-specific guanylyl cyclase.

Yoshikawa S, Miyamoto I, Aruga J, Furuichi T, Okano H, Mikoshiba K
J Neurochem. 1993; 60: 1570-3

We have isolated and characterized a new guanylyl cyclase gene (dgc1) in Drosophila. The deduced amino acid sequence (683 amino acids) most closely resembled the mammalian soluble-type guanylyl cyclase alpha subunit. The cyclase catalytic domain was highly conserved between the mammalian and Drosophila guanylyl cyclases. The dgc1 mRNA was detected in wild-type heads but not in bodies, and its level was reduced in the mutant eyes absent (eya), indicating that dgc1 is preferentially expressed in the CNS and in the eye. The enriched distribution in the eye suggests that dgc1 may have a role in phototransduction.

Irreversible inactivation of adenylyl cyclase by the "P"-site agonist 2',5'-dideoxy-,3'-p-fluorosulfonylbenzoyl adenosine.

Yeung SM, Johnson RA
J Biol Chem. 1990; 265: 16745-50

2',5'-Dideoxy,3'-p-fluorosulfonylbenzoyl Adenosine (2',5'-dd3'-FSBA) was synthesized and found to be an agonist and affinity label for the "P"-site of adenylyl cyclase. This compound irreversibly inactivated both a crude detergent-dispersed adenylyl cyclase from rat brain and the partially purified enzyme from bovine brain. The irreversible inactivation by 100 to 200 microM 2',5'-dd3'-FSBA was blocked in a concentration-dependent manner by several established P-site inhibitors of adenylyl cyclase, 2',5'-dideoxyadenosine, 2'-d3'-AMP, adenosine, and 2'-deoxyadenosine, but not by inosine, N6-(phenylisopropyl)adenosine, adenine, 2'-d3':5'-cAMP, or 5'-AMP, agents known not to act at the P-site. Moreover, irreversible inactivation by 2',5'-dd3'-FSBA occurred in the presence of ATP at concentrations up to 3 mM, making it unlikely that inactivation was due to an effect on the enzyme's catalytic site. Adenylyl cyclase was also irreversibly inactivated by 5'-FSBA, although modestly (less than 20%) and apparently nonspecifically. Dithiothreitol protected the enzyme from irreversible inactivation by 2',5'-dd3'-FSBA, but reversible inhibition of the enzyme was still observed, although with reduced potency. When 2 mM dithiothreitol was added after a 30-min preincubation with 2',5'-dd3'-FSBA, the rat brain enzyme was partially (approximately 80%) reactivated. The data suggest that 2',5'-dd3'-FSBA may irreversibly inactivate adenylyl cyclase by reacting with a cysteinyl moiety in proximity to the P-site domain of the enzyme. These data together with results of studies of P-site inhibition kinetics published elsewhere (Johnson, R. A., and Shoshani, I. (1990) J. Biol. Chem. 265, 11595-11600) strongly suggest that the P-site and catalytic site are distinct domains on the enzyme. 2',5'-dd3'-FSBA, and especially its radiolabeled analog, should prove to be a useful probe for structural studies of adenylyl cyclase, particularly with regard to the P-site.

Three discrete regions of mammalian adenylyl cyclase form a site for Gsalpha activation.

Yan SZ, Huang ZH, Rao VD, Hurley JH, Tang WJ
J Biol Chem. 1997; 272: 18849-54

The interaction between the alpha subunit of G protein Gs (Gsalpha) and the two cytoplasmic domains of adenylyl cyclase (C1 and C2) is a key step in the stimulation of cAMP synthesis by hormones. Mutational analysis reveals that three discrete regions in the primary sequence of adenylyl cyclase affect the EC50 values for Gsalpha activation and thus are the affinity determinants of Gsalpha. Based on the three-dimensional structure of C2.forskolin dimer, these three regions (C2 alpha2, C2 alpha3/beta4, and C1 beta1) are close together and form a negatively charged and hydrophobic groove the width of an alpha helix that can accommodate the positively charged adenylyl cyclase binding region of Gsalpha. Two mutations in the C2 alpha3/beta4 region decrease the Vmax values of Gsalpha activation without an increase in the EC50 values. Since these three regions are distal to the catalytic site, the likely mechanism for Gsalpha activation is to modulate the structure of the active site by controlling the orientation of the C2 alpha2 and alpha3/beta4 structures.

Conversion of forskolin-insensitive to forskolin-sensitive (mouse-type IX) adenylyl cyclase.

Yan SZ, Huang ZH, Andrews RK, Tang WJ
Mol Pharmacol. 1998; 53: 182-7

Forskolin potently activates all cloned mammalian adenylyl cyclases except type IX by interacting with two homologous cytoplasmic domains (C1 and C2) that form the catalytic core. A mutational analysis of the IIC2 protein (C2 domain from type II adenylyl cyclase) and forskolin analogs suggests that Ser942 interacts with the 7-acetyl group of forskolin. The C1/C2 complex has only one forskolin, one ATP, and one binding site for the alpha subunit of the G protein that stimulates adenylyl cyclase (Gsalpha) and its structure may be modeled using the three-dimensional structure of (IIC2/forskolin)2. The Ser942 mutation defines which forskolin in the (IIC2/forskolin)2 structure exists in C1/C2 complex. Thus, the forskolin-binding site is close to the Gsalpha-binding site but distal (15-20A) from the catalytic site. Mutation from Leu912 of IIC2 protein to tyrosine or alanine severely reduces Gsalpha activation and completely prevents forskolin activation. The corresponding residue of Leu912 is Tyr1082 at type IX isoform of adenylyl cyclase. Similar to recombinant type IX enzyme, soluble adenylyl cyclase derived from mouse-type IX adenylyl cyclase is sensitive to Gsalpha activation but not to forskolin. Changing Tyr1082 to leucine makes soluble type IX adenylyl cyclase forskolin-responsive.

Two cytoplasmic domains of mammalian adenylyl cyclase form a Gs alpha- and forskolin-activated enzyme in vitro.

Yan SZ, Hahn D, Huang ZH, Tang WJ
J Biol Chem. 1996; 271: 10941-5

Mammalian adenylyl cyclases have two homologous cytoplasmic domains (C1 and C2). The first cytoplasmic domain of type I enzyme (IC1) and the second cytoplasmic domain of type II enzyme (IIC2-delta 3, a construct in which 36 N-terminal amino acids of the C2 region are deleted) were expressed and purified to homogeneity. Alone, each had no adenylyl cyclase activity; however, mixing of the two domains in vitro resulted in Gs alpha- and forskolin-activated enzyme activity. The turnover number for Gs alpha- and forskolin-stimulated enzyme activity of the complex between IC1 and IIC2-delta 3 was 8.2 s-1. The concentration of IIC2-delta 3 to achieve half-maximal activation of IC1 was 0.8 and 1.3 microM when stimulated by forskolin and Gs alpha, respectively. The concentration of IIC2-delta 3 needed to complex with IC1 was reduced 10-fold (0.08 microM) when the enzyme was activated by both forskolin and Gs alpha, suggesting that Gs alpha and forskolin increased the affinity of the two cytoplasmic domains for each other.

The regulation of type 7 adenylyl cyclase by its C1b region and Escherichia coli peptidylprolyl isomerase, SlyD.

Yan SZ, Beeler JA, Chen Y, Shelton RK, Tang WJ
J Biol Chem. 2001; 276: 8500-6

Mammalian membrane-bound adenylyl cyclase consists of two highly conserved cytoplasmic domains (C1a and C2a) separated by a less conserved connecting region, C1b, and one of two transmembrane domains, M2. The C1a and C2a domains form a catalytic core that can be stimulated by forskolin and the stimulatory G protein subunit alpha (Galpha(s)). In this study, we analyzed the regulation of type 7 adenylyl cyclase (AC7) by C1b. The C1a, C1b, and C2a domains of AC7 were purified separately. Escherichia coli SlyD protein, a cis-trans peptidylprolyl isomerase (PPIase), copurifies with AC7 C1b (7C1b). SlyD protein can inhibit the Galpha(s)- and/or forskolin-activated activity of both soluble and membrane-bound AC7. Mutant forms of SlyD with reduced PPIase activity are less potent in the inhibition of AC7 activity. Interestingly, different isoforms of mammalian membrane-bound adenylyl cyclase can be either inhibited or stimulated by SlyD protein, raising the possibility that mammalian PPIase may regulate enzymatic activity of mammalian adenylyl cyclase. Purified 7C1b-SlyD complex has a greater inhibitory effect on AC7 activity than SlyD alone. This inhibition by 7C1b is abolished in a 7C1b mutant in which a conserved glutamic acid (amino acid residue 582) is changed to alanine. Inhibition of adenylyl cyclase activity by 7C1b is further confirmed by using 7C1b purified from an E. coli slyD-deficient strain. This inhibitory activity of AC7 is also observed with the 28-mer peptides derived from a region of C1b conserved in AC7 and AC2 but is not observed with a peptide derived from the corresponding region of AC6. This inhibitory activity exhibited by the C1b domain may result from the interaction of 7C1b with 7C1a and 7C2a and may serve to hold AC7 in the basal nonstimulated state.

Two eye guanylyl cyclases are expressed in the same photoreceptor cells and form homomers in preference to heteromers.

Yang RB, Garbers DL
J Biol Chem. 1997; 272: 13738-42

We recently described two eye guanylyl cyclases (GC-E and GC-F) that contain an apparent extracellular domain potentially capable of binding ligands (Yang, R.-B., Foster, D. C., Garbers, D. L., and Fulle, H.-J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 602-606). Here, Northern and Western analyses showed that both cyclases are expressed in the retina and enriched in photoreceptor outer segments. By the use of specific GC-E and GC-F antibodies coupled to different sized gold particles both cyclases were colocalized within the same photoreceptor cells raising the possibility of homomeric and/or heteromeric interactions. A point mutant of GC-E (D878A) was constructed and expressed; it contained no detectable cyclase activity but acted in a dominant negative fashion to abolish the activity of native GC-E and GC-F in coexpression studies. These results suggested that GC-E and GC-F could form either homomers or heteromers, at least when overexpressed in COS-7 cells. Immunoprecipitation with GC-E and GC-F antibody followed by Western analysis confirmed that both homomers and heteromers could be formed. However, similar experiments using retina or outer segments revealed that a vast majority of GC-E and GC-F were precipitated as homomers in the eye. Therefore, like other members of the membrane guanylyl cyclase subfamily, GC-E and GC-F appear to preferentially form homomers.

Two membrane forms of guanylyl cyclase found in the eye.

Yang RB, Foster DC, Garbers DL, Fulle HJ
Proc Natl Acad Sci U S A. 1995; 92: 602-6

The cDNAs for two membrane guanylyl cyclases, designated E (GC-E) and F (GC-F, were isolated from a rat eye cDNA library. Their deduced topographic structures correspond to known members of the guanylyl cyclase receptor family, containing an extracellular domain, a single membrane-spanning domain, a protein kinase-like domain, and a cyclase catalytic domain. GC-E was expressed in the eye and the pineal gland, whereas GC-F expression was confined to the eye. Overproduction of GC-E and GC-F in COS cells resulted in expression of guanylyl cyclase activity, but ligands known to activate other guanylyl cyclase receptors failed to stimulate enzyme activity. Thus, both GC-E and GC-F remain orphan receptors. Amino acid sequence similarity between GC-E and GC-F in the extracellular region and homology with a cyclase expressed in olfactory neurons and retGC, a rod outer-segment-specific cyclase, suggest that there is another subfamily of guanylyl cyclase receptors, possibly restricted to sensory tissues.

Adenylate cyclase activation and competitive binding with renal tissue using synthetic eel calcitonin analog and its fragments.

Yamamoto I, Morita R, Fukunaga M, Dokoh S, Shigeno C, Torizuka K, Noda T
Endocrinology. 1981; 108: 698-702

The structure-function relationship of calcitonin (CT) was investigated using a synthetic eel CT (E-CT) analog and its fragments. Adenylate cyclase activation and competitive binding to rat renal receptors were used as parameters of function. [Asu1,7]E-CT analog, synthesized by replacing the S-S bond of Cys1-Cys7 in the natural hormone with ethylene linkage of 1-amino suberic acid (Asu), and E-CT fragment 11-32 had about 1/5th and 1/50th the potencies of synthetic E-CT, respectively, in both adenylate cyclase activation and competitive inhibition of 125I-labeled [Asu1,7]iodo-E-CT binding on rat renal plasma membranes. The minimal chain length required to activate adenylate cyclase in rat renal plasma membranes was between 12-18 amino acids, and the minimal chain length required to affect the CT receptor binding was between 6-12 amino acids, near the C-terminus. Fragments, including the C-terminus, show a disproportionate and potent inhibition of the binding compared with the potency required for adenylate cyclase activation. Thus, the amino acid sequence near the C-terminus probably plays an important role in the binding of CT to the receptor. (Endocrinology 108: 698, 1981.)

A-type ATP binding consensus sequences are critical for the catalytic activity of the calmodulin-sensitive adenylyl cyclase from Bacillus anthracis.

Xia ZG, Storm DR
J Biol Chem. 1990; 265: 6517-20

Analysis of the predicted amino acid sequence of Bacillus anthracis adenylyl cyclase revealed sequences with homology to consensus sequences for A- and B-type ATP binding domains found in many ATP binding proteins. Based on the analysis of nucleotide binding proteins, a conserved basic amino acid residue in the A-type consensus sequence and a conserved acidic amino acid residue in the B-type consensus sequence have been implicated in the binding of ATP. The putative ATP binding sequences in the B. anthracis adenylyl cyclase possess analogous lysine residues at positions 346 and 353 within two A-type consensus sequences and a glutamate residue at position 436 within a B-type consensus sequence. The two A-type consensus sequences overlap each other and have the opposite orientation. To determine whether Lys-346, Lys-353, or Glu-436 of the B. anthracis adenylyl cyclase are crucial for enzyme activity, Lys-346 and Lys-353 were replaced with methionine and Glu-436 with glutamine by oligonucleotide-directed mutagenesis. Furthermore, Lys-346 was also replaced with arginine. The genes encoding the wild type and mutant adenylyl cyclases were placed under the control of the lac promoter for expression in Escherichia coli, and extracts were assayed for adenylyl cyclase activity. In all cases, a 90-kDa polypeptide corresponding to the catalytic subunit of the enzyme was detected in E. coli extracts by rabbit polyclonal antibodies raised against the purified B. anthracis adenylyl cyclase. The proteins with the Lys-346 to methionine or arginine mutations exhibited no adenylyl cyclase activity, indicating that Lys-346 in the A-type ATP binding consensus sequence plays a critical role for enzyme catalysis. Furthermore, the enzyme with the Lys-353 to methionine mutation was also inactive, suggesting that Lys-353 may also directly contribute to enzyme catalysis. In contrast, the protein with the Glu-436 to glutamine mutation retained 75% of enzyme activity, suggesting that Glu-436 in the B-type ATP binding consensus sequence may not be directly involved in enzyme catalysis. It is concluded that Lys-346 and Lys-353 in B. anthracis adenylyl cyclase may interact directly with ATP and contribute to the binding of the nucleotide to the enzyme.

Type I calmodulin-sensitive adenylyl cyclase is neural specific.

Xia Z, Choi EJ, Wang F, Blazynski C, Storm DR
J Neurochem. 1993; 60: 305-11

The distribution of type I calmodulin-sensitive adenylyl cyclase in bovine and rat tissues was examined by northern blot analysis and in situ hybridization. Northern blot analysis using poly(A)(+)-selected RNA from various bovine tissues indicated that mRNA for type I adenylyl cyclase was found only in brain, retina, and adrenal medulla, suggesting that this enzyme is neural specific. In situ hybridization studies using bovine, rabbit, and rat retina indicated that mRNA for type I adenylyl cyclase is found in all three nuclear layers of the neural retina and is particularly abundant in the inner segment of the photoreceptor cells. The neural-specific distribution of type I adenylyl cyclase mRNA and its restricted expression in areas of brain implicated in neuroplasticity are consistent with the proposal that this enzyme plays an important role in various neuronal functions including learning and memory.

Modification of the calcium and calmodulin sensitivity of the type I adenylyl cyclase by mutagenesis of its calmodulin binding domain.

Wu Z, Wong ST, Storms DR
J Biol Chem. 1993; 268: 23766-8

The type I adenylyl cyclase is directly stimulated by Ca2+ and calmodulin in vitro, and the enzyme is also stimulated by increases in intracellular Ca2+ in vivo. Ca2+ stimulation of the enzyme in vivo may be due to direct interactions of the enzyme with Ca2+ and calmodulin or to an indirect mechanism involving stimulation of the enzyme by Ca(2+)-activated protein kinases. In this study, we have made several point mutations within the calmodulin binding domain to determine if the Ca2+ sensitivity of the enzyme can be modified by mutagenesis. The catalytic activities of the mutant enzymes were comparable to wild type type I adenylyl cyclase. Substitution of Cys-507 with Ser-507 did not have significant effects on the calmodulin or Ca2+ sensitivity of the enzyme. However, replacement of Lys-504 with Asp caused a 4-fold decrease in sensitivity to Ca2+. Ca2+ and calmodulin stimulation were abolished by substitution of Phe-503 with Arg-503. Stimulation of type I adenylyl cyclase activity in vivo by intracellular Ca2+ was also greatly diminished with the Arg-503 mutant indicating that Ca2+ stimulation of the enzyme in vivo is due primarily to direct interactions with calmodulin and Ca2+. These data demonstrate that the Ca2+ sensitivity of this enzyme can be modulated by point mutagenesis within the putative calmodulin binding domain and indicate that the enzyme can be directly regulated by Ca2+ and calmodulin in vivo.

N-glycosylation and residues asn805 and asn890 are involved in the functional properties of type vi adenylyl cyclase.

Wu GC, Lai HL, Lin YW, Chu YT, Chern Y
J Biol Chem. 2001; 276: 35450-7

In this study, we demonstrate that type VI adenylyl cyclase (ACVI) is glycosylated in vivo. Treating HEK293 cells expressing ACVI with tunicamycin to block the addition of N-linked oligosaccharide or removing the N-linked oligosaccharide by in vitro peptidyl-N-glycosidase F digestion reduced the molecular mass of ACVI. Furthermore, tunicamycin treatment suppressed the forskolin-stimulated activity of ACVI. Mutation of either one or both potential N-glycosylation sites (Asn(805) and Asn(890), located on extracellular loops 5 and 6, respectively) also reduced the molecular mass of ACVI. Therefore, ACVI was glycosylated at both Asn(805) and Asn(890). Confocal analysis indicated that glycosylation was not required for the delivery of ACVI to the cell surface. Although no significant alterations in K(m) values for ATP or sensitivity to divalent cations were detected, the glycosylation-deficient ACVI mutant N805Q/N890Q-ACVI exhibited much lower forskolin-, Mn(2+)-, and Mg(2+)-stimulated cyclase activities than did wild-type ACVI. By contrast, the Galpha(s)-stimulated cyclase activities of wild-type ACVI and N805Q/N890Q-ACVI were indistinguishable. Furthermore, compared with wild-type ACVI, N805Q/N890Q-ACVI was less sensitive to inhibition mediated by dopamine D2 receptors or by protein kinase C. Collectively, glycosylation of ACVI not only affected its catalytic activity in an activator-dependent manner, but also altered its ability to be regulated by a Galpha(i) protein-coupled receptor or by protein kinase C.

Receptor guanylyl cyclases.

Wong SK, Garbers DL
J Clin Invest. 1992; 90: 299-305

Three different guanylyl cyclase cell receptors are known, but others will likely be discovered within the next few years. The general function of these receptors appear to relate to the regulation of fluid volume or fluid movement. New receptors, or possibly the currently known receptors, therefore, may be discovered in areas of the body where fluid volume regulation is important. Such fluids whose volume or composition might be regulated by guanylyl cyclase receptors include synovial fluid, uterine/oviductal luminal fluid, follicular fluid, aqueous humor, cerebral spinal fluid, seminiferous tubule luminal fluid, epididymal luminal fluid, seminal plasma, and airway luminal fluid. The function of the heterodimeric forms of guanylyl cyclase appear to relate to a primary regulation of nitric oxide (or similar molecules) concentrations, which are in turn regulated by a Ca2+/calmodulin-sensitive nitric oxide synthase.

Regions on adenylyl cyclase that are necessary for inhibition of activity by beta gamma and G(ialpha) subunits of heterotrimeric G proteins.

Wittpoth C, Scholich K, Yigzaw Y, Stringfield TM, Patel TB
Proc Natl Acad Sci U S A. 1999; 96: 9551-6

The two large cytoplasmic domains (C1 and C2) of adenylyl cyclases (AC), when expressed separately and mixed together, reconstitute enzyme activity that can be regulated by various modulators. Therefore, we have used the C1 or its C1a subdomain and C2 regions from type I AC (ACI) and type V AC (ACV) to identify the region on ACI that interacts with beta gamma subunits of heterotrimeric G proteins. In addition, we also used a chimeric C1 domain (VC1aIC1b) in which the C1a region was derived from ACV and the C1b region was from ACI. By mixing the C1 or C1a or VC1aIC1b domains with C2 regions of ACI or ACV, we have shown that the C1a region (amino acids 236-471) of ACI is sufficient to observe beta gamma-mediated inhibition of enzyme activity, which is stimulated by either constitutively active G(salpha) (G(salpha)*) or Ca(2+)/calmodulin (CaM). Although the C1b region and C2 domain of ACI were by themselves not sufficient for inhibition of activity by beta gamma subunits, the presence of both of these regions formed another beta gamma interaction site that was sufficient to observe G(salpha)*- or Ca(2+)/CaM-stimulated activity. Inhibition of AC activity attributable to interaction of beta gamma subunits at either of the two sites was blocked by a peptide (QEHA) that has previously been shown to inhibit the effects of beta gamma on various effectors. Moreover, the C1 region of ACI was sufficient to observe G(ialpha1)-elicited inhibition of Ca(2+)/CaM-stimulated activity. Although the C1a region of ACV was sufficient for inhibition of activity by G(ialpha1), the presence of C1b region from either ACI or ACV increased sensitivity to inhibition by the inhibitory G protein. Thus, the inhibitory influences of G(ialpha1) are mediated on the C1 regions of both ACI and ACV. The effects of beta gamma on ACI can be mediated by interactions with the C1a region and a beta gamma interacting site formed by the C1b and C2 domains of this enzyme.

Identification of sequences mediating guanylyl cyclase dimerization.

Wilson EM, Chinkers M
Biochemistry. 1995; 34: 4696-701

Deletion mutagenesis was used to identify sequences required for dimerization and enzymatic activity of the intracellular domain of the membrane guanylyl cyclase, GC-A. The intracellular domain of GC-A contains a protein kinase-like domain near its amino terminus, a guanylyl cyclase catalytic domain near its carboxyl terminus, and, between these domains, a region of unknown function predicted to form an amphipathic alpha-helix. Gel filtration analysis of deletion mutants of the GC-A intracellular domain suggested that a 43 amino acid sequence within the interdomain region was both necessary and sufficient for dimerization and was required for guanylyl cyclase catalytic activity. The ability of this sequence to mediate protein dimerization was confirmed in the yeast two-hybrid system, in which its fusion to the lexA DNA-binding domain and to the VP16 transcriptional activation domain led to their dimerization and consequent activation of a lexA-HIS3 gene. Thus, we have identified sequences responsible for dimerization of the intracellular domain of a guanylyl cyclase and shown that they are required for enzyme activity. Modulation of their interaction may be important in guanylyl cyclase activation.

Comparative biological activities of potent active-site analogues of alpha-melanotropin. Effect of tyrosine substitution at position-4.

Wilkes BC, Sawyer TK, Hruby VJ, Hadley ME
Int J Pept Protein Res. 1984; 23: 621-9

We have prepared several alpha-melanotropin (alpha-MSH) analogues with tyrosine substituted for methionine at the 4-position and determined their melanotropic activities on the frog (Rana pipiens), lizard (Anolis carolinensis) and S-91 (Cloudman) mouse melanoma adenylate cyclase bioassays. The potencies of Ac-[Tyr4]-alpha-MSH4-10-NH2 and Ac-[Tyr4]-alpha-MSH4-11-NH2 were compared with alpha-MSH and with their corresponding methionine and norleucine substituted analogues. The Tyr-4 analogues were found to be less active than the Nle-4 analogues on both the frog and lizard assays. Ac-[Tyr4]-alpha-MSH4-10-NH2 was found to be less active than Ac-[Tyr4]-alpha-MSH4-11-NH2 on the lizard bioassay, but more active than the longer fragment on the frog skin assay. Ac-[Tyr4]-alpha-MSH4-10-NH2 exhibited extremely prolonged biological activity on frog skin, but not on lizard skin, while the melanotropic activity of Ac-[Tyr4]-alpha-MSH4-11-NH2 was rapidly reversed on both assay systems. The increased potency of Ac-[Tyr4]-alpha-MSH4-10-NH2 over Ac-[Tyr4]-alpha-MSH4-11-NH2 on frog melanocytes may be related to the fact that the shorter 4-10 analogue exhibits prolonged biological activity. Interestingly, it was found that both Tyr-4 analogues were partial agonists on the mouse melanoma adenylate cyclase bioassay, and stimulated the enzyme to only about 50% of the maximal activity of alpha-MSH. We reported previously that replacement of L-Phe-7 by its D-enantiomer in [Nle4]-alpha-MSH and its Nle-4 containing analogues resulted in peptides with increased potency and in some instances prolonged activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and expression of human adenylyl cyclase type VI in normal thyroid tissues.

Wicker R, Catalan AG, Cailleux A, Starenki D, Stengel D, Sarasin A, Suarez HG
Biochim Biophys Acta. 2000; 1493: 279-83

The adenylyl cyclase type VI gene expressed in human normal thyroid tissue was cloned and sequenced. The cDNA sequence (6463 nt) is susceptible to code for a 1168 aa protein. Northern blots using specific probes showed that the expression of adenylyl cyclase type VI gene was significantly higher in one hyperfunctioning thyroid tumor than in normal thyroid tissue, in one follicular cold adenoma or in one papillary carcinoma.

Interaction of the two cytosolic domains of mammalian adenylyl cyclase.

Whisnant RE, Gilman AG, Dessauer CW
Proc Natl Acad Sci U S A. 1996; 93: 6621-5

Adenylyl cyclase activity can be reconstituted by simple mixture of the two cytosolic domains of the enzyme after their independent synthesis in Escherichia coli. We have synthesized and purified the C1a domain of type I adenylyl cyclase and the C2 domain of the type II enzyme to assess their interactions with each other and with the activators Gsalpha and forskolin. In the absence of an activator, the fragments associate with low affinity and display low catalytic activity. This basal activity can be stimulated more than 100-fold by either forskolin or activated Gsalpha. Further, the addition of these activators increases the apparent affinity of the fragments for each other. Stimulation of catalysis by Gsalpha and forskolin is synergistic. These data suggest a model wherein either Gsalpha or forskolin enhances association of the other activator with adenylyl cyclase, as well as facilitating the interaction between the C1 and C2 domains of the enzyme.

Direct demonstration of the isomerization component of the monoterpene cyclase reaction using a cyclopropylcarbinyl pyrophosphate substrate analog.

Wheeler CJ, Croteau RB
Proc Natl Acad Sci U S A. 1987; 84: 4856-9

The tightly coupled nature of the reaction sequence catalyzed by monoterpene cyclases has precluded direct observation of the topologically required isomerization step leading from geranyl pyrophosphate to the presumptive, enzyme-bound, tertiary allylic intermediate linalyl pyrophosphate, which ultimately cyclizes to the various monoterpene skeletons. By using a partially purified monoterpene cyclase preparation and 2,3-cyclopropylgeranyl pyrophosphate, a substrate analog designed to uncouple the reaction sequence, the production of the corresponding tertiary homoallylic pyrophosphate isomer was demonstrated. This provides direct evidence for the usually cryptic isomerase component of the overall catalytic cycle. A number of other related products generated by reaction of cyclase with the analog were also identified, the structures and proportions of which were consistent with the intermediacy in catalysis of a cyclopropylcarbinyl cation X pyrophosphate anion pair. Kinetic parameters for the analog were compared with those of the natural substrate geranyl pyrophosphate. The results presented confirm mechanistic similarities in the enzymatic ionization and subsequent transformation of allylic pyrophosphate and cyclopropylcarbinyl pyrophosphate intermediates of isoprenoid metabolism.

Gbeta subunit interacts with a peptide encoding region 956-982 of adenylyl cyclase 2. Cross-linking of the peptide to free Gbetagamma but not the heterotrimer.

Weng G, Li J, Dingus J, Hildebrandt JD, Weinstein H, Iyengar R
J Biol Chem. 1996; 271: 26445-8

The region encoded by amino acids 956-982 of adenylyl cyclase 2 is important for Gbetagamma stimulation. Interactions of a peptide encoding the 956-982 region of adenylyl cyclase 2 (QEHAQEPERQYMHIGTMVEFAYALVGK (QEHA peptide)) with Gbetagamma subunits were studied. QEHA peptide was covalently attached to beta subunit of free Gbetagamma by the cross-linker N-succinimidyl(4-iodoacetyl)aminobenzoate. Cross-linking was proportional to the amount of QEHA peptide added; other control peptides cross-linked minimally. When Go was used, very little cross-linking was observed with GDP and EDTA, but upon activation by guanosine 5'-3-O-(thio)triphosphate and Mg2+, specific cross-linking of the QEHA peptide to Gbeta was observed. We conclude that beta subunits of G proteins contain effector interaction domains that are occluded by Galpha subunits in the heterotrimer. Molecular modeling studies used to dock the QEHA peptide on to Gbeta indicate that amino acids 75-165 of Gbeta may be involved in effector interactions.

A functional chimera of mammalian guanylyl and adenylyl cyclases.

Weitmann S, Wursig N, Navarro JM, Kleuss C
Biochemistry. 1999; 38: 3409-13

Adenylyl and guanylyl cyclases synthesize second messenger molecules by intramolecular esterification of purine nucleotides, i.e., cAMP from ATP and cGMP from GTP, respectively. Despite their sequence homology, both families of mammalian cyclases show remarkably different regulatory patterns. In an attempt to define the functional domains in adenylyl cyclase responsible for their isotypic-common activation by Galphas or forskolin, dimeric chimeras were constructed from soluble guanylyl cyclase alpha1 subunit and the C-terminal halves of adenylyl cyclases type I, II, or V. The cyclase-hybrid generated cAMP and was inhibited by P-site ligands. The data establish structural equivalence and the ability of functional complement at the catalytic sites in both cyclases. Detailed enzymatic characterization of the chimeric cyclase revealed a crucial role of the N-terminal adenylyl cyclase half for stimulatory actions, and a major importance of the C-terminal part for nucleotide specificity.

Adenylyl cyclase type ii domains involved in gbetagamma stimulation.

Weitmann S, Schultz G, Kleuss C
Biochemistry. 2001; 40: 10853-8

Mammalian particulate adenylyl cyclases contain two transmembrane regions (M(1) and M(2)) and two cytosolic domains (C(1) and C(2)) forming the catalytic core. The cytosolic domains are subdivided into a highly conserved region (part a) and a region with lower similarity (part b). Hypothetical models exist that account for the mechanism by which Galpha(s) and forskolin stimulate mammalian adenylyl cyclase. In contrast, little is known about how Gbetagamma dimers regulate catalysis. The so-called QEHA region located in the C(2a) domain of type II adenylyl cyclase has been proposed to represent a site of interaction. Here we show (i) that the QEHA region directly interacts with Gbetagamma but (ii) that it is of minor importance for the stimulation of type II adenylyl cyclase because it can be replaced by corresponding, nonidentical regions of other adenylyl cyclase isoforms without altering the stimulatory effect of Gbetagamma and (iii) that the C(1b) region is necessary for Gbetagamma to exert a stimulatory effect on adenylyl cyclase type II as in a C(1b) deletion mutant the Gbetagamma regulation was specifically impeded whereas the Galpha(s)- and forskolin-mediated stimulation was maintained.

A mutation of the atrial natriuretic peptide (guanylyl cyclase-A) receptor results in a constitutively hyperactive enzyme.

Wedel BJ, Foster DC, Miller DE, Garbers DL
Proc Natl Acad Sci U S A. 1997; 94: 459-62

Mutation of an invariant glutamate residue found within the catalytic domain of guanylyl cyclases resulted in a dramatic 14-fold increase in the activity of the guanylyl cyclase-A receptor. Even in the presence of Mn2+/Triton X-100, a treatment previously thought to yield hormone-independent and maximum cyclase activity, the mutant enzyme remained 7-fold more active; to our knowledge, this is the first example of a protein modification or of an added agent that significantly increases cyclase activity in the presence of Mn2+/Triton X-100. Intracellular concentrations of cGMP in cells expressing the mutant (E974A) cyclase were only marginally elevated by the addition of atrial natriuretic peptide, and in broken-cell preparations, the mutant enzyme also was relatively insensitive to ligand/regulatory nucleotide. The marked increase in cyclase activity was not due to a relief of protein kinase domain inhibition, since the point mutation caused 7- to 13-fold elevations in guanylyl cyclase-A activity when the protein kinase homology domain was deleted. The E974A mutation also altered the kinetics from positive cooperative to linear with respect to MnGTP, suggesting disruption of subunit-subunit interactions. Thus, a single point mutation within the catalytic domain of a guanylyl cyclase results in a constitutively hyperactive enzyme that is independent of protein kinase domain regulation.

Functional domains of soluble guanylyl cyclase.

Wedel B, Harteneck C, Foerster J, Friebe A, Schultz G, Koesling D
J Biol Chem. 1995; 270: 24871-5

Soluble guanylyl cyclase is a heterodimer consisting of an alpha and beta subunit and stimulation occurs upon binding of NO to a prosthetic group. Little is known about the localization of catalytic and regulatory domains within the subunits of soluble guanylyl cyclase. We used deletion mutagenesis to identify the regions of alpha 1 and beta 1 subunits that are responsible for cGMP production or NO-heme-mediated activation. The amino terminus of the beta 1 subunit was necessary for NO stimulation since deletion of the 64 NH2-terminal amino acids resulted in a mutant with intact basal activity but complete loss of NO activation. The amino terminus of the alpha 1 subunit also appeared to be essential for NO sensitivity since deletion of 131 NH2-terminal amino acids of alpha 1 led to markedly reduced NO activation. These results suggest that NH2-terminal regions of alpha 1 and beta 1 are involved in NO-heme-mediated signal transduction. The NH2 terminally truncated beta 1 subunit exerted a dominant negative effect exclusively on the NO-stimulated activity of the wild type enzyme, further underlining that the regulatory domain is located within the NH2 terminus of the enzyme. Aside for the structural implications, the mutant represents a powerful tool to investigate nitric oxide-sensitive signaling pathways. Coexpression of the COOH-terminal halves of alpha 1 and beta 1 were sufficient for basal cGMP production while either of the halves expressed alone was inactive. Therefore the COOH-terminal regions appear to contain sufficient information for dimerization and basal enzymatic activity. Thus, we provide the first evidence that the regulatory and catalytic properties of soluble guanylyl cyclase can be attributed to different regions of the subunits and that the catalytic domain can be functionally expressed separately from the NH2-terminal regulatory domain. Taken together with findings on the membrane bound enzyme form, guanylyl cyclases, appear to resemble fusion proteins where different regulatory domains have been joined with a common cGMP-forming segment.

The guanylyl cyclase family at Y2K.

Wedel B, Garbers D
Annu Rev Physiol. 2001; 63: 215-33

During the 1980s the purification, cloning, and expression of various forms of guanylyl cyclase (GC) revealed that they served as receptors for extracellular signals. Seven membrane forms, which presumably exist as homodimers, and four subunits of apparent heterodimers (commonly referred to as the soluble forms) are known, but in animals such as nematodes, much larger numbers of GCs are expressed. The number of transmembrane segments (none, one, or multiple) divide the GC family into three groups. Those with no or one transmembrane segment bind nitric oxide/carbon monoxide (NO/CO) or peptides. There are no known ligands for the multiple transmembrane segment class of GCs. Mutational and structural analyses support a model where catalysis requires a shared substrate binding site between the subunits, whether homomeric or heteromeric in nature. Because some cyclases or cyclase ligand genes lack specific GC inhibitors, disruption of either has been used to define the functions of individual cyclases, as well as to define human genetic disease counterparts.

Molecular cloning and characterization of the type VII isoform of mammalian adenylyl cyclase expressed widely in mouse tissues and in S49 mouse lymphoma cells.

Watson PA, Krupinski J, Kempinski AM, Frankenfield CD
J Biol Chem. 1994; 269: 28893-8

We have isolated a 5199-nucleotide cDNA from a mouse library containing an open reading frame encoding the 1099-amino acid type VII adenylyl cyclase protein. The type VII protein is most closely related in primary structure to an unpublished human adenylyl cyclase clone (GenBank accession no. D25538) and type II adenylyl cyclase. Northern blot analysis demonstrates that the type VII mRNA is most abundant in mouse heart, spleen, and lung. cAMP content rises rapidly in HEK 293 cells overexpressing type VII adenylyl cyclase following treatment with phorbol ester, peaking by 4 min, while cells expressing the type II adenylyl cyclase reach peak accumulation only after 20 min. Increases in intracellular calcium through treatment of type VII-293 cells with either ATP or A23187 alone failed to increase intracellular cAMP content. Phorbol ester treatment acted synergistically with beta-adrenergic stimulation to increase cAMP content in type VII-transformed cells. Pretreatment of type VII-transformed cells with pertussis toxin fails to prevent phorbol ester potentiation of isoproterenol stimulation. Thus the ability of phorbol ester to increase basal and isoproterenol-stimulated type VII activity appears to be a direct effect on this adenylyl cyclase isoform and not the result of modification of the inhibitory G protein, Gi.

Type I and type VIII adenylyl cyclases constitute a family whose activation is coupled to cellular deformation through the action of calcium-calmodulin.

Watson PA, Giger KE, Kempinski AM
Biochem Cell Biol. 1995; 73: 367-72

In certain tissues and cells, increases in concentrations of the second messenger cAMP are seen in response to mechanical or deformational stimuli. Type I and type VIII adenylyl cyclases, representing members of a family of calcium-calmodulin-stimulated adenylyl cyclases, and type VII adenylyl cyclase were each stably expressed in human embryonal kidney (HEK) 293 cells. HEK 293 cells exogenously expressing either type I adenylyl cyclase or any one of three type VIII adenylyl cyclase splice variants respond to swelling with increases in cAMP, requiring the presence of calcium in the extracellular medium for such responsiveness. Type VII expressing HEK 293 cells failed to respond to swelling with increased cAMP but demonstrated potentiation of isoproterenol-stimulated activity. This is characteristic of the influence of protein kinase C on the activity of the type VII protein. The relative swelling responsiveness of HEK 293 cells expressing splice variants of the type VIII adenylyl cyclase is consistent with the relative EC50 values for calcium-calmodulin stimulation of these splice variants. This is consistent with the involvement of calmodulin and the requirement for increases in intracellular calcium in mediating swelling-induced acceleration of type VIII adenylyl cyclase activity.

Cell-free synthesis of functional type IV adenylyl cyclase.

Warner DR, Basi NS, Rebois RV
Anal Biochem. 1995; 232: 31-6

Type IV adenylyl cyclase was synthesized in a cell-free coupled transcription and translation system. Radiolabeled type IV adenylyl cyclase was specifically immune precipitated with anti-ACIV antibodies. The molecular weight of in vitro translated type IV adenylyl cyclase was 110,000, similar to that for type IV adenylyl cyclase produced in the baculovirus system [B. Gao and A. G. Gilman, (1991) Proc. Natl. Acad. Sci. USA 88, 10178-10182]. Dimyristoyl phosphatidylcholine was required for efficient stimulation of activity by both forskolin and GS, with a maximum specific activity of 700 +/- 100 nmol cAMP.min-1.mg-1 attained with both effectors combined. Both bovine brain GS and in vitro translated GS alpha activated in vitro translated type IV adenylyl cyclase; however, G beta gamma only enhanced stimulation in the presence of in vitro translated GS alpha. Forskolin maximally activated at concentrations from 200 to 400 microM in the absence or presence of GS. The in vitro translated product was very stable as production of cAMP by forskolin/GS activated type IV adenylyl cyclase was linear for up to 90 min at 30 degrees C.

Analysis of the function of the 70-kilodalton cyclase-associated protein (CAP) by using mutants of yeast adenylyl cyclase defective in CAP binding.

Wang J, Suzuki N, Nishida Y, Kataoka T
Mol Cell Biol. 1993; 13: 4087-97

In Saccharomyces cerevisiae, adenylyl cyclase forms a complex with the 70-kDa cyclase-associated protein (CAP). By in vitro mutagenesis, we assigned a CAP-binding site of adenylyl cyclase to a small segment near its C terminus and created mutants which lost the ability to bind CAP. CAP binding was assessed first by observing the ability of the overproduced C-terminal 150 residues of adenylyl cyclase to sequester CAP, thereby suppressing the heat shock sensitivity of yeast cells bearing the activated RAS2 gene (RAS2Val-19), and then by immunoprecipitability of adenylyl cyclase activity with anti-CAP antibody and by direct measurement of the amount of CAP bound. Yeast cells whose chromosomal adenylyl cyclase genes were replaced by the CAP-nonbinding mutants possessed adenylyl cyclase activity fully responsive to RAS2 protein in vitro. However, they did not exhibit sensitivity to heat shock in the RAS2Val-19 background. When glucose-induced accumulation of cyclic AMP (cAMP) was measured in these mutants carrying RAS2Val-19, a rapid transient rise indistinguishable from that of wild-type cells was observed and a high peak level and following persistent elevation of the cAMP concentration characteristic of RAS2Val-19 were abolished. In contrast, in the wild-type RAS2 background, similar cyclase gene replacement did not affect the glucose-induced cAMP response. These results suggest that the association with CAP, although not involved in the in vivo response to the wild-type RAS2 protein, is somehow required for the exaggerated response of adenylyl cyclase to activated RAS2.

Repression of adipogenesis by adenylyl cyclase stimulatory G-protein alpha subunit is expressed within region 146-220.

Wang Hy, Johnson GL, Liu X, Malbon CC
J Biol Chem. 1996; 271: 22022-9

The heterotrimeric G-protein alpha subunit stimulatory with respect to adenylyl cyclase (Gsalpha) represses adipogenesis of 3T3-L1 mouse embryonic fibroblasts. Derepression occurs in response to inducers, to oligodeoxynucleotides antisense to Gsalpha, and to overexpression of heterotrimeric G-protein alpha subunit 2, inhibitory with respect to adenylyl cyclase (Gialpha2). Constitutive expression of Gsalpha blocks adipogenesis and was exploited as an assay, in which chimeras of Gialpha2 and Gsalpha were expressed stably in 3T3-L1 cells to define the region controlling adipogenesis. N-terminal analysis revealed region 146-220 of Gsalpha as a repressor of adipogenesis; substitution of Gialpha2 abolished the ability of the chimera to repress adipogenesis in response to inducers. Expression of a chimera in which the 146-235 region of Gsalpha was embedded in Gialpha2 fully repressed adipogenesis in response to the inducers. C-terminal analysis revealed no loss of function for truncated Gsalpha, lacking the terminal 38 residues. The repressor domain for adipogenesis maps to a region that includes switch domains I and II and is spatially distinct from the regions mapped for control of adenylyl cyclase.

Molecular cloning and expression of a novel type V adenylyl cyclase from rabbit myocardium.

Wallach J, Droste M, Kluxen FW, Pfeuffer T, Frank R
FEBS Lett. 1994; 338: 257-63

A cDNA of a novel form of type V adenylyl cyclase has been cloned from rabbit myocardium using oligonucleotide probes derived from peptides that were produced by enzymatic cleavage of purified heart cyclase. A corresponding mRNA (6 kb) has been detected in rabbit myocardial tissue by Northern blot analysis. The cDNA encodes a protein of 1,264 amino acids exhibiting 12 putative membrane-spanning regions in its hydrophilicity profile. Sequence comparison to two other previously published type V adenylyl cyclase reveals amino-terminal domains of different length and low correlative homology, whereas the rest of the sequences is almost identical. The nonconserved amino-terminal region of the subtype consists of 214 amino acids and exceeds the length of the others by 40 and 80 residues, respectively. Its presence in membrane preparations from different tissues has been confirmed immunologically using an antibody directed against a synthetic peptide. The cloned adenylyl cyclase was functionally expressed in COS-1 cells to attain an enzymatic activity 3.5- to 14-fold above control in the presence of forskolin.

Identification of ligand recognition sites in heat-stable enterotoxin receptor, membrane-associated guanylyl cyclase C by site-directed mutational analysis.

Wada A, Hirayama T, Kitaura H, Fujisawa J, Hasegawa M, Hidaka Y, Shimonishi Y
Infect Immun. 1996; 64: 5144-50

Guanylyl cyclase C (STaR), a receptor protein for heat-stable enterotoxin (STa) elaborated by Escherichia coli, is associated with and spans the plasma membrane of mammalian intestinal cells. The extracellular domain functions in the binding of STa and the association of each domain to an oligomeric form. Two amino acid residues, Arg-136 and Asp-347, were identified as the residues binding to STa in the extracellular domain of pig STaR by site-directed mutagenesis and analysis of expression on 293T cells. Replacement of these residues by other amino acid residues resulted in the loss of binding of pig STaR to STa, and as a result, STa-induced guanylyl cyclase activity was eliminated. Furthermore, mutation in a region (from Asp-347 to Val-401) which is close to the transmembrane domain caused a significant reduction in both STa-binding activity and guanylyl cyclase catalytic activity. These results suggest that the region adjacent to the transmembrane domain plays an important role in facilitating a favorable conformation of STaR for STa binding.

Cloning and expression of a bovine adenylyl cyclase type VII specific to the retinal pigment epithelium.

Volkel H, Beitz E, Klumpp S, Schultz JE
FEBS Lett. 1996; 378: 245-9

A cDNA of a type 7 adenylyl cyclase isoform was cloned from a bovine retinal pigment epithelium cDNA library using oligonucleotides developed to conserved regions common to mammalian adenylyl cyclases. A 6.7 kb mRNA of very high abundance was uniquely present on Northern blots containing mRNA or total RNA from the pigment epithelium. This transcript was undetectable in all other tissues examined. The cDNA encoded a protein of 1,097 amino acids and exhibited the known doublet of 6 transmembrane-spanning regions in a hydrophobicity plot. The novel member of the type 7 adenylyl cyclase isoform was expressed in COS-1 cells. It was stimulated 10- and 20-fold by 10 microM GTP gamma S and 100 microM forskolin, respectively. The high expression rate exclusively in the retinal pigment epithelium suggests that this adenylyl cyclase isoform is involved in processes specific to this functionally exceedingly important subretinal cell layer.

Cloning, chromosomal mapping, and expression of human fetal brain type I adenylyl cyclase.

Villacres EC, Xia Z, Bookbinder LH, Edelhoff S, Disteche CM, Storm DR
Genomics. 1993; 16: 473-8

The neural-specific calmodulin-sensitive adenylyl cyclase (type I), which was first cloned from bovine brain, has been implicated in learning and memory. The objective of this study was to clone and determine the chromosomal localization of human fetal brain type I adenylyl cyclase. A 3.8-kb cDNA clone was isolated that contained sequence coinciding with the 3' end 2553 nucleotides of the bovine open reading frame. This clone shows 87% nucleotide and 92% translated amino acid sequence identity to the bovine clone. The most significant sequence differences were in the carboxy-terminal 100 amino acid residues. This region contains one of several possible calmodulin binding domains and the only putative cAMP-dependent protein kinase A phosphorylation site. A chimera was constructed that contained the 5' half of the bovine type I adenylyl cyclase and the 3' half of the human type I adenylyl cyclase. The activity of the chimeric gene product and its sensitivity to calmodulin and calcium were indistinguishable from those of the bovine type I adenylyl cyclase. In situ hybridization was used to localize the human type I adenylyl cyclase gene to the proximal portion of the short arm of chromosome 7.

Biochemical characterization of the intracellular domain of the human guanylyl cyclase C receptor provides evidence for a catalytically active homotrimer.

Vijayachandra K, Guruprasad M, Bhandari R, Manjunath UH, Somesh BP, Srinivasan N, Suguna K, Visweswariah SS
Biochemistry. 2000; 39: 16075-83

Guanylyl cyclase C (GCC) is the receptor for the family of guanylin peptides and bacterial heat-stable enterotoxins (ST). The receptor is composed of an extracellular, ligand-binding domain and an intracellular domain with a region of homology to protein kinases and a guanylyl cyclase catalytic domain. We have expressed the entire intracellular domain of GCC in insect cells and purified the recombinant protein, GCC-IDbac, to study its catalytic activity and regulation. Kinetic properties of the purified protein were similar to that of full-length GCC, and high activity was observed when MnGTP was used as the substrate. Nonionic detergents, which stimulate the guanylyl cyclase activity of membrane-associated GCC, did not appreciably increase the activity of GCC-IDbac, indicating that activation of the receptor by Lubrol involved conformational changes that required the transmembrane and/or the extracellular domain. The guanylyl cyclase activity of GCC-IDbac was inhibited by Zn(2+), at concentrations shown to inhibit adenylyl cyclase, suggesting a structural homology between the two enzymes. Covalent cross-linking of GCC-IDbac indicated that the protein could associate as a dimer, but a large fraction was present as a trimer. Gel filtration analysis also showed that the major fraction of the protein eluted at a molecular size of a trimer, suggesting that the dimer detected by cross-linking represented subtle differences in the juxtaposition of the individual polypeptide chains. We therefore provide evidence that the trimeric state of GCC is catalytically active, and sequences required to generate the trimer are present in the intracellular domain of GCC.

Signal transduction: activation of the guanylate cyclase-cyclic guanosine-3'-5' monophosphate system by hormones and free radicals.

Vesely DL
Am J Med Sci. 1997; 314: 311-23

Intracellular communication and transmission of messages for many hormones and free radicals occur after the hormones and free radicals bind to their receptors by enhancing the activity of guanylate cyclase, the enzyme that catalyzes the conversion of guanosine triphosphate to the intracellular messenger cyclic guanosine-3'-5' monophosphate (cyclic GMP). The guanylate cyclase-linked receptors exist intracellularly (ie, cytoplasmic) and in membrane-bound forms. Enhancement of guanylate cyclase by hormones or free radicals increases intracellular cyclic GMP, which closes cation channels in the kidney while activating cation channels in the retina and olfactory cilia, either directly or by cyclic GMP-dependent protein kinase. Cyclic GMP also has potent blood pressure lowering properties. Cyclic GMP promotes growth by increasing DNA, RNA, and protein synthesis. Overactivity of this system is observed in Traveler's diarrhea, whereas underactivity occurs in Chediak-Higashi syndrome in which lysosomal enzyme release and chemotaxis are defective and can be corrected in vitro by addition of cyclic GMP.

Identification of adenylyl cyclase isoenzymes in CHO and B82 cells.

Varga EV, Stropova D, Rubenzik M, Wang M, Landsman RS, Roeske WR, Yamamura HI
Eur J Pharmacol. 1998; 348: 12-12

The identification of adenylyl cyclase isoenzymes in mammalian host cells is important for the interpretation of data obtained from cell lines heterologously expressing G-protein coupled receptors. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to amplify adenylyl cyclase cDNAs from Chinese hamster ovary (CHO) and mouse fibroblast (B82) cells. The isolated fragments were identified by restriction analyses and by sequencing. We found mRNAs for adenylyl cyclases VI and VII in CHO and adenylyl cyclases IX and VII in B82 cells.

Adenylate and guanylate cyclases in Tetrahymena.

Umeki S, Nozawa Y
Prog Mol Subcell Biol. 1996; 17: 40-60

Biochemical analysis of a dimerization domain mutation in RetGC-1 associated with dominant cone-rod dystrophy.

Tucker CL, Woodcock SC, Kelsell RE, Ramamurthy V, Hunt DM, Hurley JB
Proc Natl Acad Sci U S A. 1999; 96: 9039-44

Mutations in the photoreceptor membrane guanylyl cyclase RetGC-1 have been linked to autosomal dominant cone-rod dystrophy. Three mutations were identified that alter strictly conserved residues within the RetGC-1 dimerization domain, a region predicted to form an amphipathic alpha-helical coil. Here we report on a biochemical characterization of one of the mutations, a substitution of cysteine for arginine at residue 838. We generated this mutation in vitro and measured its catalytic activity and sensitivity to guanylyl cyclase activating protein 1 (GCAP-1) and GCAP-2. The R838C substitution has several effects. It reduces the overall catalytic ability of RetGC-1 and dramatically reduces stimulation by GCAP-2, although GCAP-2 still appears to interact with the protein. The R838C substitution also increases the apparent affinity of RetGC-1 for GCAP-1 and alters the Ca(2+) sensitivity of the GCAP-1 response, allowing the mutant to be stimulated by GCAP-1 at higher Ca(2+) concentrations than wild type. The diminished response to GCAP-2, which we propose is not likely the cause of cone-rod degeneration in these patients, is interesting mechanistically because it separates the ability to bind a specific GCAP from the ability to be stimulated by it, and it also discriminates between the mechanisms of activation of GCAP-1 vs. GCAP-2. We suggest that the gain-of-function effects of R838C on RetGC-1 stimulated by GCAP-1, which are dominant in vitro and may cause an abnormal increase in cGMP synthesis in dark-adapted photoreceptors, may be the cause of the cone-rod degeneration.

Domain-specific stabilization of photoreceptor membrane guanylyl cyclase by adenine nucleotides and guanylyl cyclase activating proteins (GCAPs).

Tucker CL, Laura RP, Hurley JB
Biochemistry. 1997; 36: 11995-2000

In photoreceptor outer segments, particulate guanylyl cyclase (RetGC) is stimulated at low intracellular Ca2+ concentrations by guanylyl cyclase activating protein (GCAP), a Ca2+-sensitive activator, to resynthesize light-depleted cGMP. In washed outer segment membranes, we find that GCAP-stimulable RetGC is rapidly inactivated at physiological temperatures (30-37 degrees C). Under the same conditions, RetGC remains competent for stimulation by S-100 protein preparations or Mn2+/Triton X-100, indicating that the cyclase catalytic domain remains functional. GCAPs and adenine nucleotides protect against inactivation. Protection by GCAPs is independent of Ca2+ concentration, suggesting that there is a Ca2+-independent interaction between GCAP and RetGC. Protection by ATP (EC50 = 150 microM) is not due to phosphorylation, since the nonhydrolyzable analogue adenylyl imidodiphosphate (AMP-PNP) protects equally well. In addition to their roles in protection, ATP and AMP-PNP also slowly stimulate cyclase activity. In parallel with the functional change in RetGC at physiological temperatures, we also observe a structural change. A 62-kDa intracellular fragment of RetGC-1 becomes more sensitive to cleavage by trypsin after preincubation at 30 degrees C unless ATP, AMP-PNP, or GCAP is present. Adenine nucleotides and GCAPs thus protect RetGC structurally, as well as functionally.

Forskolin derivatives with increased selectivity for cardiac adenylyl cyclase.

Toya Y, Schwencke C, Ishikawa Y
J Mol Cell Cardiol. 1998; 30: 97-108

The current study was undertaken to examine whether we can target adenylyl cyclase to regulate beta-adrenergic signaling with increased cardiac selectivity. Forskolin, a natural diterpene compound, interacts directly with adenylyl cyclase. We studied the adenylyl cyclase isoform-selectivity of forskolin derivatives using insect cell membranes overexpressing type II, III, and V adenylyl cyclase isoforms. 6-[3-(dimethylamino) propionyl] forskolin (NKH477) stimulated type V more potently (1.87 +/- 0.02-fold) than type II (1.04 +/- 0.02-fold) and type III (0.89 +/- 0.03-fold) relative to forskolin (50 microM, P < 0.05). Similarly, 6-[3-(dimethylamino)propionyl]-14,15-dihydro-forskolin (DMAPD) stimulated type V (1.39 +/- 0.02-fold) more potently than types II (0.66 +/- 0.02-fold) and type III (0.31 +/- 0.02-fold) relative to forskolin (P < 0.05). This selectivity was maintained under different assay conditions--i.e. with different forskolin (0.1-100 microM) and Mg (1-10 mM) concentrations, with or without Gs alpha. NKH477 increased cAMP accumulation in HEK293 cells stably overexpressing type V more than forskolin (1.57 +/- 0.13-fold) (P < 0.05). Examination of multiple tissue homogenates revealed that DMAPD and NKH477 stimulated cardiac adenylyl cyclase more potently than the other tissue adenylyl cyclases (lung, brain, and kidney) relative to forskolin. Our results suggest that a particular side-chain modification of forskolin enhanced the selectivity for the cardiac isoform stimulation. Adenylyl cyclase isoforms may be targeted to increase tissue selectivity in future drug therapy for beta-adrenergic regulation.

Inhibition of adenylyl cyclase by caveolin peptides.

Toya Y, Schwencke C, Couet J, Lisanti MP, Ishikawa Y
Endocrinology. 1998; 139: 2025-31

Caveolae and their principal component caveolin have been implicated in playing a major role in G protein-mediated transmembrane signaling. We examined whether caveolin interacts with adenylyl cyclase, an effector of G protein signaling, using a 20-mer peptide derived from the N-terminus scaffolding domain of caveolin-1. When tissue adenylyl cyclases were examined, cardiac adenylyl cyclase was inhibited more potently than other tissue adenylyl cyclases. The caveolin-1 peptide inhibited type V, as well as type III adenylyl cyclase, overexpressed in insect cells, whereas the same peptide had no effect on type II. The caveolin-3 scaffolding domain peptide similarly inhibited type V adenylyl cyclase. In contrast, peptides derived from the caveolin-2 scaffolding domain and a caveolin-1 nonscaffolding domain had no effect. Kinetic studies showed that the caveolin-1 peptide decreased the maximal rate (Vmax) value of type V without changing the Michaelis constant (Km) value for the substrate ATP. Studies with various truncations and point mutations of this peptide revealed that a minimum of 16 amino acid residues and intact aromatic residues are important for the inhibitory effect. The potency of inhibition was greater when adenylyl cyclase was in stimulated condition vs. basal condition. Thus, caveolin may be another cellular component that regulates adenylyl cyclase catalytic activity. Our results also suggest that the caveolin peptide may be used as an isoform-selective inhibitor of adenylyl cyclase.

Molecular cloning and expression of an adenylyl cyclase from Xenopus laevis oocytes.

Torrejon M, Echeverria V, Retamales G, Herrera L, Hinrichs MV, Olate J
FEBS Lett. 1997; 404: 91-4

We have cloned a cDNA that encodes a novel Xenopus laevis oocyte adenylyl cyclase (xlAC) using oligonucleotides against conserved mammalian adenylyl cyclase regions. The isolated cDNA is 4372 bp long with an open reading frame of 4065 nucleotides which encodes a protein of 1355 amino acids. Comparison of the deduced amino acid sequence with previously cloned mammalian adenylyl cyclases shows a low identity, 19.7% with type 2 rat adenylyl cyclase and 24.2% with type 4 rat adenylyl cyclase, indicating that this Xenopus isoform represents a new member of this protein family. Gene expression studies of the xlAC by reverse PCR showed that this gene is expressed in all oogenesis stages but not during early embryogenesis. Expression of the xlAC in COS-7 cells resulted in increased basal AC activity, that was stimulated by forskolin, Gpp(NH)p and aluminium fluoride, and was insensitive to calcium and calcium-calmodulin (Ca2(+)-CaM).

Dominant negative mutations of the guanylyl cyclase-A receptor. Extracellular domain deletion and catalytic domain point mutations.

Thompson DK, Garbers DL
J Biol Chem. 1995; 270: 425-30

Guanylyl cyclase-A (GC-A), a receptor for A-type natriuretic peptide (ANP), contains an extracellular ligand-binding domain, a single transmembrane domain, and intracellular protein kinase-like and cyclase catalytic domains. Expression of the putative cyclase catalytic region (HCAT) resulted in the formation of an active enzyme that migrated as a homodimer on gel filtration columns; treatment with sodium trichloroacetate caused dissociation of the dimer and a loss of cyclase activity. Co-transfection of HCAT and full-length GC-A led to elevated basal intact cell cGMP concentrations and increased cell homogenate guanylyl cyclase activity. However, atrial natriuretic peptide-induced elevations of cGMP and cyclase activity were inhibited by the introduction of HCAT. Alanine scanning mutagenesis of highly conserved residues within HCAT identified one mutation (D893A) that destroyed enzyme activity but not the ability of the mutant subunit to form homodimers. The mutant subunit inhibited the cyclase activity of wild-type HCAT (approximately 70%) as well as that of full-length GC-A (approximately 85%) in co-expression studies where the amount of wild-type HCAT or full-length GC-A was not altered. Unlike co-transfection with wild-type HCAT, co-transfection of HCA-TD893A and GC-A did not result in elevated basal intact cell cGMP concentrations. For the first time we describe deletion and point mutations within the plasma membrane family of guanylyl cyclase receptors that result in the formation of effective dominant negative proteins.

Two-metal-Ion catalysis in adenylyl cyclase.

Tesmer JJ, Sunahara RK, Johnson RA, Gosselin G, Gilman AG, Sprang SR
Science. 1999; 285: 756-60

Adenylyl cyclase (AC) converts adenosine triphosphate (ATP) to cyclic adenosine monophosphate, a ubiquitous second messenger that regulates many cellular functions. Recent structural studies have revealed much about the structure and function of mammalian AC but have not fully defined its active site or catalytic mechanism. Four crystal structures were determined of the catalytic domains of AC in complex with two different ATP analogs and various divalent metal ions. These structures provide a model for the enzyme-substrate complex and conclusively demonstrate that two metal ions bind in the active site. The similarity of the active site of AC to those of DNA polymerases suggests that the enzymes catalyze phosphoryl transfer by the same two-metal-ion mechanism and likely have evolved from a common ancestor.

Crystal structure of the catalytic domains of adenylyl cyclase in a complex with Gsalpha.GTPgammaS.

Tesmer JJ, Sunahara RK, Gilman AG, Sprang SR
Science. 1997; 278: 1907-16

The crystal structure of a soluble, catalytically active form of adenylyl cyclase in a complex with its stimulatory heterotrimeric G protein alpha subunit (Gsalpha) and forskolin was determined to a resolution of 2.3 angstroms. When P-site inhibitors were soaked into native crystals of the complex, the active site of adenylyl cyclase was located and structural elements important for substrate recognition and catalysis were identified. On the basis of these and other structures, a molecular mechanism is proposed for the activation of adenylyl cyclase by Gsalpha.

The structure, catalytic mechanism and regulation of adenylyl cyclase.

Tesmer JJ, Sprang SR
Curr Opin Struct Biol. 1998; 8: 713-9

The recent structure determinations of the mammalian effector enzyme adenylyl cyclase reveal the structure of its catalytic core, provide new insights into its catalytic mechanism and suggest how diverse signaling molecules regulate its activity.

Molecular basis for P-site inhibition of adenylyl cyclase.

Tesmer JJ, Dessauer CW, Sunahara RK, Murray LD, Johnson RA, Gilman AG, Sprang SR
Biochemistry. 2000; 39: 14464-71

P-site inhibitors are adenosine and adenine nucleotide analogues that inhibit adenylyl cyclase, the effector enzyme that catalyzes the synthesis of cyclic AMP from ATP. Some of these inhibitors may represent physiological regulators of adenylyl cyclase, and the most potent may ultimately serve as useful therapeutic agents. Described here are crystal structures of the catalytic core of adenylyl cyclase complexed with two such P-site inhibitors, 2'-deoxyadenosine 3'-monophosphate (2'-d-3'-AMP) and 2',5'-dideoxyadenosine 3'-triphosphate (2',5'-dd-3'-ATP). Both inhibitors bind in the active site yet exhibit non- or uncompetitive patterns of inhibition. While most P-site inhibitors require pyrophosphate (PP(i)) as a coinhibitor, 2',5'-dd-3'-ATP is a potent inhibitor by itself. The crystal structure reveals that this inhibitor exhibits two binding modes: one with the nucleoside moiety bound to the nucleoside binding pocket of the enzyme and the other with the beta and gamma phosphates bound to the pyrophosphate site of the 2'-d-3'-AMP.PP(i) complex. A single metal binding site is observed in the complex with 2'-d-3'-AMP, whereas two are observed in the complex with 2', 5'-dd-3'-ATP. Even though P-site inhibitors are typically 10 times more potent in the presence of Mn(2+), the electron density maps reveal no inherent preference of either metal site for Mn(2+) over Mg(2+). 2',5'-dd-3'-ATP binds to the catalytic core of adenylyl cyclase with a K(d) of 2.4 microM in the presence of Mg(2+) and 0.2 microM in the presence of Mn(2+). Pyrophosphate does not compete with 2',5'-dd-3'-ATP and enhances inhibition.

An adenylate cyclase, Cya1, regulates cell motility in the cyanobacterium Synechocystis sp. PCC 6803.

Terauchi K, Ohmori M
Plant Cell Physiol. 1999; 40: 248-51

The cyanobacterium Synechocystis sp. PCC 6803 contained two genes, cya1 and cya2, encoding putative adenylate cyclases. The wild type cells are motile, whereas the disruption mutants of cya1, but not cya2, were immotile. Disruption of the cya1 gene also caused reduction of cellular cAMP level. The cya1 mutant cells regained motility when cAMP was added exogenously.

Trypanosoma cruzi adenylyl cyclase is encoded by a complex multigene family.

Taylor MC, Muhia DK, Baker DA, Mondragon A, Schaap PB, Kelly JM
Mol Biochem Parasitol. 1999; 104: 205-17

The parasitic protozoan Trypanosoma cruzi undergoes several differentiation events during its life cycle. Some of these transitions are thought to involve activation of adenylyl cyclase via the binding of peptide ligands to the cell surface. Here we describe the characterisation of the adenylyl cyclase gene family of T. cruzi. Two complete genes and one pseudogene have been sequenced. The protein products appear to have a large extracellular domain, a single transmembrane helix and a cytosolic catalytic domain. The adenylyl cyclase genes are present on at least six chromosomes and are scattered rather than clustered. They form a large polymorphic family in which the extracellular domain is particularly variable. An Escherichia coli adenylyl cyclase mutant could be complemented by expression of the catalytic domain of the T. cruzi enzyme. The recombinant protein had adenylyl cyclase activity in vitro, which was enhanced by increasing concentrations of divalent cations (Mn2+ > Mg2+). This constitutively active recombinant protein will be a useful tool for dissecting the catalytic mechanism of adenylyl cyclase.

Hypoxia inhibits cyclic nucleotide-stimulated epithelial ion transport: role for nucleotide cyclases as oxygen sensors.

Taylor CT, Lisco SJ, Awtrey CS, Colgan SP
J Pharmacol Exp Ther. 1998; 284: 568-75

Decreased oxygen delivery to cells (hypoxia) is prevalent in a number of important diseases. Little is known about mechanisms of oxygen sensing at the cellular level or about whether functional correlates of oxygen sensing exist. In this study, we examined the impact of hypoxia on stimulated epithelial ion transport function. T84 cells, a model of intestinal epithelia, were grown on permeable supports, exposed to hypoxia (range 1-21% O2) for periods of time between 0 and 72 h and assessed for stimulated ion transport. Hypoxia evoked a specific decrease in cyclic nucleotide-stimulated (cAMP and cGMP) but not Ca++-stimulated ion transport. 86Rb (K+ tracer) uptake and 125I (Cl- tracer) efflux were reduced in hypoxic cells by >50% and >40%, respectively, fluid movement was reduced by hypoxia (>50% decrease) and reoxygenation resulted in partial recovery of the ion transport responses. Stimulated and basal levels of both cAMP and cGMP were decreased in response to hypoxia, although intracellular ATP levels were unaltered under similar conditions. Exogenous addition of cobalt, nickel or manganese, all of which compete for oxygen binding on heme-containing proteins, mimicked hypoxia. Because guanylate cyclase is a heme protein, we measured the influence of cobalt on activity of guanylate cyclase in purified plasma membrane preparations and found cobalt to inhibit stimulated cGMP levels in this cell-free system. Finally, pharmacological lowering of intracellular cGMP (using LY83583) resulted in decreased cAMP-stimulated Cl- secretion, and direct elevation of cGMP (using 8-bromo-cGMP or dibutyryl-cGMP) restored this hypoxia-induced activity. We conclude that a potential oxygen-sensing mechanism of epithelial cells involves the cooperation of heme-containing proteins such as guanylate cyclase and that biochemical cross-talk between cAMP- and cGMP-stimulated pathways may be important in such responses.

Type-specific regulation of mammalian adenylyl cyclases by G protein pathways.

Taussig R, Zimmermann G
Adv Second Messenger Phosphoprotein Res. 1998; 32: 81-98

Distinct patterns of bidirectional regulation of mammalian adenylyl cyclases.

Taussig R, Tang WJ, Hepler JR, Gilman AG
J Biol Chem. 1994; 269: 6093-100

The capacities of the alpha subunits of pertussis toxin-sensitive guanine nucleotide-binding regulatory proteins (G proteins) to inhibit different isoforms of mammalian adenylyl cyclases were assessed. Membranes from Sf9 cells infected with recombinant baculoviruses encoding either type I, II, V, or VI adenylyl cyclase were reconstituted with purified G protein subunits. Types V and VI adenylyl cyclase are most sensitive to inhibition by Gi alpha 1, Gi alpha 2, and Gi alpha 3; type I adenylyl cyclase can be inhibited by these three Gi alpha proteins and by G(o) alpha as well. Type II adenylyl cyclase appears to be immune to inhibition by these proteins. Examination of the effects of native and mutant Gi alpha proteins, as well as analysis of competition for binding of Gs alpha to adenylyl cyclases, indicate that at least certain adenylyl cyclases have independent sites for interaction with Gs alpha (site 1, stimulatory) and Gi alpha (site 2, inhibitory). High concentrations of Gi alpha can interact with site 1 on types I and II adenylyl cyclase and activate the enzymes. Types I and II adenylyl cyclase also appear to have independent sites for interaction with G protein beta gamma subunits. The type I enzyme is strongly inhibited, while type II adenylyl cyclase is activated if Gs alpha is also present.

Mammalian membrane-bound adenylyl cyclases.

Taussig R, Gilman AG
J Biol Chem. 1995; 270: 1-4

Forskolin derivatives. I. Synthesis, and cardiovascular and adenylate cyclase-stimulating activities of water-soluble forskolins.

Tatee T, Narita A, Narita K, Izumi G, Takahira T, Sakurai M, Fujita A, Hosono M, Yamashita K, Enomoto K, Shiozawa A
Chem Pharm Bull (Tokyo). 1996; 44: 2274-9

Water-soluble forskolin and 7-deacetylforskolin derivatives with an aminoacetyl, a 3-aminopropionyl, or a 4-aminobutyryl group at the 6- or 7-position were prepared, and their positive inotropic as well as vasodilative activities were evaluated in anesthetized dogs. 7-Deacetylforskolin (2) and 7-deacetyl-1-silylforskolin (6) were converted to the corresponding 7-chloroacylderivatives (3, 7, 10), which were reacted with amines to obtain 7-aminoacyl-7-deacetylforskolins (4a-f, 9a, b, 11). The 7-acyl substituents migrated to the 6-position with sodium hydroxide in acetonitrile-water to afford 6-aminoacyl-7-deacetylforskolins (12a-f). The 7-position of 12a, d-f was selectively acetylated with acetyl chloride to obtain the corresponding 6-aminoacylforskolins (13a-d). Among the 6-aminoacylforskolins, 6-(3-dimethylaminopropionyl)forskolin (13b) and 6-(4-dimethylaminobutyryl)forskolin (13d) exhibited potent positive inotropic and vasodilative activities comparable to those of forskolin (1). The activities of 13b and 13d were approximately ten times more potent than those of 7-aminoacyl- and 6-aminoacyl-7-deacetylforskolins (4a-f, 9a, 12a-c, f). 6-Dimethylaminoacetylforskolin (13a) and 6-(3-diethylaminopropionyl)forskolin (13c) were less potent than 1. The effects of the soluble forskolins on adenylate cyclase activity were also examined in vitro. 6-Aminoacylforskolins (13a-d) exhibited potent adenylate cyclase-stimulating activity, comparable to that of 1.

Class III adenylyl cyclases: regulation and underlying mechanisms.

Tang WJ, Yan S, Drum CL
Adv Second Messenger Phosphoprotein Res. 1998; 32: 137-51

Truncation and alanine-scanning mutants of type I adenylyl cyclase.

Tang WJ, Stanzel M, Gilman AG
Biochemistry. 1995; 34: 14563-72

A variety of truncated constructs of type I and type II adenylyl cyclase have been synthesized in Sf9 cells using recombinant baculoviruses, as have a number of type I adenylyl cyclases with point mutations. Truncations indicate that the nonconserved C1b and C2b domains of adenylyl cyclase are not necessary for regulation of enzymatic activity by Gs alpha and forskolin. Point mutations demonstrate the requirement for both of the conserved (and homologous) domains of adenylyl cyclase (C1a and C2a) and the nonequivalence of these domains. Linkage of certain effects of mutations on the Km for substrate with alterations of the characteristics of P-site inhibition suggest that ATP and P-site inhibitors may bind to different conformations of the same site. However, other mutations affected only P-site inhibition. Although the mutations studied have not permitted assignment of unique functions to the two homologous domains, they have revealed novel phenotypes that appear to reflect the regulatory complexity of mammalian membrane-bound adenylyl cyclases, including the possibility of oligomerization of the enzymes.

Catalytic mechanism and regulation of mammalian adenylyl cyclases.

Tang WJ, Hurley JH
Mol Pharmacol. 1998; 54: 231-40

Construction of a soluble adenylyl cyclase activated by Gs alpha and forskolin.

Tang WJ, Gilman AG
Science. 1995; 268: 1769-72

A soluble adenylyl cyclase was constructed by linkage of portions of the cytosolic domains of the mammalian type I and type II enzymes. The soluble enzyme was stimulated by both forskolin and the alpha subunit of the heterotrimeric guanine nucleotide-binding protein (G protein) Gs (Gs alpha). Expression of the construct complemented the catabolic defect in a strain of Escherichia coli that is deficient in adenylyl cyclase activity. The active, approximately 60-kilodalton enzyme accumulated in the cytoplasmic fraction of E. coli to yield activities in excess of 1 nanomole per minute per milligram of protein. The two sets of transmembrane helices of mammalian adenylyl cyclases are thus not necessary for the catalytic or the most characteristic regulatory activities of the enzyme. This system may be useful for both genetic and biochemical analysis of G protein-regulated adenylyl cyclases.

The third cytoplasmic domain of the GLP-1[7-36 amide] receptor is required for coupling to the adenylyl cyclase system.

Takhar S, Gyomorey S, Su RC, Mathi SK, Li X, Wheeler MB
Endocrinology. 1996; 137: 2175-8

Truncated forms of glucagon-like peptide-1 (tGLP-1) are potent endogenous stimuli of insulin secretion from pancreatic beta cells and have powerful antidiabetogenic effects. In the present study we sought to determine the precise regions of the tGLP-1 receptor (R) that are required for its efficient coupling to the adenylyl cyclase (AC) system since it is well established that cAMP is the primary second messenger activated by tGLP-1. The predicted third intracellular loop (IC3) of the rat tGLP-1R was systemically scanned using a mutagenic based strategy. The resulting receptor mutants were expressed in COS-7 cells and examined for cAMP formation in response to tGLP-1 stimulation (10nM) and [125I] tGLP-1(7-36) amide binding. A single block deletion (IC3-1) within the N-terminal region of IC3 (K334-L335-K336) resulted in a dramatic reduction in the cAMP response to tGLP-1 (7.1 +/- 1.4% of the wild type (wt) tGLP-1R response, n = 3, p < or = 0.01), while displaying comparable levels of expression, (expressed as the %Bmax of the wt-tGLP-1R (101 +/- 13%, n = 3, p > or = 0.05). This receptor mutation was further analyzed by stable expression in CHO-K1 cells. In agreement with the COS model, IC3-1 displayed comparable levels of receptor expression (97 +/- 16% Bmax of wt tGLP-1R, n = 3, p > or = 0.05) and affinity for tGLP-1(Kd of 460 +/- 15pM vs. 450 +/- 12pM wt tGLP-1R, n = 3, p > or = 0.05), but was unable to effectively stimulate cAMP production (7.7 +/- 0.4% of wt tGLP-1R, n = 3, p < or = 0.01) in response to tGLP-1 (10nM), No other mutation examined within the IC3 domain displayed a lack of correlation between binding activity and cAMP accumulation. Further analysis of the K334-L335-K336 sequence by substitution analysis revealed that a K334 to A substitution was the only modification to result in a striking attenuation of the cAMP response (28 +/- 1.9% of wt tGLP-1, n = 3, p < or = 0.01). These results strongly suggest that within the IC3 domain the N-terminal KLK sequence or a portion thereof (specifically K-334) is required for the efficient coupling of the tGLP-1 receptor to the AC system.

Adenylate cyclase-the more membrane associated, the less radiosensitive.

Takats A, Binh VH, Offenmuller K, Bodo K, Bertok L, Koteles GJ
J Radiat Res (Tokyo). 1993; 34: 141-7

A dose dependent but not parallel decreases were observed both in SH content and catalytic activity of "free" catalytic subunit after irradiation (0-3200 Gy), while SH groups of membrane-associated adenylate cyclase were insensitive (under 3200 Gy). An initial "radioactivation" of membrane-associated enzyme was found under 800 Gy, then an inhibition above 1600 Gy. The SH alkylating agent, N-ethylmaleimide resulted in a complete inactivation, both of membrane associated form of adenylate cyclase and "free" catalytic subunit with similar inactivation profiles. These data indicate that in the radiosensitivity or "radioprotection" of adenylate cyclase, its membrane association/integration might play a more important role than the SH groups themselves.

A pitfall in the interpretation of data on adenylate cyclase inactivation by irradiation.

Swillens S, Dumont JE
FEBS Lett. 1981; 134: 29-31

Changes in the expression of adenylyl cyclases in the rat uterus during the course of pregnancy.

Suzuki Y, Shen T, Miyamoto N, Defer N, Matsuoka I, Hanoune J
Biol Reprod. 1997; 57: 778-82

The changes in the expression of the various adenylyl cyclases (ACs) in the rat uterus during the course of pregnancy and after delivery were examined by Northern blot analysis and AC assay. Northern blot analysis revealed that five isoforms of ACs (AC2, AC4, AC6, AC7, and AC9) were expressed in the rat uterus, AC6 being the most abundant. The level of expression of these ACs increased 1.7- to 3.4-fold during the course of pregnancy and diminished near term and after delivery. The highest level of expression in each type of AC was consistently seen on Day 17 of pregnancy, and the relative increase of expression, as compared to that in nonpregnant rats, was as follows: AC2 > AC4 > AC7 > AC9 > AC6. In agreement with these findings, both basal and forskolin-stimulated AC activities exhibited a 2- to 3-fold increase during the course of pregnancy, followed by a decrease near term. Our data indicate that post-receptor events, namely marked changes in the level of AC mRNA (and presumably proteins) occur during pregnancy and after delivery and that they contribute to the essential role of cAMP in maintaining uterus quiescence.

Antibody mimicking the action of RAS proteins on yeast adenylyl cyclase: implication for RAS-effector interaction.

Suzuki N, Tsujino K, Minato T, Nishida Y, Okada T, Kataoka T
Mol Cell Biol. 1993; 13: 769-74

Polyclonal antisera were raised against various subregions of Saccharomyces cerevisiae adenylyl cyclase in order to examine the molecular mechanism of interaction between adenylyl cyclase and RAS proteins. One of the antisera was found to activate adenylyl cyclase to an extent comparable to that activated by saturating amounts of yeast RAS2 protein produced in Escherichia coli. The stimulatory effect of this antiserum was shown to be additive with RAS2 protein when both antisera and RAS2 protein were present at low concentrations. At saturating amounts of RAS2 protein, the antisera did not exhibit additional stimulatory effects, suggesting that the actions of RAS2 protein and the antisera are complementary with each other. The antigenic determinant for the antibody involved in the activation was mapped to a 14-amino-acid segment, 1452-NSVDNGADVANLSY-1465, located between the leucine-rich repeats and the catalytic domain of adenylyl cyclase. Certain missense mutations affecting this 14-amino acid segment significantly reduced the response of adenylyl cyclase to both activating antibody and RAS proteins. These results suggest that this segment of adenylyl cyclase is intimately involved in the mechanism by which RAS proteins activate this downstream effector.

Irreversible inhibition of forskolin interactions with type I adenylyl cyclase by a 6-isothiocyanate derivative of forskolin.

Sutkowski EM, Robbins JD, Tang WJ, Seamon KB
Mol Pharmacol. 1996; 50: 299-305

Forskolin (Fsk) has been demonstrated to interact directly with the enzyme adenylyl cyclase (EC 4.6.1.1) in diverse tissues. However, the ability of Fsk to bind to and activate adenylyl cyclase varies depending on the tissue being studied. Different adenylyl cyclase subtypes have been cloned and expressed in a recombinant Sf9 expression system. This provides an opportunity to study the effects of chemically reactive derivatives of Fsk on individual adenylyl cyclase subtypes in the absence of Gs alpha. Reaction of type I adenylyl cyclase with an isothiocyanate derivative of Fsk (6-[[N-(2-isothiocyanatoethyl)amino]carbonyl]forskolin) causes irreversible inhibition of Fsk binding with an IC50 of 300 nM and irreversible inhibition of Fsk activation with an IC50 of 10 microM, suggesting that there are two sites of 6-[[N-(2-isothiocyanatoethyl)amino]carbonyl]forskolin interaction. These studies establish the usefulness of the isothiocyanate derivative of Fsk in localizing the site(s) of Fsk interaction with type I adenylyl cyclase.

Interaction of 7-bromoacetyl-7-desacetylforskolin, and alkylating derivative of forskolin, with bovine brain adenylyl cyclase and human erythrocyte glucose transporter.

Sutkowski EM, Maher F, Laurenza A, Simpson IA, Seamon KB
Biochemistry. 1993; 32: 2415-22

7-Bromoacetyl-7-desacetylforskolin (BrAcFsk), an alkylating derivative of forskolin, activated adenylyl cyclase and irreversibly blocked high affinity forskolin binding sites in human platelet membranes and rat brain membranes (Laurenza et al., 1990). Photoincorporation of an iodinated arylazido derivative of forskolin, 125I-6-AIPP-Fsk, into adenylyl cyclase in bovine brain membranes was irreversibly inhibited by BrAcFsk but not by 1,9-dideoxy-BrAcFsk, suggesting that BrAcFsk was reacting specifically with a nucleophilic group(s) at the forskolin binding site of adenylyl cyclase. Immunoblotting with antiforskolin antiserum demonstrated that partially purified bovine brain adenylyl cyclase had incorporated BrAcFsk. The interaction of BrAcFsk with the glucose transporter in human erythrocyte membranes was examined in a similar manner. Photoincorporation of 125I-7-AIPP-Fsk, an iodinated arylazido derivative of forskolin which is specific for the glucose transporter, into the glucose transporter was not irreversibly inhibited by BrAcFsk, suggesting that, in contrast to adenylyl cyclase, there is no reactive nucleophilic group at the forskolin binding site on the human erythrocyte glucose transporter. The immunoblotting procedure with antiforskolin antiserum confirmed that BrAcFsk was not covalently attached to human erythrocyte glucose transporter.

Crystal structure of the adenylyl cyclase activator Gsalpha.

Sunahara RK, Tesmer JJ, Gilman AG, Sprang SR
Science. 1997; 278: 1943-7

The crystal structure of Gsalpha, the heterotrimeric G protein alpha subunit that stimulates adenylyl cyclase, was determined at 2.5 A in a complex with guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). Gsalpha is the prototypic member of a family of GTP-binding proteins that regulate the activities of effectors in a hormone-dependent manner. Comparison of the structure of Gsalpha.GTPgammaS with that of Gialpha.GTPgammaS suggests that their effector specificity is primarily dictated by the shape of the binding surface formed by the switch II helix and the alpha3-beta5 loop, despite the high sequence homology of these elements. In contrast, sequence divergence explains the inability of regulators of G protein signaling to stimulate the GTPase activity of Gsalpha. The betagamma binding surface of Gsalpha is largely conserved in sequence and structure to that of Gialpha, whereas differences in the surface formed by the carboxyl-terminal helix and the alpha4-beta6 loop may mediate receptor specificity.

Interaction of Gsalpha with the cytosolic domains of mammalian adenylyl cyclase.

Sunahara RK, Dessauer CW, Whisnant RE, Kleuss C, Gilman AG
J Biol Chem. 1997; 272: 22265-71

Forskolin- and Gsalpha-stimulated adenylyl cyclase activity is observed after mixture of two independently-synthesized approximately 25-kDa cytosolic fragments derived from mammalian adenylyl cyclases (native Mr approximately 120,000). The C1a domain from type V adenylyl cyclase (VC1) and the C2 domain from type II adenylyl cyclase (IIC2) can both be expressed in large quantities and purified to homogeneity. When mixed, their maximally stimulated specific activity, 150 micromol/min/mg protein, substantially exceeds values observed previously with the intact enzyme. A soluble, high-affinity complex containing one molecule each of VC1, IIC2, and guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-Gsalpha is responsible for the observed enzymatic activity and can be isolated. In addition, GTPgammaS-Gsalpha interacts with homodimers of IIC2 to form a heterodimeric complex (one molecule each of Gsalpha and IIC2) but not detectably with homodimers of VC1. Nevertheless, Gsalpha can be cross-linked to VC1 in the activated heterotrimeric complex of VC1, IIC2, and Gsalpha, indicating its proximity to both components of the enzyme that are required for efficient catalysis. These results and those in the accompanying report (Dessauer, C. W., Scully, T. T., and Gilman, A. G. (1997) J. Biol. Chem. 272, 22272-22277) suggest that activators of adenylyl cyclase facilitate formation of a single, high-activity catalytic site at the interface between C1 and C2.

Exchange of substrate and inhibitor specificities between adenylyl and guanylyl cyclases.

Sunahara RK, Beuve A, Tesmer JJ, Sprang SR, Garbers DL, Gilman AG
J Biol Chem. 1998; 273: 16332-8

The active sites of guanylyl and adenylyl cyclases are closely related. The crystal structure of adenylyl cyclase and modeling studies suggest that specificity for ATP or GTP is dictated in part by a few amino acid residues, invariant in each family, that interact with the purine ring of the substrate. By exchanging these residues between guanylyl cyclase and adenylyl cyclase, we can completely change the nucleotide specificity of guanylyl cyclase and convert adenylyl cyclase into a nonselective purine nucleotide cyclase. The activities of these mutant enzymes remain fully responsive to their respective stimulators, sodium nitroprusside and Gsalpha. The specificity of nucleotide inhibitors of guanylyl and adenylyl cyclases that do not act competitively with respect to substrate are similarly altered, indicative of their action at the active sites of these enzymes.

Structure-activity studies of hydrophobic amino acid replacements at positions 9, 11 and 16 of glucagon.

Sturm NS, Hutzler AM, David CS, Azizeh BY, Trivedi D, Hruby VJ
J Pept Res. 1997; 49: 293-9

We have designed and synthesized eight compounds 2-9 which incorporate neutral, hydrophobic amino acid residues in positions 9, 11 and 16 of the glucagon molecule: (2) [desHis1, Val9. Ile11,16] glucagon amide, (3) [desHis1, Val9,11,16] glucagon amide, (4) [desHis1, Val9, Leu11,16]glucagon amide, (5) [desHis1, Nle9, Ile11,16]glucagon amide, (6) [desHis1, Nle9, Val11,16] glucagon amide, (7) [desHis1,-Nle9, Leu11,16] glucagon amide, (8) [desHis1, Val9, Leu11,16, Lys17,18, Glu21] glucagon amide and (9) [desHis1, Nle9, Leu11,16, Lys17,18, Glu21] glucagon amide. The effect of neutral, hydrophobic residues at positions 9, 11 and 16 led to good binding to the glucagon receptor. Compared to glucagon (IC50 = 1.5 nM), analogues 2-9 were found to have IC50 values of 6.0, 6.0, 11.0, 9.0, 2.5, 2.8, 6.5 and 7.0 nM, respectively. When these compounds were tested for their ability to block adenylate cyclase (AC) activity, they were found to be antagonists having no stimulation of adenyl cyclase, with pA2 values of 6.15, 6.20, 6.30, 7.25, 6.10, 7.30, 6.25 and 7.25, respectively.

Three-state and two-state models.

Strange PG
Trends Pharmacol Sci. 1998; 19: 85-6

The substrate specificity of tocopherol cyclase.

Stocker A, Fretz H, Frick H, Ruttimann A, Woggon WD
Bioorg Med Chem. 1996; 4: 1129-34

The substrate specificity of the enzyme tocopherol cyclase from the blue-green algae Anabaena variabilis (Cyanobacteria) was investigated with 11 substrate analogues revealing the significance of three major recognition sites: (i) the OH group at C(1) of the hydroquinone, (ii) the (E) configuration of the double bond, and (iii) the length of the lipophilic side chain. Experiments with two affinity matrices suggest that substrates approach the enzyme's active site with the hydrophobic tail.

Different chromosomal localization of two adenylyl cyclase genes expressed in human brain.

Stengel D, Parma J, Gannage MH, Roeckel N, Mattei MG, Barouki R, Hanoune J
Hum Genet. 1992; 90: 126-30

Recently, we characterized a cDNA clone that encodes a human brain adenylyl cyclase (HBAC1). In the present study, we identified a second population of mRNA suspected to encode a new brain adenylyl cyclase (HBA C2). The amino acid sequence of HBA C2 displays significant homology with HBA C1 in the highly conserved adenylyl cyclase domain (250 aminio acids), found in the 3' cytoplasmic domain of all mammalian adenylyl cyclases. However, outside this domain, the homology is extremely low, suggesting that the corresponding mRNA originates from a different gene. We report here the first chromosomal localization of the adenylyl cyclase genes determined by in situ hybridization of human metaphase chromosomal spreads using human brain cDNA probes specific for each mRNA. The probe corresponding to HBA C1 exhibited a strong specific signal on chromosome 8q24, with a major peak in the band q24.2. In contrast, the HBA C2 probe hybridized to chromosome 5p15, with a major peak in the band p15.3. The two cDNAs hybridized at the two loci without any cross reactivity. Thus, in human brain, a heterogeneous population of adenylyl cyclase mRNAs is expressed, and the corresponding genes might be under the control of independent regulatory mechanisms.

Proposed structure for brain adenylate cyclase purified using blue dextran-Sepharose chromatography.

Stellwagen E, Baker B
Nature. 1976; 261: 719-20

Identification of a guanylyl cyclase-activating protein-binding site within the catalytic domain of retinal guanylyl cyclase 1.

Sokal I, Haeseleer F, Arendt A, Adman ET, Hargrave PA, Palczewski K
Biochemistry. 1999; 38: 1387-93

Regulation of cAMP and cGMP production is a fundamental step in a broad range of signal transduction systems, including phototransduction. To identify regions within photoreceptor guanylyl cyclase 1 (GC1) that interact with GC-activating proteins (GCAPs), we synthesized the intracellular fragment of GC1, residues 491-1110, as a set of 15 amino acid long, partially overlapping peptides on the surface of individual pins arranged in a microtiter plate format. This pin assay identified 8 peptides derived from different regions of the GC1 intracellular domain that bind GCAPs. Peptide variants containing these sequences were synthesized as free peptides and tested for their ability to inhibit GC1 stimulation by GCAPs. A free peptide,968GTFRMRHMPEVPVRIRIG, from the catalytic domain of GC1 was the strongest inhibitor of GCAP1/GCAP2-mediated activation. In native GC1, this polypeptide fragment is likely to form a loop between alpha-helix 3 and beta-strand 4. When this region in GC1 was replaced by the corresponding sequence of GCAP-insensitive GC type A, GCAPs did not stimulate the GC1 mutant. The corresponding loops in related adenylyl cyclase (AC) are involved in the activating and inhibiting interactions with Gs alpha and Gi alpha, respectively. Thus, despite interacting with different activating proteins, both AC and GC activity may be modulated through their respective regions within catalytic domains.

An adenylyl cyclase that functions during late development of Dictyostelium.

Soderbom F, Anjard C, Iranfar N, Fuller D, Loomis WF
Development. 1999; 126: 5463-71

A variety of extracellular signals lead to the accumulation of cAMP which can act as a second message within cells by activating protein kinase A (PKA). Expression of many of the essential developmental genes in Dictyostelium discoideum are known to depend on PKA activity. Cells in which the receptor-coupled adenylyl cyclase gene, acaA, is genetically inactivated grow well but are unable to develop. Surprisingly, acaA(-) mutant cells can be rescued by developing them in mixtures with wild-type cells, suggesting that another adenylyl cyclase is present in developing cells that can provide the internal cAMP necessary to activate PKA. However, the only other known adenylyl cyclase gene in Dictyostelium, acgA, is only expressed during germination of spores and plays no role in the formation of fruiting bodies. By screening morphological mutants generated by Restriction Enzyme Mediated Integration (REMI) we discovered a novel adenylyl cyclase gene, acrA, that is expressed at low levels in growing cells and at more than 25-fold higher levels during development. Growth and development up to the slug stage are unaffected in acrA(-) mutant strains but the cells make almost no viable spores and produce unnaturally long stalks. Adenylyl cyclase activity increases during aggregation, plateaus during the slug stage and then increases considerably during terminal differentiation. The increase in activity following aggregation fails to occur in acrA(-) cells. As long as ACA is fully active, ACR is not required until culmination but then plays a critical role in sporulation and construction of the stalk.

The adenylcyclase complex: a model of its mechanism of action in relation to the - and -adrenergic receptors.

Smythies JR
Eur J Pharmacol. 1972; 19: 18-24

Mammalian adenylyl cyclases.

Smit MJ, Iyengar R
Adv Second Messenger Phosphoprotein Res. 1998; 32: 1-21

How Gsalpha activates adenylyl cyclase.

Skiba NP, Hamm HE
Nat Struct Biol. 1998; 5: 88-92

Aeromonas hydrophila adenylyl cyclase 2: a new class of adenylyl cyclases with thermophilic properties and sequence similarities to proteins from hyperthermophilic archaebacteria.

Sismeiro O, Trotot P, Biville F, Vivares C, Danchin A
J Bacteriol. 1998; 180: 3339-44

Complementation of an Escherichia coli cya mutant with a genomic library from Aeromonas hydrophila allowed isolation of clones containing two different cya genes. Whereas one of these genes (cyaA) coded for an adenylyl cyclase (AC1) belonging to the previously described class I adenylyl cyclases (ACs), the second one (cyaB) coded for a protein (AC2) that did not match any previously characterized protein when compared to protein sequence databases. In particular, it did not align with any of members of the three known classes of ACs. The purified AC2 enzyme exhibited remarkable biochemical characteristics, namely, an optimum activity at a high temperature (65 degrees C) and at an alkalinic pH (9.5). In order to investigate the functions of both cyclases in A. hydrophila, each gene was inactivated in the chromosome and the resulting mutant strains were examined for physiological alterations. It was shown that, in contrast to cyaA, the cyaB gene was not expressed under usual laboratory growth conditions. However, introduction of a plasmid harboring the cyaB gene in a cyaA mutant, as well as in a cyaA cyaB mutant, allowed cyclic AMP production. AC2 is the first member of a new class of previously unrecognized ACs, and to date, no functional counterpart has been demonstrated in other organisms. However, scanning databases revealed a significant similarity between AC2 and the gene product of three hyperthermophilic archaebacteria: Methanobacterium thermoautotrophicum, Archaeglobus fulgidus, and Methanococcus jannaschii. The possibility of a gene transfer between such phylogenetically divergent bacteria is discussed.

Identification of a novel guanylyl cyclase that is related to receptor guanylyl cyclases, but lacks extracellular and transmembrane domains.

Simpson PJ, Nighorn A, Morton DB
J Biol Chem. 1999; 274: 4440-6

We have identified a novel guanylyl cyclase, named MsGC-I, that is expressed in the nervous system of Manduca sexta. MsGC-I shows highest sequence identity with receptor guanylyl cyclases throughout its catalytic and dimerization domains but does not contain the ligand-binding, transmembrane, or kinase-like domains characteristic of receptor guanylyl cyclases. In addition, MsGC-I contains a C-terminal extension of 149 amino acids that is not present in other receptor guanylyl cyclases. The sequence of MsGC-I contains no regions that show similarity to the regulatory domain of soluble guanylyl cyclases. Thus, MsGC-I appears to represent a member of a new class of guanylyl cyclases. We show that both a transcript and a protein of the sizes predicted from the MsGC-I cDNA are present in the nervous system of Manduca and that MsGC-I is expressed in a small population of neurons within the abdominal ganglia. When expressed in COS-7 cells, MsGC-I appears to exist as a soluble homodimer with high levels of basal guanylyl cyclase activity that is insensitive to stimulation by nitric oxide. Western blot analysis, however, shows that MsGC-I is localized to the particulate fraction of nervous system homogenates, suggesting that it may be membrane-associated in vivo.

G protein regulation of adenylate cyclase.

Simonds WF
Trends Pharmacol Sci. 1999; 20: 66-73

Adenylate cyclase integrates positive and negative signals that act through G protein-coupled cell-surface receptors with other extracellular stimuli to finely regulate levels of cAMP within the cell. Recently, the structures of the cyclase catalytic core complexed with the plant diterpene forskolin, and a cyclase-forskolin complex bound to an activated form of the stimulatory G protein subunit Gs alpha have been solved by X-ray crystallography. These structures provide a wealth of detail about how different signals could converge at the core cyclase domains to regulate catalysis. In this article, William Simonds reviews recent advances in the molecular and structural biology of this key regulatory enzyme, which provide new insight into its ability to integrate multiple signals in diverse cellular contexts.

Molecular cloning of a retina-specific membrane guanylyl cyclase.

Shyjan AW, de Sauvage FJ, Gillett NA, Goeddel DV, Lowe DG
Neuron. 1992; 9: 727-37

We have isolated and characterized cDNA clones encoding the human retinal guanylyl cyclase (retGC), a novel member of the membrane guanylyl cyclase gene family. Like other membrane guanylyl cyclases, the 1101 aa retGC is predicted to have a hydrophobic amino-terminal signal sequence followed by a large extracellular domain, a single membrane spanning domain, a kinase homology domain, and a guanylyl cyclase catalytic domain. In contrast to other membrane guanylyl cyclases, such as natriuretic peptide receptors, retGC has a relatively high basal level of activity when expressed in human 293 cells. cGMP production by retGC is unaffected by any of the known natriuretic peptides. In situ hybridization analysis of a variety of rhesus monkey tissues showed retGC transcripts to be localized exclusively along the retinal outer nuclear layer, corresponding to the nuclei of the rod and cone photoreceptor cells. Our results suggest that retGC may synthesize cGMP required for recovery of the dark state after phototransduction.

[Internal symmetry of the mirror type and repeats in molecules of the membrane-bound forms of adenylate cyclase]

Shpakov AO, Denisov EV
Tsitologiia. 1999; 41: 628-36

The analysis of mirror type internal symmetry distribution in primary structures of different types of mammalian membrane-bound adenylyl cyclases was made. The transmembrane domain clusters determining enzyme topology in membrane, a highly conservative region of cytoplasmic domains forming both catalytic and regulatory centres of adenylyl cyclases, and the functionally important regions in variable parts of their molecules (in particular, calmodulin binding regions) are shown to have symmetrical structures. These data are in conformity with a hypothesis put forward by the authors: the centres of internal symmetry may commonly either coincide with sites responsible for protein biological activity, or be spaced in the immediate vicinity of these sites. In different types of adenylyl cyclases long repeating sequences were identified. The segmentary structures were established for some enzyme subdomains. The regions containing repeats usually displayed a symmetrical structure which confirms a positive correlation between internal symmetry of amino acid sequence and its repeat distribution.

[The helices with heptad regularity in cytoplasmic domains of membrane-bound adenylyl cyclases and their possible role in interaction with other adenylyl cyclase signaling system components]

Shpakov AO
Tsitologiia. 1999; 41: 667-74

The helices with heptad regularity in C1 and C2 cytoplasmic domains of membrane-bound adenylyl cyclases (AC) of mammals were identified. The most helices were localized in N-terminal and central regions of high conservative C1a and C2a subdomains of AC. The regions are responsible for regulation of enzyme functional activity. The amino acid regions, corresponding to these helices, were homologous to G-protein beta and gamma subunit regions, which participate in coupling with alpha subunits and in forming the heterotrimeric alpha beta gamma complex. The similarity was found both primary and secondary structure levels. On the basis of obtained data the next supposition was made. The regular helices of C1a and C2a subdomains of AC can interact with G protein alpha-helices the by coiled-coil mechanism and thus regulate the AC catalytic activity. Additionally, the regular helices were identified in variable C1b and C2b subdomains of several AC types (in particular, I and III types). Some of the helices are similar in the secondary structure level to amphipatic helices of bacterial AC, which participate in calmodulin binding, and can carry out also the calmodulin-binding function.

Synthesis and use of 3'-(azidoiodosalicyl) derivatives of 2', 5'-dideoxyadenosine as photoaffinity ligands for adenylyl cyclase.

Shoshani I, Taussig R, Iyengar R, Johnson RA
Arch Biochem Biophys. 2000; 376: 221-8

3'-[(4-Azidosalicyl)glycyl]-2',5'-dideoxyadenosine (1), 3'- [(4-azidosalicyl)-gamma-aminobutyryl]-2',5'-dideoxyadenosine (2), and the (125)I-labeled mono- and diiodinated analogs of 1 were synthesized and tested as photoaffinity probes for adenylyl cyclases. Kinetics for inhibition of purified type I enzyme by 1 was noncompetitive with respect to Mn(*)5'-ATP in the absence of light, implying a P-site mechanism of inhibition. In a UV-dependent manner both 1 and 2 and the iodinated derivative of 1 irreversibly inactivated membrane-bound and purified forms of recombinant type I bovine adenylyl cyclase expressed in ovarian cells of either the fall armyworm (Sf9) or Trichoplasia ni (High Five). Irreversible inactivation was independent of 5'-ATP and was prevented by 2', 5'-dideoxyadenosine. Adenylyl cyclase, whether purified from bovine brain or in membranes from High Five cells expressing type I enzyme, when subjected to UV irradiation in the presence of (125)I-labeled 1 resulted in radioactive incorporation into protein migrating at approximately 116 kDa. The cross-linking of 1 and its iodinated derivative with adenylyl cyclase suggests potential for such compounds to be useful in structural studies of adenylyl cyclases or of other proteins for which adenine nucleosides are substrates or allosteric regulators.

Azido-iodo-phenyl-analogs of 2',5'-dideoxy-adenosine as photoaffinity ligands for adenylyl cyclase.

Shoshani I, Qui H, Johnson F, Taussig R, Johnson RA
Biochim Biophys Acta. 1995; 1245: 37-42

Azidoiodophenyl-analogs of 2',5'-dideoxyadenosine were synthesized and tested as potential 'P'-site selective affinity probes for adenylyl cyclases. The 3'-substituted analogs included: 1: 3'-[(4-nitrophenyl)-acetyl]-2',5'-dideoxy-adenosine 2: 3'-[(4-nitrophenyl)-butyryl]-2',5'-dideoxyadenosine 3: 3'-[(4-azido-3-iodophenyl)-acetyl]-2',5'-dideoxyadenosine and 4: 3'-[(4-azido-3-iodophenyl)-butyryl]-2',5'-dideoxyadenosine. The azidoiodo-phenyl-analogs inactivated adenylyl cyclase irreversibly and in a light-dependent manner. This was observed with detergent-dispersed enzyme from rat brain, purified native enzyme from bovine brain, and recombinant Type I bovine adenylyl cyclase expressed in membranes from fall army worm ovarian (Sf9) cells. Inactivation of the recombinant enzyme was inversely dependent on ATP concentration and was not completely prevented by 2',5'-dideoxyadenosine. Inhibition kinetics with the recombinant enzyme in the absence of light suggested two sites of inhibition, whereas with the native Type I enzyme inhibition kinetics exhibited a straightforward noncompetitive mechanism. Occupation of either or both sites by ligand protected the enzyme against denaturation by UV-irradiation per se. The data are consistent with inactivation of the recombinant enzyme occurring both through the 'P'-site and the catalytic active site, but suggest that this is a characteristic of the recombinant enzyme and is not dependent on the probes per se. The data suggest the potential for independent interactions of such ligands with different sites on a given enzyme and also with other enzymes containing adenosine or adenine nucleotide binding domains.

Lys-Ala mutations of type I adenylyl cyclase result in altered susceptibility to inhibition by adenine nucleoside 3'-polyphosphates.

Shoshani I, Bianchi G, Desaubry L, Dessauer CW, Johnson RA
Arch Biochem Biophys. 2000; 374: 389-94

Native and recombinant wild type and mutant forms of type I adenylyl cyclase, expressed in fall army worm ovarian cells (Sf9) cells, with mutations Lys-923-Ala, Lys-921-Ala, and Lys-350-Ala, retained the characteristic noncompetitive inhibition by adenine nucleoside 3'-polyphosphates, but exhibited substantially different sensitivities to inhibition by them. The type I K923A enzyme resulted in increased IC(50) values, e.g., >100-fold for 2'-deoxyadenosine-3'-monophosphate, but the shift diminished as the number of 3'-phosphates increased. The K921A mutation increased IC(50) values approximately 5-fold for all adenine nucleosides tested, whereas the K350A mutation increased IC(50) values approximately 6- to 8-fold for all adenine nucleosides tested except 2'-deoxyadenosine-3'-diphosphate, which was increased >/=2-fold. The data suggest that 3'-phosphates sufficiently increase binding affinity of these ligands to compensate for the reduced coordination of the adenine moiety induced by the K923A mutation. Moreover, the altered structures induced by both K350A and K921A mutations impair ligand binding in general, but paradoxically those resulting from the K350A change minimally affected nucleoside 3'-diphosphate binding, implying that selective changes in ligand binding can be induced by this site-specific mutation.

A mRNA for membrane form of guanylyl cyclase is expressed exclusively in the testis of the sea urchin Hemicentrotus pulcherrimus.

Shimizu T, Takeda K, Furuya H, Hoshino K, Nomura K, Suzuki N
Zoolog Sci. 1996; 13: 285-94

A cDNA clone encoding the membrane form of guanylyl cyclase was isolated from a Hemicentrotus pulcherrimus testis cDNA library and its nucleotide sequence was determined. The cDNA was 4123 bp long and an open reading frame predicted a protein of 1125 amino acids including an apparent signal peptide of 21 residues; a single transmembrane domain of 25 amino acids which divides the mature protein into an amino-terminal, extracellular domain of 485 amino acids and a carboxyl-terminal, intracellular domain of 594 amino acids. Three potential N-linked glycosylation sites were present in the extracellular domain. Northern blot analysis of poly(A)+RNA from testes, ovaries, eggs and embryos at various developmental stages showed that the cDNA encoding guanylyl cyclase hybridized to a mRNA of 4.4 kb from the testes. We developed a large scale purification method of the phosphorylated (131 kDa) and dephosphorylated (128 kDa) forms of the membrane-bound guanylyl cyclase from H. pulcherrimus spermatozoa. The purified 131 kDa and 128 kDa forms of the guanylyl cyclase contained 26.0 +/- 1.3 and 4.3 +/- 0.7 moles of phosphate per mol protein (mean +/- S.D.; n = 6), respectively. The amino-terminal amino acids of both the 131 kDa and 128 kDa forms of the guanylyl cyclase could not be detected, suggesting that they were blocked.

Effect of association with adenylyl cyclase-associated protein on the interaction of yeast adenylyl cyclase with Ras protein.

Shima F, Yamawaki-Kataoka Y, Yanagihara C, Tamada M, Okada T, Kariya K, Kataoka T
Mol Cell Biol. 1997; 17: 1057-64

Posttranslational modification of Ras protein has been shown to be critical for interaction with its effector molecules, including Saccharomyces cerevisiae adenylyl cyclase. However, the mechanism of its action was unknown. In this study, we used a reconstituted system with purified adenylyl cyclase and Ras proteins carrying various degrees of the modification to show that the posttranslational modification, especially the farnesylation step, is responsible for 5- to 10-fold increase in Ras-dependent activation of adenylyl cyclase activity even though it has no significant effect on their binding affinity. The stimulatory effect of farnesylation is found to depend on the association of adenylyl cyclase with 70-kDa adenylyl cyclase-associated protein (CAP), which was known to be required for proper in vivo response of adenylyl cyclase to Ras protein, by comparing the levels of Ras-dependent activation of purified adenylyl cyclase with and without bound CAP. The region of CAP required for this effect is mapped to its N-terminal segment of 168 amino acid residues, which coincides with the region required for the in vivo effect. Furthermore, the stimulatory effect is successfully reconstituted by in vitro association of CAP with the purified adenylyl cyclase molecule lacking the bound CAP. These results indicate that the association of adenylyl cyclase with CAP is responsible for the stimulatory effect of posttranslational modification of Ras on its activity and that this may be the mechanism underlying its requirement for the proper in vivo cyclic AMP response.

Plasma membrane guanylate cyclase. A multimodule transduction system.

Sharma RK, Duda T
Adv Exp Med Biol. 1997; 407: 271-9

Two Drosophila genes that encode the alph and beta subunits of the brain soluble guanylyl cyclase.

Shah S, Hyde DR
J Biol Chem. 1995; 270: 15368-76

We identified two Drosophila genes (dgc alpha 1 and dgc beta 1) that encode the soluble guanylyl cyclase alpha and beta subunits, respectively. The putative Dgc alpha 1 protein is 76 kDa, has 35% amino acid identity with previously isolated alpha subunits, and was immunolocalized to the adult retina, to the optic lobes, and throughout the brain neuropil. The Dgc beta 1 protein is 86 kDa and exhibits 59% amino acid identity with the rat beta 1 protein. However, the Dgc beta 1 protein has an additional 118 amino acids inserted near the amino terminus, which makes it significantly larger than the rat beta 1. The Dgc beta 1 protein was immunolocalized to the optic lobes and throughout the brain neuropil, with no detectable expression in the retina. The Dgc alpha 1 and Dgc beta 1 cDNAs were stably transfected into human kidney 293 cells. Expression of the individual subunits and mixing of the individually expressed subunits failed to generate significant guanylyl cyclase activity. Only coexpression of the subunits resulted in significant guanylyl cyclase activity. Our results indicate that Dgc alpha 1 and Dgc beta 1 are soluble guanylyl cyclase alpha and beta subunits that are capable of forming a functional guanylyl cyclase heterodimer.

Primary structure and differential gene expression of three membrane forms of guanylyl cyclase found in the eye of the teleost Oryzias latipes.

Seimiya M, Kusakabe T, Suzuki N
J Biol Chem. 1997; 272: 23407-17

Three cDNAs (OlGC3, OlGC4, and OlGC5) encoding membrane guanylyl cyclases were isolated from a medaka (Oryzias latipes) eye cDNA library. An open reading frame for OlGC3 predicted a protein of 1057 amino acids, and those for OlGC4 and OlGC5, 1134 and 1151, respectively. These proteins consist of an apparent signal peptide (21 residues for OlGC3, 50 residues for OlGC4, and 48 residues for OlGC5) and a single transmembrane domain that divides the protein into an amino-terminal extracellular domain and a carboxyl-terminal intracellular domain that further divides into a kinase-like domain and a cyclase catalytic domain. Phylogenetic analysis with amino acid sequences of OlGC3, OlGC4, and OlGC5, as well as those of other membrane guanylyl cyclases, indicated that OlGC3, OlGC4, and OlGC5 are members of the sensory organ-specific guanylyl cyclase family. Reverse transcription-polymerase chain reaction and Northern blot analyses demonstrated that OlGC3, OlGC4, and OlGC5 transcripts are present in the eye, which contains more cGMP than the other organs. In addition to being expressed in the eye, OlGC3 transcripts are also present in the brain, heart, liver, pancreas, and ovary, while OlGC4 is present in the liver and OlGC5 in the heart. Reverse transcription-polymerase chain reaction analysis with RNA from unfertilized eggs and embryos showed that OlGC3 and OlGC5 are expressed both maternally and zygotically, while OlGC4 is expressed only zygotically, and that the zygotic expression of these three genes is differentially activated. These results suggest a structural and functional diversity of sensory organ-specific guanylyl cyclases in vertebrates.

An isoform-specific interaction of the membrane anchors affects mammalian adenylyl cyclase type V activity.

Seebacher T, Linder JU, Schultz JE
Eur J Biochem. 2001; 268: 105-10

The nine membrane-bound mammalian adenylyl cyclases (ACs) contain two highly diverged membrane anchors, M1 and M2, with six transmembrane spans each and two conserved cytosolic domains which coalesce into a pseudoheterodimeric catalytic unit. Previously, the catalytic segments, bacterially expressed as soluble proteins, were characterized extensively whereas the function of the membrane domains remained unexplored. Using the catalytic C1 and C2 domains of AC type V we employed the membrane anchors from type V and VII ACs for construction of enzymes with duplicated, inverted, fully swapped and chimeric membrane anchors. Further, in the M1 membrane domain individual transmembrane spans were removed or exchanged between type V and VII ACs. The constructs were expressed in HEK293 cells, the expression levels and membrane localization was assessed by Western blotting. Cell-free basal, forskolin-, GTP gamma S-and G(s alpha)/GTP gamma S-stimulated AC activities were determined. The results demonstrate that enzymatic activities were only maintained when the M1 and M2 membrane domains were derived from either AC V or VII. Constructs with chimeric membrane domains, i.e. M1 from type V and M2 from type VII AC or vice versa, were essentially inactive although the expression levels and membrane localization appeared to be normal. The data indicate a functionally important interaction of the membrane domains of ACs in that they seem to interact in a pair-like, isoform delimited manner. This interaction directly impinges on the formation of the catalytic interface. We propose that protein-protein interactions of the AC membrane domains may constitute another, yet unexplored level of AC regulation.

The expanding family of guanylyl cyclases.

Schulz S, Yuen PS, Garbers DL
Trends Pharmacol Sci. 1991; 12: 116-20

The guanylyl cyclase receptor family contains members that exist in both the particulate and soluble fractions of cell homogenates. Soluble forms of the enzyme recognize EDRF (nitric oxide) or similar molecules, while diversity within the extracellular domain of the plasma membrane forms has resulted in a series of guanylyl cyclases that are specifically activated by different ligands. David Garbers and colleagues describe the recent cloning of some of these molecules.

The cloning and expression of a new guanylyl cyclase orphan receptor.

Schulz S, Wedel BJ, Matthews A, Garbers DL
J Biol Chem. 1998; 273: 1032-7

A novel membrane form of guanylyl cyclase (GC-G) has been identified through the isolation of a full-length cDNA clone; it is predicted to contain an extracellular ligand binding domain, a single transmembrane segment, and intracellular protein kinase-like and cyclase catalytic domains. That GC-G represents a guanylyl cyclase was confirmed by both transient expression in COS-7 cells and stable expression in H293 cells. Endogenous cyclic GMP concentrations of transfected or stable cells, however, were much higher than control cells, suggesting an inability of the cells to effectively regulate GC-G cyclase activity. Of six Cys residues found within the extracellular domain of guanylyl cyclase-A (GC-A), the receptor for atrial natriuretic peptide, five are conserved within GC-G. Ligands for the other cyclase receptors, nevertheless, failed to stimulate GC-G expressed in transient or stable cells, suggesting that the unknown ligands possess a structure different from the natriuretic peptides or heat-stable enterotoxins. 125I-ANP also failed to bind to H293 cells overexpressing GC-G. Based on Northern hybridization, mRNA for GC-G was predominantly expressed in lung, intestine, and skeletal muscle. Using the candidate gene approach to potentially define function, the gene for GC-G was mapped to the distal region of mouse chromosome 19 (syntenic with human chromosome 10q), but no human genetic defect has been ascribed to the GC-G locus. The finding of a new membrane form of guanylyl cyclase in peripheral tissues suggests the existence of another family or subfamily of ligands that signal through elevations of cGMP.

Identification of a domain in guanylyl cyclase-activating protein 1 that interacts with a complex of guanylyl cyclase and tubulin in photoreceptors.

Schrem A, Lange C, Beyermann M, Koch KW
J Biol Chem. 1999; 274: 6244-9

The membrane-bound guanylyl cyclase in rod photoreceptors is activated by guanylyl cyclase-activating protein 1 (GCAP-1) at low free [Ca2+]. GCAP-1 is a Ca2+-binding protein and belongs to the superfamily of EF-hand proteins. We created an oligopeptide library of overlapping peptides that encompass the entire amino acid sequence of GCAP-1. Peptides were used in competitive screening assays to identify interaction regions in GCAP-1 that directly bind the guanylyl cyclase in bovine photoreceptor cells. We found four regions in GCAP-1 that participate in regulating guanylyl cyclase. A 15-amino acid peptide located adjacent to the second EF-hand motif (Phe73-Lys87) was identified as the main interaction domain. Inhibition of GCAP-1-stimulated guanylyl cyclase activity by the peptide Phe73-Lys87 was completely relieved when an excess amount of GCAP-1 was added. An affinity column made from this peptide was able to bind a complex of photoreceptor guanylyl cyclase and tubulin. Using an anti-GCAP-1 antibody, we coimmunoprecipitated GCAP-1 with guanylyl cyclase and tubulin. Complex formation between GCAP-1 and guanylyl cyclase was observed independent of [Ca2+]. Our experiments suggest that there exists a tight association of guanylyl cyclase and tubulin in rod outer segments.

Identification of an intramolecular interaction between small regions in type V adenylyl cyclase that influences stimulation of enzyme activity by Gsalpha.

Scholich K, Wittpoth C, Barbier AJ, Mullenix JB, Patel TB
Proc Natl Acad Sci U S A. 1997; 94: 9602-7

Using the full-length and two engineered soluble forms (C1-C2 and Cla-C2) of type V adenylyl cyclase (ACV), we have investigated the role of an intramolecular interaction in ACV that modulates the ability of the alpha subunit of the stimulatory GTP-binding protein of AC (Gsalpha) to stimulate enzyme activity. Concentration-response curves with Gsalpha suggested the presence of high and low affinity sites on ACV, which interact with the G protein. Activation of enzyme by Gsalpha interaction at these two sites was most apparent in the C1a-C2 form of ACV, which lacks the C1b region (K572-F683). Yeast two-hybrid data demonstrated that the C1b region interacted with the C2 region and its 64-aa subdomain, C2I. Using peptides corresponding to the C2I region of ACV, we investigated the role of the C1b/C2I interaction on Gsalpha-mediated stimulation of C1-C2 and full-length ACV. Our data demonstrate that a 10-aa peptide corresponding to L1042-T1051 alters the profile of the activation curves of full-length and C1-C2 forms of ACV by different Gsalpha concentrations to mimic the activation profile observed with C1a-C2 ACV. The various peptides used in our studies did not alter forskolin-mediated stimulation of full-length and C1-C2 forms of ACV. We conclude that the C1b region of ACV interacts with the 10-aa region (L1042-T1051) in the C2 domain of the enzyme to modulate Gsalpha-elicited stimulation of activity.

Characterization of soluble forms of nonchimeric type V adenylyl cyclases.

Scholich K, Barbier AJ, Mullenix JB, Patel TB
Proc Natl Acad Sci U S A. 1997; 94: 2915-20

Type V adenylyl cyclase (ACV) belongs to the family of Ca2+-inhibited cyclases. We have generated two soluble forms of the enzyme containing the C1 or C1a region (which lacks the C-terminal 112 amino acids) linked to the C2 domain and compared their regulation with the full-length ACV. All three forms of ACV were stimulated by the alpha subunit of the stimulatory G protein Gs (G(s alpha)) and forskolin. However, the synergistic stimulation by both these activators was markedly enhanced in the soluble enzymes. Moreover, the alpha subunit of the inhibitory G protein Gi (G(i alpha)) inhibited all forms of the enzyme, indicating that the regions for G(s alpha) and G(i alpha) interaction are preserved in the soluble forms. Ca2+ inhibited forskolin-stimulated adenylyl cyclase (AC) activity of the full-length and C1-C2 forms of ACV but did not alter the activity of the C1a-C2 form. Maximal stimulation of AC activity by combination of G(s alpha) and forskolin obliterated the Ca2+-mediated inhibition of the full-length and C1-C2 forms of ACV. In 45Ca2+ overlay experiments, the C1-C2 but not the C1a-C2 soluble ACV bound Ca2+. Moreover, proteins corresponding to the C1a and C2 domains did not bind calcium. On the other hand, the proteins corresponding to C1 and its C-terminal 112 amino acids (C1b) bound 45Ca2+. To our knowledge, this is the first report of nonchimeric soluble forms of AC in which regulation by G(s alpha) and G(i alpha) is preserved. Moreover, we demonstrate that the 112 amino acid C1b region of ACV is responsible for the binding of Ca2+ and inhibition of enzyme activity.

Guanylate cyclase activity in permeabilized Dictyostelium discoideum cells.

Schoen CD, Schulkes CC, Arents JC, van Driel R
J Cell Biochem. 1996; 60: 411-23

Dictyostelium discoideum cells respond to chemoattractants by transient activation of guanylate cyclase. Cyclic GMP is a second messenger that transduces the chemotactic signal. We used an electropermeabilized cell system to investigate the regulation of guanylate cyclase. Enzyme activity in permeabilized cells was dependent on the presence of a nonhydrolysable GTP analogue (e.g., GTP gamma S), which could not be replaced by GTP, GDP, or GMP. After the initiation of the guanylate cyclase reaction in permeabilized cells only a short burst of activity is observed, because the enzyme is inactivated with a t1/2 of about 15 s. We show that inactivation is not due to lack of substrate, resealing of the pores in the cell membrane, product inhibition by cGMP, or intrinsic instability of the enzyme. Physiological concentrations of Ca2+ ions inhibited the enzyme (half-maximal effect at 0.3 microM), whereas InsP3 had no effect. Once inactivated, the enzyme could only be reactivated after homogenization of the permeabilized cells and removal of the soluble cell fraction. This suggests that a soluble factor is involved in an autonomous process that inactivates guanylate cyclase and is triggered only after the enzyme is activated. The initial rate of guanylate cyclase activity in permeabilized cells is similar to that in intact, chemotactically activated cells. Moreover, the rate of inactivation of the enzyme in permeabilized cells and that due to adaptation in vivo are about equal. This suggests that the activation and inactivation of guanylate cyclase observed in this permeabilized cell system is related to that of chemotactic activation and adaptation in intact cells.

Studies of fluoride-preactivated rabbit liver adenylyl cyclase.

Schmidt JJ, Najjar VA
Biochim Biophys Acta. 1978; 526: 276-88

Rabbit liver adenylyl (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) cyclase was stimulated by preincubation with F- and Mg2+ and the stimulation persisted despite extensive washing and/or detergent solubilization. Optimum preactivation conditions were found to be 4 mM NaF and 2 mM MgCl2; higher or lower concentrations produced submaximum stimulation regardless of preincub ation time. In addition to an enhanced catalytic acticity, the activated enzyme also exhibited different responses to Ca2+ and Cu2+ when compared to the basal enzyme. ATP caused a time-dependent inhibition that could be partially prevented or reversed by F-, but was not completely reversed by washing. This inhibition was not observed when 5'-adenosine(beta, gamma-imide) triphosphate blocks inhibition by ATP. The results support, but do not prove, the proposed molecular basis of F- activation which entails a phosphorylation-dephosphorylation mechanism.

Structure, glycosylation, and localization of rat intestinal guanylyl cyclase C: modulation by fasting.

Scheving LA, Russell WE, Chong KM
Am J Physiol. 1996; 271: 95968-95968

Guanylyl cyclase C (GC-C), an intestinal receptor guanylyl cyclase, binds diarrhea-producing bacterial ligands such as the Escherichia coli heat stable enterotoxin. We examined the regulatory influence of feeding and fasting on the expression, structure, and biochemical properties of GC-C. When solubilized at 4 degrees C under nonreducing conditions, GC-C from both fed and fasted rats migrated on 7% sodium dodecyl sulfate-polyacrylamide electrophoretic gels as two extremely large aggregates that barely penetrated the stacking and resolving gels. Chemical reduction of disulfide linkages disaggregated GC-C in fed but not fasted rat samples, causing it to migrate as smaller forms (approximately 220 and 240 kDa). Although GC-C aggregates from fasted rats resisted this disaggregating effect of chemical reduction, they rapidly acquired it within 90 min of refeeding. When solubilized at denaturing temperatures (95 degrees C) under reducing conditions, GC-C aggregates largely disassembled into four smaller proteins (relative molecular weight approximately 140,000, 131,000, 85,000, and 65,000). However, the 131-kDa glycoprotein was disproportionately increased in fasted rat membranes. This unit and the 220-kDa unit were sensitive to endoglycosidase H. Subcellular fractionation and immunohistochemical studies revealed a major redistribution of GC-C from surface to intracellular enterocyte sites during fasting.

Myocardial adenylyl cyclase type V and VI mRNA: differential regulation with age.

Scarpace PJ, Matheny M, Tumer N
J Cardiovasc Pharmacol. 1996; 27: 86-90

Myocardial beta-adrenergic signal transduction diminishes with age. This decrease is not due to a decrease in the number of beta-adrenoceptors, but may be a result of an impaired capacity to activate adenylyl cyclase (AC). Forskolin-stimulated AC activity is diminished, and the number of forskolin binding sites is decreased, suggesting that the decrease in signal transduction with age is a result of fewer AC catalytic units. To investigate whether the decrease in AC with age is associated with diminished AC mRNA, we assessed AC type V and type VI mRNA in ventricles from 6-, 11-, and 24-month-old F-344 rats. The predominant mRNA species, type V, increased by 45% between 6 and 11 months of age but decreased to just below the 6-month level by age 24 months. In contrast, type VI mRNA decreased by 44% between 6 and 11 months of age and then increased to the 6-month level at age 24 months. The changes in type V and type VI mRNA did not parallel the decreases in the AC activity or forskolin binding sites with senescence. These data indicate that the steady-state levels of type V and VI AC mRNA are not reliable predictors of the efficacy of forskolin stimulation of AC activity at different ages.

Carbon-11-forskolin: a ligand for visualization of the adenylate cyclase-related second messenger system.

Sasaki T, Enta A, Nozaki T, Ishii S, Senda M
J Nucl Med. 1993; 34: 1944-8

To visualize the adenylate cyclase (AC)-related second messenger system, [11C]forskolin, [11C]1-acetyl-7-deacetylforskolin, [11C]1,9-dideoxyforskolin and [11C]1-deoxyforskolin were synthesized by acetylation of the respective deacetyl-precursors using [11C]acetylchloride and dimethylaminopyridine. The radiochemical yield of [11C]forskolin, [11C]1-acetyl-7-deacetylforskolin, [11C]1,9-dideoxyforskolin and [11C]1-deoxyforskolin calculated from trapped [11C]CO2 were 5%, 10%, 15% and 18%, respectively. Since the 1- and 9-OH groups on the forskolin structure are critical for specific binding to AC (active type), we considered [11C]1-acetyl-7-deacetylforskolin, [11C]1,9-dideoxyforskolin and [11C]1-deoxyforskolin to be nonspecific forskolin analogs. A comparative study of [11C]forskolin and its analogs on the n-octanol/phosphate buffer (pH 7.4) partition ratio showed that [11C]1-acetyl-7-deacetylforskolin has similar physical properties to [11C]forskolin. In the mouse heart, kidneys, liver and lungs, more [11C]forskolin accumulated than [11C]1-acetyl-7-deacetylforskolin. Moreover, simultaneous [11C]forskolin with forskolin (10 micrograms) administration reduced the accumulation of [11C]forskolin particularly in the heart to the level of [11C]1-acetyl-7-deacetylforskolin. These results indicate that [11C]forskolin would be a useful imaging agent for the AC-related second messenger system.

Activation of adenylyl cyclase in Chlamydomonas reinhardtii by adhesion and by heat.

Saito T, Small L, Goodenough UW
J Cell Biol. 1993; 122: 137-47

Adhesion between Chlamydomonas reinhardtii gametes generates a rapid rise in cAMP levels which stimulates mating responses and zygotic cell fusion (Pasquale and Goodenough, 1987). We show here that sexual adhesion in vivo results in a twofold stimulation of flagellar adenylyl cyclase activity when the enzyme is subsequently assayed in vitro, a stimulation that is specifically blocked by Cd2+. A twofold stimulation is also elicited by the in vitro presentation of soluble cross-linking reagents (antisera and concanavalin A). In contrast, the 10-30-fold stimulation of the flagellar cyclase by in vitro exposure to 40 degrees C, first described by Zhang et al. (1991), is insensitive to Cd2+ but sensitive to such drugs as trifluoperizine and dibucaine. The capacity for twofold stimulation is displayed by the vegetative and gametic enzymes but is lost when gametes fuse to form zygotes; in contrast, the 10-fold stimulation is displayed by the gametic and zygotic enzymes but not the vegetative enzyme. The signal-defective mutant imp-3 fails to generate the normal mating-triggered cAMP production and can be rescued by exogenous dibutyryl cAMP. It displays normal basal rates of flagellar cyclase activity and a normal twofold stimulation by sexual adhesion and by soluble cross-linkers, but it is defective in 40 degrees C activation. The gametic cell-body adenylyl cyclase is stimulated when wild-type flagella, but not imp-3 flagella, undergo adhesive interactions in vivo, and it can be directly stimulated in vitro by cAMP presentation. We propose that the two levels of flagellar cyclase stimulation reflect either sequential steps in the activation of a single cyclase enzyme, with imp-3 blocked in the second step, or else the sequential activation of two different flagellar enzymes, with imp-3 defective in the second enzyme. We further propose that the cell-body enzyme is activated by the cAMP that is generated when flagellar cyclase activity is fully stimulated.

Classification of protein folds.

Russell RB
Methods Mol Biol. 2000; 143: 305-24

Biogenesis, cellular localization, and functional activation of the heat-stable enterotoxin receptor (guanylyl cyclase C).

Rudner XL, Nicchitta C, Almenoff JS
Biochemistry. 1996; 35: 10680-6

Enterotoxigenic Escherichia coli elaborate a peptide called heat-stable enterotoxin (ST), which binds to and activates the intestinal ST receptor (STaR). STaR, also known as guanylyl cyclase C (GC-C), is a member of the transmembrane guanylyl cyclase receptor family. The mRNA for STaR encodes an approximately 120 kDa protein with the N-terminal ligand binding domain on the cell surface. Ligand affinity cross-linking studies have previously demonstrated several species of potential ST binding proteins, ranging in size from approximately 50 to 160 kDa. Although these smaller forms of STaR (50-80 kDa) have been proposed to act in vivo as toxin binding proteins, their biogenesis and localization have not previously been examined. Using pulse labeling in vivo and synchronized translation in vitro, we demonstrate that these smaller forms represent incomplete translational products and are not formed through limited proteolysis of the full-length receptor, as had previously been believed. We determined, using fluorescence confocal microscopy and surface labeling, that only approximately 25% of cellular receptors are expressed at the surface, while the remaining population is retained within the endoplasmic reticulum. Only full-length receptor is found at the surface of the cell, indicating this to be the biologically active form of STaR responsible for interacting with the heat-stable enterotoxin and other luminal intestinal peptides. The large intracellular receptor population, and potential for function before translocation to the cell surface, may impact on how pharmacologic modulators of this clinically important receptor are designed.

Complete abolition of the retinal-specific guanylyl cyclase (retGC-1) catalytic ability consistently leads to leber congenital amaurosis (LCA).

Rozet JM, Perrault I, Gerber S, Hanein S, Barbet F, Ducroq D, Souied E, Munnich A, Kaplan J
Invest Ophthalmol Vis Sci. 2001; 42: 1190-2

PURPOSE. Leber congenital amaurosis (LCA) is the earliest and the most severe form of all inherited retinal dystrophies. In 1996, the current investigators ascribed the disease in families linked to the LCA1 locus on chromosome 17p13.1 to mutations in the photoreceptor-specific guanylyl cyclase (retGC-1) gene. So far, 22 different mutations, of which 11 are missense mutations, have been identified in 25 unrelated families. This is a report of the functional analyses of nine of the missense mutations. METHODS. cDNA constructs were generated that contained the retGC-1 missense mutations identified in patients related to the LCA1 locus. Mutants were expressed in COS7 cells and assayed for their ability to hydrolyze guanosine triphosphate (GTP) into cyclic guanosine monophosphate (cGMP). RESULTS. All mutations lying in the catalytic domain showed a complete abolition of cyclase activity. In contrast, only one mutation lying in the extracellular domain also resulted in a severely reduced catalytic activity, whereas the others showed completely normal activity. CONCLUSIONS. More than half the mutations identified in patients related to the LCA1 locus are truncating mutations expected to result in a total abolition of retGC-1 activity. Concerning missense mutations, half of them lying in the catalytic domain of the protein also result in the complete inability of the mutant cyclases to hydrolyze GTP into cGMP in vitro. In contrast, missense mutations lying in the extracellular domain, except one affecting the initiation codon, showed normal catalytic activity of retGC-1. Nevertheless, considering that all patients related to the LCA1 locus displayed the same phenotype, it can be assumed that all missense mutations would have the same dramatic consequences on protein activity in vivo as truncation mutations.

Evidence for a novel adenylyl cyclase in human epididymal sperm.

Rojas FJ, Patrizio P, Do J, Silber S, Asch RH, Moretti-Rojas I
Endocrinology. 1993; 133: 3030-3

Since cAMP is considered to play a major role in the acquisition of maturation and fertilizing capacity of mammalian sperm, we investigated the expression of cAMP-synthesizing adenylyl cyclase (AC) in sperm retrieved directly from the human epididymis. Particulate fractions were prepared from purified epididymal sperm samples and AC was monitored by the direct conversion of ATP into cAMP. We report that in great contrast to human ejaculated sperm and other mammalian sperm cells, the human epididymal sperm do not express a Mn(2+)-sensitive AC. However, a functional AC was readily detectable in these sperm cells in the presence of saturating concentrations of Ca2+ (50mM) and bicarbonate (HCO3-, 50mM), a combination that causes maximal activation in human ejaculated sperm. Using these conditions, human epididymal sperm AC showed similar capacity to generate cAMP compared to human ejaculated sperm AC. When assays were performed in the presence of Mg2+ and a saturating concentration of GMP-P(NH)P (50 microM), the hydrolysis-resistant GTP analog, and forskolin (100 microM), no activity was detected indicating that the epididymal sperm AC differs from that in somatic cells. These data demonstrate that human epididymal sperm contain an AC that is unique and different from the enzyme system described in somatic cells and other mammalian sperm cells, including human ejaculated sperm.

Guanylate cyclase in Dictyostelium discoideum with the topology of mammalian adenylate cyclase.

Roelofs J, Snippe H, Kleineidam RG, Van Haastert PJ
Biochem J. 2001; 354: 697-706

The core of adenylate and guanylate cyclases is formed by an intramolecular or intermolecular dimer of two cyclase domains arranged in an antiparallel fashion. Metazoan membrane-bound adenylate cyclases are composed of 12 transmembrane spanning regions, and two cyclase domains which function as a heterodimer and are activated by G-proteins. In contrast, membrane-bound guanylate cyclases have only one transmembrane spanning region and one cyclase domain, and are activated by extracellular ligands to form a homodimer. In the cellular slime mould, Dictyostelium discoideum, membrane-bound guanylate cyclase activity is induced after cAMP stimulation; a G-protein-coupled cAMP receptor and G-proteins are essential for this activation. We have cloned a Dictyostelium gene, DdGCA, encoding a protein with 12 transmembrane spanning regions and two cyclase domains. Sequence alignment demonstrates that the two cyclase domains are transposed, relative to these domains in adenylate cyclases. DdGCA expressed in Dictyostelium exhibits high guanylate cyclase activity and no detectable adenylate cyclase activity. Deletion of the gene indicates that DdGCA is not essential for chemotaxis or osmo-regulation. The knock-out strain still exhibits substantial guanylate cyclase activity, demonstrating that Dictyostelium contains at least one other guanylate cyclase.

The Dictyostelium homologue of mammalian soluble adenylyl cyclase encodes a guanylyl cyclase.

Roelofs J, Meima M, Schaap P, Van Haastert PJ
EMBO J. 2001; 20: 4341-8

A new Dictyostelium discoideum cyclase gene was identified that encodes a protein (sGC) with 35% similarity to mammalian soluble adenylyl cyclase (sAC). Gene disruption of sGC has no effect on adenylyl cyclase activity and results in a >10-fold reduction in guanylyl cyclase activity. The scg- null mutants show reduced chemotactic sensitivity and aggregate poorly under stringent conditions. With Mn(2+)/GTP as substrate, most of the sGC activity is soluble, but with the more physiological Mg(2+)/GTP the activity is detected in membranes and stimulated by GTPgammaS. Unexpectedly, orthologues of sGC and sAC are present in bacteria and vertebrates, but absent from Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana and Saccharomyces cerevisiae.

Relationship between local myocardial adenylyl cyclase activity and local coronary blood flow in the dog heart.

Rodriguez E, Weiss HR
J Auton Pharmacol. 1993; 13: 95-103

1. This study tested the hypothesis that heterogeneity of myocardial adenylyl cyclase activity exists and contributes to the heterogeneity of blood flow in canine myocardium, in which flow varies in different areas at any one moment or in a single area at different times. 2. Tissue adenylyl cyclase activity was measured simultaneously with coronary blood flow in the left ventricle of eight anaesthetized open-chest dogs. Coronary flow was measured using radioactive microspheres under control conditions and after sympathetic stimulation. The ventricle was cut into 15 subepicardial and 15 subendocardial sections in which both flow and adenylyl cyclase (basal and forskolin-stimulated) activity were assayed. 3. Control coronary flow was 73.1 +/- 19.9 ml min-1 100 g-1 and ansa subclavia nerve stimulation increased flow to 93.2 +/- 26.1. Baseline adenylyl cyclase activity averaged 51.9 +/- 38.8 pmol min-1 mg protein-1, while forskolin-stimulated activity was 588.2 +/- 393.6. Method variability was approximately 10% of the mean, much less than the biological variability of 70%. 4. Regression analysis of control flow vs baseline adenylyl cyclase activity produced the equation: flow = 0.25 (Adenylyl Cyclase)+60.6, (r = 0.47, P < 0.0001). The equation for stimulated flow vs stimulated adenylyl cyclase was: flow = 0.03 (Adenylyl Cyclase)+73.1, (r = 0.51, P < 0.0001). 5. Thus, significant heterogeneity of tissue adenylyl cyclase activity existed in the heart. There was also a direct linear relationship between adenylyl cyclase activity and coronary flow heterogeneity in canine left ventricle.

Characteristics of glucagon action on the hepatic adenylate cyclase system.

Rodbell M, Birnbaumer L, Pohl SL
Biochem J. 1971; 125: 5859-5859

Structure-function problems with the adenylate cyclase system.

Rodbell M
Adv Cyclic Nucleotide Protein Phosphorylation Res. 1984; 17: 207-14

Evidence for possible involvement of guanylate cyclase in diarrhoea induced by castor oil in mice.

Robert FS, Rao JP
J Diarrhoeal Dis Res. 1996; 14: 218-9

Forskolin carbamates: binding and activation studies with type I adenylyl cyclase.

Robbins JD, Boring DL, Tang WJ, Shank R, Seamon KB
J Med Chem. 1996; 39: 2745-52

Three series of analogs were regioselectively prepared from a protected forskolin precursor to afford 7-carbamoyl-7-desacetylforskolins (series 1), 6-carbamoyl-7-desacetylforskolins (series 2), and 6-carbamoylforskolins (series 3). The analogs were pharmacologically evaluated for binding (IC50) to and activation (EC50) of type I adenylyl cyclase in membranes from stably transfected Sf9 cell lines expressing a single adenylate cyclase subtype. The following ranges were determined for the IC50's and EC50's of each individual series: series 1, IC50 = 43-1600 nM, EC50 = 0.5-9.6 microM; series 2, IC50 = 65-680 nM, EC50 = 0.63-6.5 microM; series 3, IC50 = 21-271 nM, EC50 = 0.5-8.1 microM (forskolin IC50 = 41 nM and EC50 = 0.5 microM). Activation paralleled binding; however, some analogs exhibited poor binding and good activation whereas others demonstrated good binding but poor activation. Steric bulk tended to diminish binding and activation when at the 6- or 7-position, although bulk was accommodated at the 6-position if the 7-site was reacetylated. Acylation of the 7-position by the carbamoyl linker or acetyl was important for obtaining good binding and activation; however, the effect was more pronounced with binding. For both binding and activation, small, linear, lipophilic substituents (propyl, allyl, isopropyl) are well tolerated at the 7-position but less so in the 6-position, even when the 7-site is reacetylated. Planar aromatic moieties (phenyl and 2-pyridinyl) demonstrated moderate to good potency for binding and activation when located at either the 6- or 7-positions. There is an overall trend toward increasing potency for both binding and activation with polar substituents.

[Mechanism of action of hormones at the level of the regulator proteins of adenylate cyclase]

Reyl-Desmars FJ
Gastroenterol Clin Biol. 1988; 12: 149-55

Stimulation of yeast adenylyl cyclase activity by lysophospholipids and fatty acids. Implications for the regulation of Ras/effector function by lipids.

Resnick RJ, Tomaska L
J Biol Chem. 1994; 269: 32336-41

A reconstituted system containing membranes prepared from various Saccharomyces cerevisiae strains (CYR1 ras1 ras2) and a recombinant RAS2 protein was used to evaluate the effect of lipids on adenylyl cyclase activity. Incubation of wild-type membranes with lysophosphatidylinositol, lysophosphatidylserine, or lysophosphatidylcholine stimulated adenylyl cyclase activity in the absence and presence of RAS between 2-10-fold depending upon the individual lipid. Unsaturated fatty acids preferentially increased activity 2-3-fold in the presence of RAS. Other phospholipids as well as several detergents had only marginal effects on adenylyl cyclase activity. Lipids had no effect on either the binding or hydrolysis of GTP by RAS. LysoPI decreased both the Km for ATP and the amount of RAS required for enzyme activation. Four catalytically active deletion mutants of the CYR1 protein including one containing only the C-terminal 417 amino acids were similarly responsive to lysoPI when compared to the wild-type enzyme. These data suggest that lysophospholipids and fatty acids, metabolites of the mitogenically responsive enzyme phospholipase A2, may represent a novel mechanism for modulating the activity of downstream effector molecules and their interaction with Ras proteins.

Eukaryotic-like Adenylyl Cyclases in Mycobacterium tuberculosis H37Rv. CLONING AND CHARACTERIZATION.

Reddy SK, Kamireddi M, Dhanireddy K, Young L, Davis A, Reddy PT
J Biol Chem. 2001; 276: 35141-9

Screening the Mycobacterium tuberculosis H37Rv genomic library for complementation of catabolic defect for cAMP-dependent expression of maltose operon produced the adenylyl cyclase gene (Mtb cya, GenBank(TM) accession no. (1997)) annotated later as Rv1625c (Cole, S. T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S. V., Eiglmeier, K., Gas, S., Barry, C. E., III, et al. (1998) Nature 393, 537-544). The deduced amino acid (aa) sequence (443 aa) encoded by Mtb cya contains a single hydrophobic domain of six transmembrane helices (152 aa) in the amino-terminal half of the protein. Flanking this domain are an arginine-rich (17%) amino-terminal cytoplasmic tail (46 aa) and a carboxyl-terminal cytoplasmic domain (245 aa) with extensive homology to the catalytic core of eukaryotic adenylyl cyclases. Site-directed mutagenesis of Arg(43) and Arg(44) to alanine/glycine showed a loss of adenylyl cyclase activity, whereas mutagenesis to lysine restored the activity. Hence it is proposed that the formation of the catalytic site in Mtb adenylyl cyclase requires an interaction between Arg(43) and Arg(44) residues in the distal cytoplasmic tail and the carboxyl-terminal cytoplasmic domain. Mtb adenylyl cyclase activity at the physiological concentration of ATP (1 mm) was 475 nmol of cAMP/min/mg of membrane protein in the presence of Mn(2+) but only 10 nmol of cAMP/min/mg of membrane protein in the presence of Mg(2+). The physiological significance of the activation of Mtb adenylyl cyclase by Mn(2+) is discussed in view of the presence of manganese transporter protein in mycobacteria and macrophages wherein mycobacteria reside.

Voltage-sensitive adenylyl cyclase activity in cultured neurons. A calcium-independent phenomenon.

Reddy R, Smith D, Wayman G, Wu Z, Villacres EC, Storm DR
J Biol Chem. 1995; 270: 14340-6

Catalytic subunits of mammalian adenylyl cyclases have been proposed to contain 12 transmembrane domains, a property shared with some voltage-sensitive ion channels. Here we report that adenylyl cyclase activity in cerebellar neurons is synergistically stimulated by depolarizing agents and beta-adrenergic receptor activation. This phenomenon is Ca(2+)-independent and not attributable to Ca(2+)-stimulated adenylyl cyclase activity. Cholera toxin and forskolin also synergistically stimulate adenylyl cyclase activity in combination with depolarizing agents. We hypothesize that conformational changes in the catalytic subunit of the enzymes caused by changes in the membrane potential may enhance stimulation of adenylyl cyclases by the guanylyl nucleotide stimulatory protein. This novel mechanism for regulation of adenylyl cyclases generates robust cAMP signals that may contribute to various neuromodulatory events including some forms of neuroplasticity.

Mapping domains in proteins: dissection and expression of Escherichia coli adenylyl cyclase.

Reddy P, Hoskins J, McKenney K
Anal Biochem. 1995; 231: 282-6

We have used the pRE expression vector containing the Escherichia coli adenylyl cyclase gene (cya) with the unique NdeI restriction site CATATG at the initiation codon in conjunction with a family of self-complimentary oligonucleotides to create amino- and carboxyl-terminal domains in adenylyl cyclase. The three sets of oligonucleotides contain a TAA translation stop codon in all reading frames flanked by the NdeI restriction endonuclease sequence and one or two nucleotides (5' NNCATATGTTAATTAATTAACATATGNN 3'). Ligation of one of these annealed oligonucleotides into a restriction site or creation of 5' TAA/CATATG 3' translation stop/NdeI restriction site along a gene in the pRE expression vector facilitates the premature termination of protein synthesis thus yielding amino-terminal domains. Removal of a fragment of the gene corresponding to the amino-terminal portion by NdeI restriction and ligation brings the 3' end of the gene in frame with the initiator ATG. With this strategy, expression of the carboxyl-terminal domain of a protein is possible which is otherwise not as simple as the expression of the amino-terminal domain. The feasibility of expression of any domain of a protein is demonstrated using the cya gene to create several amino- and carboxyl-terminal domains of adenylyl cyclase.

Cerium ions in the histochemical demonstration of second-messenger enzymes.

Rechardt L
Microsc Res Tech. 1998; 40: 440-5

Second-messenger systems are involved in the regulation of numerous cellular processes. Adenylate cyclase (AC) and guanylate cyclase (GC) enzymes are in key positions in the regulation of these systems. The cerium method has been successfully applied to demonstrate amine- and neuropeptide-stimulated AC in rat nervous and adipose tissues and human sweat glands at the electron microscopic level. AC was also localized in cultured neurons. Nitric oxide compounds stimulated GC were demonstrated in rat hippocampal areas. Enzyme reactions were located in neurons pre- and postsynaptically in synapses; in addition, GC activity was seen intraneuronally and in glial cells. Adipocytes and eccrine glandular cells exhibited reaction products in their plasmalemmas. Optimal histochemical conditions are described, combined with control experiments. Some handicaps, related to the sensitivity of the enzymes to the fixatives, penetration problems of cerium salts, and especially the specificity of the method in phosphatase enzyme histochemistry in general are discussed.

Cloning and sequence of partial cDNAs encoding the human type V and VI adenylyl cyclases and subsequent RNA-quantification in various tissues.

Raimundo S, Giray J, Volff JN, Schwab M, Altenbuchner J, Ratge D, Wisser H
Clin Chim Acta. 1999; 285: 155-61

Adenylyl cyclases (Acs) and VI are the predominant form of Acs in mammalian heart where they are part of the beta-adrenergic pathway. Up to now, the sequences for both enzymes from human tissues have not yet been reported. We investigated the mRNA expression AC V and VI in human colon, heart, liver, lung and MNL. Partial cDNAs of human types V and VI Acs were amplified by PCR using oligonucleotides derived from the cytoplasmatic domain sequences of the corresponding enzymes from dog heart. Primers derived from the human sequence were used to detect the mRNAs corresponding to both Acs. For quantification of mRNAs we constructed internal standards for competitive quantitative reverse transcriptase PCR (RT-PCR). Both types of transcripts could be found in all investigated tissues except MNL where only type VI could be detected. Further we demonstrated a more than 60 times higher amount of AC V-mRNA in human heart compared to AC VI-mRNA.

Guanylate cyclase activity in male pattern baldness. Stimulating effect of 3-beta-androstanediol.

Puerto AM, Mallol J
Rev Esp Fisiol. 1993; 49: 1-5

Guanylate cyclase (GC) activity was measured in the cytosolic fraction of sebaceous gland-enriched skin samples obtained from alopecic and non-alopecic areas of subjects affected by male pattern baldness. GC activity was significantly higher in alopecic scalp (7.13 +/- 2.7 pmol/min/mg prot; n = 22) than in non-alopecic samples (3.91 +/- 0.48 pmol/min/mg prot; n = 8). 3-beta-androstanediol was able to increase GC activity up to 15% when added to the incubation medium (1 x 10(-5) M). The regional differences observed should be due to the larger size of the sebaceous glands in alopecic areas. The higher production of dihydrotestosterone and 3-beta-androstanediol in alopecic areas may also contribute to determine the level of GC in scalp skin. The role of second messenger systems in sebaceous glands to understand certain aspects of the action mechanism of androgens is discussed.

Identification and characterization of a widely expressed form of adenylyl cyclase.

Premont RT, Matsuoka I, Mattei MG, Pouille Y, Defer N, Hanoune J
J Biol Chem. 1996; 271: 13900-7

A novel mammalian adenylyl cyclase was identified by reverse transcription-polymerase chain reaction amplification using degenerate primers based on a conserved region of previously described adenylyl cyclases (Premont, R. T. (1994) Methods Enzymol. 238, 116-127). The full-length cDNA sequence obtained from mouse brain predicts a 1353-amino acid protein possessing a 12-membrane span topology, and containing two regions of high similarity with the catalytic domains of adenylyl cyclases. Comparison of this novel adenylyl cyclase with the eight previously described mammalian enzymes indicates that this type 9 adenylyl cyclase sequence is the most divergent, defining a sixth distinct subclass of mammalian adenylyl cyclases. The AC9 gene has been localized to human chromosome band 16p13.3-13.2. The 8.5-kb mRNA encoding the type 9 adenylyl cyclase is widely distributed, being readily detected in all tissues tested, and is found at very high levels in skeletal muscle and brain. AC9 mRNA is found throughout rat brain but is particularly abundant in hippocampus, cerebellum, and neocortex. An antiserum directed against the carboxyl terminus of the type 9 adenylyl cyclase detects native and expressed recombinant AC9 protein in tissue and cell membranes. Levels of the AC9 protein are highest in mouse brain membranes. Characterization of expressed recombinant AC9 reveals that the protein is a functional adenylyl cyclase that is stimulated by Mg2+, forskolin, and mutationally activated Gsalpha. AC9 activity is not affected by Ca2+/calmodulin or by G protein betagamma-subunits. Thus AC9 represents a functional G protein-regulated adenylyl cyclase found in brain and in most somatic tissues.

Lowered responsiveness of the catalyst of adenylyl cyclase to stimulation by GS in heterologous desensitization: a role for adenosine 3',5'-monophosphate-dependent phosphorylation.

Premont RT, Jacobowitz O, Iyengar R
Endocrinology. 1992; 131: 2774-84

Treatment of chick hepatocytes with glucagon results in homologous and heterologous desensitization of the receptor-stimulated adenylyl cyclase. The loci of postreceptor heterologous desensitization was studied. The addition of excess purified GS to glucagon-desensitized hepatocyte membranes did not fully restore fluoride stimulation of adenylyl cyclase, even though the absolute activity was increased at least 2-fold. Treatment of chick hepatocytes with 8-bromo-cAMP resulted in a similar reduction of fluoride stimulation that could not be restored by the addition of purified GS. When membranes from control and glucagon-treated hepatocytes were treated with purified catalytic subunit of protein kinase-A (PKA), fluoride stimulation was lowered in control, but not glucagon-treated, membranes. Treatment of membranes from S49 kin- lymphoma cells with PKA also resulted in decreased fluoride- and forskolin-stimulated adenylyl cyclase activity, but activity stimulated by Mn2+ was not altered. Since previous studies from our laboratory had shown that GS and G(i) are not substrates for protein kinase-A, it appears that the catalyst of adenylyl cyclase is the likely locus of modulation. To determine if both chick hepatocytes and S49 cells contain similar types of adenylyl cyclase that could account for the similar PKA regulatory properties, we used polymerase chain reaction-based techniques to identify GS-stimulated adenylyl cyclases present in these systems. The chick liver contains both type 5 and type 6 adenylyl cyclases, while S49 cells contain the type 6 enzyme. Type 5 and 6 adenylyl cyclases are members of one widely expressed subfamily of mammalian GS-responsive adenylyl cyclases and share a predicted PKA phosphorylation site in the central cytoplasmic loop. This site is not found in other known adenylyl cyclases (types 1-4), although the olfactory-specific type 3 enzyme has a predicted site nearby. These data indicate that one component of hormone-induced desensitization of the adenylyl cyclase system can be at the level of the catalyst, where PKA-mediated phosphorylation could result in lowered responsiveness. The types 5 and 6 adenylyl cyclases are likely candidates for such regulation.

Two members of a widely expressed subfamily of hormone-stimulated adenylyl cyclases.

Premont RT, Chen J, Ma HW, Ponnapalli M, Iyengar R
Proc Natl Acad Sci U S A. 1992; 89: 9809-13

cDNA encoding a hormone- and guanine nucleotide-stimulated adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] (type 6) from rat liver and kidney has been cloned and expressed. This enzyme is stimulated by forskolin, guanosine 5'-[gamma-thio]triphosphate, and isoproterenol plus GTP but is not stimulated by beta gamma subunits of guanine nucleotide-binding proteins. A second form (type 5), which is 75% similar to type 6, has also been cloned. Both types 5 and 6 cDNAs have multiple messages. PCR-based detection of the mRNA for the type 5 and 6 enzymes indicates that both are widely distributed. Homology analyses indicate at least four distinct subfamilies of guanine nucleotide stimulatory protein-regulated adenylyl cyclases. Types 5 and 6 enzymes define one distinct subfamily of mammalian adenylyl cyclases. Diversity of one guanine nucleotide-binding protein-regulated effector may allow different modes of regulation of cell-surface signal transmission.

Specific MG2+ and adenosine sites involved in a bireactant mechanism for adenylate cyclase inhibition and their probable localization on this enzyme's catalytic component.

Premont J, Guillon G, Bockaert J
Biochem Biophys Res Commun. 1979; 90: 513-9

Biochemical methods for detection and measurement of cyclic AMP and adenylyl cyclase activity.

Post SR, Ostrom RS, Insel PA
Methods Mol Biol. 2000; 126: 363-74

Alterations in plasma membrane proteins associated with the proteolytic activation of adenylate cyclase.

Pinkett MO, Anderson WB
Arch Biochem Biophys. 1980; 200: 261-8

Type 5 adenylyl cyclase distribution.

Pieroni JP, Miller D, Premont RT, Iyengar R
Nature. 1993; 363: 679-80

Signal recognition and integration by Gs-stimulated adenylyl cyclases.

Pieroni JP, Jacobowitz O, Chen J, Iyengar R
Curr Opin Neurobiol. 1993; 3: 345-51

Six Gs-stimulated adenylyl cyclases have been cloned. Two additional forms have been identified as partial cDNAs. These adenylyl cyclases have distinct functional properties and are differentially regulated by protein kinases. The adenylyl cyclases have distinct patterns of distribution in peripheral tissues and various brain regions. The unique functional characteristics of the members of this effector family may allow each cell type and/or brain region to customize the signal-recognition and integrative properties of its cAMP-generation system by varying the ratios of the various adenylyl cyclases.

Distinct characteristics of the basal activities of adenylyl cyclases 2 and 6.

Pieroni JP, Harry A, Chen J, Jacobowitz O, Magnusson RP, Iyengar R
J Biol Chem. 1995; 270: 21368-73

Regulation of basal activities of adenylyl cyclase (AC) 2 and 6, expressed in Sf9 cells by infection with recombinant baculovirus, was studied. An antipeptide antibody that recognizes AC2 and AC6 with equal sensitivity was used to establish that equivalent levels were expressed. Basal activities of AC2 and AC6 were compared at varying concentrations of Mg2+ or Mn2+ ions; AC2 had 15- and 10-fold greater activity than AC6, respectively. At 20 mM Mg2+, the Km values for ATP were 88 and 39 microM for AC2 and AC6, respectively, whereas their Vmax values were 281 and 11 pmol/mg protein.min. With 100 microM forskolin and either Mg2+ or Mn2+, the difference in activities between AC2 and AC6 was reduced to approximately 2-fold. Forskolin stimulated AC6 greater than 40-fold at 0.5-2 mM Mg2+, whereas AC2 was stimulated 4-6-fold. At 20 mM Mg2+, AC2 was stimulated 2-fold by forskolin, whereas AC6 was stimulated 18-fold. With Mg2+ alone, activities of AC2 and AC6 were not saturable up to 20 mM and yielded curvilinear Hofstee transformations. With forskolin, activities of both AC2 and AC6 were saturable by 10 mM Mg2+ and yielded linear Hofstee transformations. These data indicate that there are substantial differences in the basal enzymatic activities of adenylyl cyclase isoforms, due to differential regulation by Mg2+ ions rather than intrinsic catalytic capabilities. Thus the presence and relative abundance of adenylyl cyclase subtypes could greatly affect the resting cellular cAMP levels with consequent effects on important biological functions, such as differentiation and proliferation.

Topology of the GTP-binding site of adenylyl cyclase from pigeon erythrocytes.

Pfeuffer T, Eckstein F
FEBS Lett. 1976; 67: 354-8

Characterization and localization of adenylyl cyclase in membrane vesicles and intact boar and human spermatozoa.

Peterson RN, Russell L, Hook L, Bundman D, Freund M
J Cell Sci. 1980; 43: 93-102

The enzymic properties of adenylyl cyclase in purified membrane vesicles from human and boar spermatozoa are described. Plasma membrane vesicles, which appear to be right-side-out, show a marked increase in activity in the presence of the detergent Triton X-100, manganous ion and alkaline pH. Electron-microscope cytochemical assays indicated the presence of adenylyl cyclase in boar and human sperm plasma membranes and also within the axoneme of intact human spermatozoa. The significance and precautions in the evaluation of the cytochemical data are discussed.

A pyruvate-stimulated adenylate cyclase has a sequence related to the fes/fps oncogenes and to eukaryotic cyclases.

Peters EP, Wilderspin AF, Wood SP, Zvelebil MJ, Sezer O, Danchin A
Mol Microbiol. 1991; 5: 1175-81

The pyruvate-stimulated adenylate cyclase from Brevibacterium liquefaciens produces up to 450 microM cyclic AMP in the culture medium when the bacterium is grown on glucose and alanine. In this paper we report the cloning, expression and sequencing of the gene for this enzyme. Residues were identified, within the C-terminal domain, which are conserved in adenylate and guanylate cyclase sequences from eukaryotes and in the adenylate cyclase of the prokaryote Rhizobium meliloti. We have also identified a sequence of 30 residues near the N-terminus of the protein which is homologous to part of the regulatory domain of the cellular homologues of the oncogenes fes and fps; this sequence is also present in the avian Fujinami sarcoma virus fps gene.

Bacterial adenylyl cyclases.

Peterkofsky A, Reizer A, Reizer J, Gollop N, Zhu PP, Amin N
Prog Nucleic Acid Res Mol Biol. 1993; 44: 31-65

The Escherichia coli adenylyl cyclase complex: stimulation by GTP and other nucleotides.

Peterkofsky A, Gollop N
Protein Sci. 1993; 2: 498-505

Escherichia coli cells permeabilized by treatment with low concentrations of toluene contain an adenylyl cyclase activity that can be stimulated 3.6-7.6-fold by GTP. The stimulatory effect of GTP is maximal at concentrations of the nucleotide in the physiological range (above 0.7 mM). Studies of the dependence of velocity on substrate (ATP) concentration indicate that the velocity vs. substrate plots are sigmoid in the absence of GTP but hyperbolic in the presence of GTP, suggesting an allosteric regulatory site that can be occupied by either ATP or GTP. Replacement of ATP by AMPPNP as substrate results in velocity vs. substrate plots that are hyperbolic in the absence or presence of GTP, although GTP increases the Vmax by a factor of 2.2; these findings indicate that AMPPNP specifically occupies the substrate site and GTP exclusively occupies the regulatory site. A test of the capacity of other guanine nucleotides to stimulate adenylyl cyclase activity showed that 2'-deoxy-GTP was almost as effective as GTP, but that GDP, GMP, ppGpp, and 3',5'-cGMP were not stimulatory effectors; GTP-gamma-S and GMPPNP stimulated adenylyl cyclase activity but to a lesser degree than did GTP. In addition to the previous indication that ATP can occupy the regulatory site on adenylyl cyclase, it was found that CTP and UTP were potent stimulators. Thus, all the naturally occurring RNA precursor nucleoside triphosphates are capable of stimulating adenylyl cyclase activity. In contrast, PPPi inhibits adenylyl cyclase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

A temperature-sensitive adenylyl cyclase mutant of Dictyostelium.

Patel H, Guo K, Parent C, Gross J, Devreotes PN, Weijer CJ
EMBO J. 2000; 19: 2247-56

Dictyostelium development starts with the chemotactic aggregation of up to 10(6) amoebae in response to propagating cAMP waves. cAMP is produced by the aggregation stage adenylyl cyclase (ACA) and cells lacking ACA (aca null) cannot aggregate. Temperature-sensitive mutants of ACA were selected from a population of aca null cells transformed with a library of ACA genes, a major segment of which had been amplified by error-prone PCR. One mutant (tsaca2) that can complement the aggregation null phenotype of aca null cells at 22 degrees C but not at 28 degrees C was characterized in detail. The basal catalytic activity of the enzyme in this mutant was rapidly and reversibly inactivated at 28 degrees C. Using this mutant strain we show that cell movement in aggregates and mounds is organized by propagating waves of cAMP. Synergy experiments between wild-type and tsaca2 cells, shifted to the restrictive temperature at various stages of development, showed that ACA plays an important role in the control of cell sorting and tip formation.

Sequence of a human brain adenylyl cyclase partial cDNA: evidence for a consensus cyclase specific domain.

Parma J, Stengel D, Gannage MH, Poyard M, Barouki R, Hanoune J
Biochem Biophys Res Commun. 1991; 179: 455-62

A cDNA coding for a human brain adenylyl cyclase was isolated and sequenced. The deduced partial 675 amino-acid sequence was compared with those of other known adenylyl and guanylyl cyclases. Comparison of this predicted amino-acid sequence with that of bovine brain (type I) and rat olfactory (type III) adenylyl cyclase indicated a significant homology with the carboxyl-terminal halves of both enzymes. The homology between the human adenylyl cyclase and the other two mammalian adenylyl cyclase also appears at the topographic level. Indeed, the human enzyme includes a extremely hydrophobic region containing six potential membrane-spanning segments followed by a large hydrophilic domain. At the beginning of the hydrophilic domain, there is a 250 amino-acid region which shows not only a striking homology with the bovine and rat adenylyl cyclase (86% of similarity and 57% of identity), but also a significant homology with non-mammalian adenylyl cyclase and guanylyl cyclases. We found that this 250 amino-acid domain contains a sequence of about 165 amino-acids which is highly conserved in most of the known nucleotide cyclases suggesting that it includes residues that are critical for the function of the enzymes.

A nuclear location for Ca2+-activated adenylyl cyclases I and III in neurones.

Parkinson NA, Bolsover SR
Brain Res Mol Brain Res. 2001; 91: 43-9

Calcium/calmodulin-sensitive adenylyl cyclases are increasingly recognised as important factors in memory formation and synaptic plasticity. We have examined the distributions of adenylyl cyclases types I, III, and VIII within rat primary sensory neurons. Immunofluorescence revealed distinct staining for adenylyl cyclases type I and III, but not adenylyl cyclase type VIII, within the cell nucleus. Western blots suggest that a processed form of adenylyl cyclase type III may be found within primary neurons and PC12 cells as a 70-kDa protein. We propose that the observed nuclear adenylyl cyclases are soluble forms of the cyclases.

Constitutively active adenylyl cyclase mutant requires neither G proteins nor cytosolic regulators.

Parent CA, Devreotes PN
J Biol Chem. 1996; 271: 18333-6

Receptor-mediated G protein-linked adenylyl cyclase systems are universal signal transducers. We exploited the essential role of this cascade in Dictyostelium development to screen for random mutations in the catalytic component, ACA. This enzyme is activated by G protein betagamma-subunits acting in concert with a novel cytosolic regulator, CRAC. By suppression of the CRAC-null phenotype, we isolated constitutively active versions of the enzyme that require neither exogenous stimuli nor internal regulators. One mutant displayed a 15-fold increase in its Vmax. It harbors a single amino acid substitution (L394S) affecting a conserved residue located in the first cytoplasmic loop near the N-terminal hydrophobic domain of ACA. The screening procedure can be adapted for isolation of constitutive mutations in mammalian adenylyl cyclases.

A novel Myb homolog initiates Dictyostelium development by induction of adenylyl cyclase expression.

Otsuka H, Van Haastert PJ
Genes Dev. 1998; 12: 1738-48

Dictyostelium development is induced by starvation. The adenylyl cyclase gene ACA is one of the first genes expressed upon starvation. ACA produces extracellular cAMP that induces chemotaxis, aggregation, and differentiation in neighboring cells. Using insertional mutagenesis we have isolated a mutant that does not aggregate upon starvation but is rescued by adding extracellular cAMP. Sequencing of the mutated locus revealed a new gene, DdMYB2, whose product contains three Myb repeats, the DNA-binding motif of Myb-related transcription factors. Ddmyb2-null cells show undetectable levels of ACA transcript and no cAMP production. Ectopic expression of ACA from a constitutive promotor rescues differentiation and morphogenesis of Ddmyb2-null mutants. The results suggest that development in Dictyostelium starts by starvation-mediated DdMyb2 activation, which induces adenylyl cyclase activity producing the differentiation-inducing signal cAMP.

Persistence of the interaction of calmodulin with adenylyl cyclase: implications for integration of transient calcium stimuli.

Onyike CU, Lin AH, Abrams TW
J Neurochem. 1998; 71: 1298-306

Ca2+/calmodulin-sensitive adenylyl cyclase plays a role in several forms of synaptic plasticity and learning. To understand how cellular signals from neuronal activity during behavioral stimuli might be integrated by adenylyl cyclase, we have characterized the response of type I adenylyl cyclase to transient Ca2+ stimuli. Stimulation by a several second Ca2+ stimulus is delayed, rising to a peak after the Ca2+ stimulus has ended. We attempted to identify the site of the persistent Ca2+ signal that enabled adenylyl cyclase stimulation to increase after free Ca2+ had declined. Free calmodulin itself displayed no persistent activation by Ca2+ and was unable to activate adenylyl cyclase if exposed to low Ca2+ solution <1 s before reaching adenylyl cyclase. In contrast, activation of the calmodulin-adenylyl cyclase complex persisted for seconds after Ca2+ stimulus. Activation decayed with a time constant of 6 or 13 s depending on assay conditions. These results suggest that the calmodulin-adenylyl cyclase complex can serve as a site of cellular memory for a Ca2+ transient that has ended even before adenylyl cyclase is fully activated.

Identification of a domain in Bordetella pertussis adenylyl cyclase important for subunit interactions and cell invasion activity.

Oldenburg DJ, Storm DR
Microb Pathog. 1993; 15: 153-7

Bordetella pertussis produces a calmodulin-stimulated adenylyl cyclase that invades animal cells and raises intracellular cAMP levels. The enzyme does not enter animal cells by receptor-mediated endocytosis, but the mechanism for invasion of animal cells has not been defined. We have proposed that the 177 kDa adenylyl cyclase is proteolyzed to a 45 kDa catalytic subunit and one or more polypeptides (invasive factor) that facilitate entry of the catalytic subunit into animal cells. In this study, we report the identification of a sequence of amino acids within the adenylyl cyclase catalytic subunit that is important for entry of the enzyme into eukaryotic cells. A synthetic peptide corresponding to amino acids 313-339 within the catalytic subunit was shown to inhibit invasion of neuroblastoma cells by the adenylyl cyclase. In addition, this peptide inhibited the association of the catalytic subunit with invasive factor. We propose that this domain is a site for interaction between the catalytic subunit and invasive factor.

Identification of A2a adenosine receptor domains involved in selective coupling to Gs. Analysis of chimeric A1/A2a adenosine receptors.

Olah ME
J Biol Chem. 1997; 272: 337-44

Responses to adenosine are governed by selective activation of distinct G proteins by adenosine receptor (AR) subtypes. The A2aAR couples via Gs to adenylyl cyclase stimulation while the A1AR couples to Gi to inhibit adenylyl cyclase. To determine regions of the A2aAR that selectively couple to Gs, chimeric A1/A2aARs were expressed in Chinese hamster ovary cells and ligand binding and adenylyl cyclase activity analyzed. Replacement of the third intracellular loop of the A2aAR with that of the A1AR reduced maximal adenylyl cyclase stimulation and decreased agonist potency. Restricted chimeras indicated that the NH2-terminal portion of intracellular loop 3 was predominantly responsible for this impairment. Reciprocal chimeras composed primarily of A1AR sequence with limited A2aAR sequence substitution stimulated adenylyl cyclase and thus supported these findings. A lysine and glutamic acid residue were identified as necessary for efficient A2aAR-Gs coupling. Analysis of chimeric receptors in which sequence of intracellular loop 2 was substituted indicated that the nature of amino acids in this domain may indirectly modulate A2aAR-Gs coupling. Replacement of the cytoplasmic tail of the A2aAR with the A1AR tail did not affect adenylyl cyclase stimulation. Thus, selective activation of Gs is predominantly dictated by the NH2-terminal segment of the third intracellular loop of the A2aAR.

Synechocystis strain PCC 6803 cya2, a prokaryotic gene that encodes a guanylyl cyclase.

Ochoa De Alda JA, Ajlani G, Houmard J
J Bacteriol. 2000; 182: 3839-42

Synechocystis strain PCC 6803 exhibits similar levels of cyclic AMP (cAMP) and cyclic GMP (cGMP). A thorough analysis of its genome showed that Cya2 (Sll0646) has all the sequence determinants required in terms of activity and purine specificity for being a guanylyl cyclase. Insertional mutagenesis of cya2 caused a marked reduction in cGMP content without altering the cAMP content. Thus, Cya2 represents the first example of a prokaryotic guanylyl cyclase.

Coiled-coil interaction of N-terminal 36 residues of cyclase-associated protein with adenylyl cyclase is sufficient for its function in Saccharomyces cerevisiae ras pathway.

Nishida Y, Shima F, Sen H, Tanaka Y, Yanagihara C, Yamawaki-Kataoka Y, Kariya K, Kataoka T
J Biol Chem. 1998; 273: 28019-24

In the budding yeast Saccharomyces cerevisiae, association with the 70-kDa cyclase-associated protein (CAP) is required for proper response of adenylyl cyclase to Ras proteins. We show here that a small segment comprising the N-terminal 36 amino acid residues of CAP is sufficient for association with adenylyl cyclase as well as for its function in the Ras-adenylyl cyclase pathway as assayed by the ability to confer RAS2(Val-19)-dependent heat shock sensitivity to yeast cells. The CAP-binding site of adenylyl cyclase was mapped to a segment of 119 amino acid residues near its C terminus. Both of these regions contained tandem repetitions of a heptad motif alphaXXalphaXXX (where alpha represents a hydrophobic amino acid and X represents any amino acid), suggesting a coiled-coil interaction. When mutants of CAP defective in associating with adenylyl cyclase were isolated by screening of a pool of randomly mutagenized CAP, they were found to carry substitution mutations in one of the key hydrophobic residues in the heptad repeats. Furthermore, mutations of the key hydrophobic residues in the heptad repeats of adenylyl cyclase also resulted in loss of association with CAP. These results indicate the coiled-coil mechanism as a basis of the CAP-adenylyl cyclase interaction.

The novel guanylyl cyclase MsGC-I is strongly expressed in higher-order neuropils in the brain of Manduca sexta.

Nighorn A, Simpson PJ, Morton DB
J Exp Biol. 2001; 204: 305-14

Guanylyl cyclases are usually characterized as being either soluble (sGCs) or receptor (rGCs). We have recently cloned a novel guanylyl cyclase, MsGC-I, from the developing nervous system of the hawkmoth Manduca sexta that cannot be classified as either an sGC or an rGC. MsGC-I shows highest sequence identity with receptor guanylyl cyclases throughout its catalytic and dimerization domains, but does not contain the ligand-binding, transmembrane or kinase-like domains characteristic of receptor guanylyl cyclases. In addition, MsGC-I contains a C-terminal extension of 149 amino acid residues. In this paper, we report the expression of MsGC-I in the adult. Northern blots show that it is expressed preferentially in the nervous system, with high levels in the pharate adult brain and antennae. In the antennae, immunohistochemical analyses show that it is expressed in the cell bodies and dendrites, but not axons, of olfactory receptor neurons. In the brain, it is expressed in a variety of sensory neuropils including the antennal and optic lobes. It is also expressed in structures involved in higher-order processing including the mushroom bodies and central complex. This complicated expression pattern suggests that this novel guanylyl cyclase plays an important role in mediating cyclic GMP levels in the nervous system of Manduca sexta.

Spontaneous dimerization and leucine-zipper induced activation of the recombinant catalytic domain of a new adenylyl cyclase of Trypanosoma brucei, GRESAG4.4B.

Naula C, Schaub R, Leech V, Melville S, Seebeck T
Mol Biochem Parasitol. 2001; 112: 19-28

In this study, we describe the isolation and characterization of a new adenylyl cyclase from Trypanosoma brucei and its activation by dimerization of the catalytic domain. In agreement with the current nomenclature of trypanosomal adenylyl cyclases, this new gene is termed GRESAG4.4B. The complete ORF of the GRESAG4.4B gene encodes a protein of 1291 amino acids. Its predicted protein structure is consistent with the structure of other trypanosomal cyclases, and with the cyclases of L. donovani. GRESAG 4.4B is constitutively expressed during the life cycle of trypanosomes. GRESAG4.4B is a member of a gene family, which contains at least six members, which are all clustered on chromosome IV. The catalytic domain of GRESAG4.4B is able to dimerize spontaneously. However, these spontaneously formed, stable dimers only show minimal enzymatic activity. The addition of a leucine zipper (LZ) derived from the S. cerevisiae GCN 4 gene to the N-terminus of the catalytic domain of GRESAG4.4B strongly activated its enzymatic activity. The LZ appears to enforce a distinct conformation of the dimer, which leads to an increased enzymatic activity, and thus may mimic the effect of ligand-induced dimerization of adenylyl cyclase in vivo.

Cloning of guanylyl cyclase isoforms.

Nakane M, Murad F
Adv Pharmacol. 1994; 26: 7-18

The cloning of particulate and soluble guanylyl cyclases is summarized in Table I. With respect to transmembrane signal transduction systems, guanylyl and adenylyl cyclases can be grouped together with some protein tyrosine kinases and protein tyrosine phosphatases to form a diverse protein family with various structural and functional similarities (Garbers, 1989, 1991, 1992; Koesling et al., 1991; Chinkers and Garbers, 1991; Fig. 1). Particulate guanylyl cyclase contains a single transmembrane domain, and the peptide-binding portion (ligand receptor) is on the exterior surface and the catalytic region on the interior, similar to the protein tyrosine kinase/receptor and the protein tyrosine phosphatase/receptor families (Yarden et al., 1986; Charbonneau et al., 1988; Tonks et al., 1988). Protein tyrosine kinases and phosphatases are also activated by ligand binding to the extracellular domain, which in turn results in phosphorylation or dephosphorylation. On the other hand, soluble guanylyl cyclase exists as a heterodimer with two putative catalytic domains, and both subunits are essential for enzyme activity and activation by nitric oxide. It is thus particularly interesting that adenylyl cyclase also contains two catalytic domains, which are both necessary for catalytic activity (Tang et al., 1991). It is possible that particulate guanylyl cyclase may also dimerize on hormonal stimulation and two catalytic domains from two monomers form a functional catalytic center capable of forming cyclic GMP. The catalytic core of GC-A expressed in bacteria was shown to form a homodimer with positively cooperative kinetics (Thorpe et al., 1991). The physiological significance of the existence of multiple forms of soluble guanylyl cyclase subunits remains unclear. Future studies should reveal the differences in tissue distribution and activation by nitrovasodilators in various heterodimers of soluble guanylyl cyclase.

Construction of a full-length Ca2+-sensitive adenylyl cyclase/aequorin chimera.

Nakahashi Y, Nelson E, Fagan K, Gonzales E, Guillou JL, Cooper DM
J Biol Chem. 1997; 272: 18093-7

Ca2+-sensitive adenylyl cyclases are key integrators of Ca2+ and cAMP signaling. To selectively probe dynamic changes in [Ca2+]i at the plasma membrane where adenylyl cyclases reside, a full-length, Ca2+-inhibitable type VI adenylyl cyclase/aequorin chimera has been constructed by a two-stage polymerase chain reaction method. The expressed adenylyl cyclase/aequorin chimera was appropriately localized to the plasma membrane, as judged by biochemical fractionation and functional analysis. The chimera retained full adenylyl cyclase activity and sensitivity to inhibition by physiological [Ca2+]i elevation. The aequorin portion of the chimeric construct was also capable of measuring changes in [Ca2+] both in vitro and in vivo. When the plasma membrane-tagged aequorin and cytosolic aequorin were compared in their measurement of [Ca2+]i, they showed contrasting sensitivities depending on whether the [Ca2+]i originated from internal stores or capacitative entry. This is the first full-length enzyme-aequorin chimera that retains the full biological properties of both aequorin and a Ca2+-sensitive adenylyl cyclase. This novel chimeric Ca2+ sensor provides the unique ability to directly report the dynamics of [Ca2+]i that regulates this Ca2+-sensitive enzyme under a variety of physiological conditions. Since this chimera is localized to the plasma membrane, it can also be used to assess local changes in [Ca2+]i at the plasma membrane as distinct from global changes in [Ca2+]i within the cytosol.

Structural flexibility of the calmodulin-binding locus in Bordetella pertussis adenylate cyclase. Reconstitution of catalytically active species from fragments or inactive forms of the enzyme.

Munier H, Bouhss A, Gilles AM, Krin E, Glaser P, Danchin A, Barzu O
Eur J Biochem. 1993; 217: 581-6

The catalytic domain of Bordetella pertussis adenylate cyclase, a calmodulin-activated enzyme with toxic properties, is a modular construct cleaved by trypsin into two subdomains of 224 (T25) and 175 (T18) amino acids. The calmodulin-binding locus of the bacterial enzyme consists of approximately 70 amino acids and overlaps the C-terminus of T25 and the N-terminus of T18. This region, exposed to the solvent or proteases, also exhibits an unusual high flexibility and allows, as demonstrated in this study, reconstitution in the presence of calmodulin of active species of adenylate cyclase from overlapping inactive fragments of the enzyme. Moreover, several combinations of inactive variants of the bacterial enzyme obtained by site-directed mutagenesis can yield active species. Heterodimers, resulting from a few selected combinations of inactive species of adenylate cyclase, exhibit specific activity similar to that of the native enzyme. Productive complementation from inactive fragments is a unique phenomenon among calmodulin-activated enzymes and represents a new and helpful tool in the understanding of the molecular mechanism of activation of B. pertussis adenylate cyclase upon binding of calmodulin.

[Adenylate cyclase]

Muller AB, Ammon HP
Pharm Unserer Zeit. 1986; 15: 49-52

Purification of bovine brain adenylyl cyclase with a novel derivative of forskolin: evidence for a high specific activity form of the enzyme.

Moos M Jr, Morris DI, Robbins J, Appel L, Seamon KB
Prep Biochem Biotechnol. 1996; 26: 155-67

An improved affinity support for the purification of adenylyl cyclase was prepared from 7-desacetyl-7-aminoethylaminocarbonyl forskolin. This analog allows convenient synthesis of an affinity matrix that is chemically stable, with-standing repeated use for up to two years, and efficient, yielding purifications of adenylyl cyclase from solubilized bovine brain membranes of 2,000-6,000 fold in a single step. Immunoblotting data suggest that the majority of the enzyme purified in this fashion differs from forms described previously. Since the specific activity of this preparation is substantially higher than that described in previous reports, it is possible that the purification described here selects, presumably on the basis of affinity for forskolin, for a form of adenylyl cyclase with higher specific activity than any described previously.

Discrete expression of Ca2+/calmodulin-sensitive and Ca(2+)-insensitive adenylyl cyclases in the rat brain.

Mons N, Yoshimura M, Cooper DM
Synapse. 1993; 14: 51-9

Physiological studies and inferences from invertebrate models implicate Ca2+/calmodulin-sensitive adenylyl cyclase with memory processes. However, Ca2+/calmodulin-insensitive adenylyl cyclase also occurs in brain, and its neuronal functions are less clear. Two oligonucleotide probes, based on rat cDNAs for Types I and II adenylyl cyclase, which appear to correspond to these functional forms, were used to conduct in situ hybridization analysis of the relative abundance and localization of these two species in the rat brain as a first step in evaluating their neuronal role. Quite discrete patterns of expression were encountered; in some areas, both species were co-expressed, but in others, little overlap was observed. The differential expression of the two mRNAs suggests that discrete roles may be fulfilled by the two adenylyl cyclases in neural tissues.

Immunohistochemical localization of adenylyl cyclase in rat brain indicates a highly selective concentration at synapses.

Mons N, Harry A, Dubourg P, Premont RT, Iyengar R, Cooper DM
Proc Natl Acad Sci U S A. 1995; 92: 8473-7

Only three isoforms of adenylyl cyclase (EC 4.6.1.1) mRNAs (AC1, -2, and -5) are expressed at high levels in rat brain. AC1 occurs predominantly in hippocampus and cerebellum, AC5 is restricted to the basal ganglia, whereas AC2 is more widely expressed, but at much lower levels. The distribution and abundance of adenylyl cyclase protein were examined by immunohistochemistry with an antiserum that recognizes a peptide sequence shared by all known mammalian adenylyl cyclase isoforms. The immunoreactivity in striatum and hippocampus could be readily interpreted within the context of previous in situ hybridization studies. However, extending the information that could be gathered by comparisons with in situ hybridization analysis, it was apparent that staining was confined to the neuropil--corresponding to immunoreactive dendrites and axon terminals. Electron microscopy indicated a remarkably selective subcellular distribution of adenylyl cyclase protein. In the CA1 area of the hippocampus, the densest immunoreactivity was seen in postsynaptic densities in dendritic spine heads. Labeled presynaptic axon terminals were also observed, indicating the participation of adenylyl cyclase in the regulation of neurotransmitter release. The selective concentration of adenylyl cyclases at synaptic sites provides morphological data for understanding the pre- and postsynaptic roles of adenylyl cyclase in discrete neuronal circuits in rat brain. The apparent clustering of adenylyl cyclases, coupled with other data that suggest higher-order associations of regulatory elements including G proteins, N-methyl-D-aspartate receptors, and cAMP-dependent protein kinases, suggests not only that the primary structural information has been encoded to render the cAMP system responsive to the Ca(2+)-signaling system but also that higher-order strictures are in place to ensure that Ca2+ signals are economically delivered and propagated.

Acylation of adenylyl cyclase catalyst is important for enzymic activity.

Mollner S, Beck K, Pfeuffer T
FEBS Lett. 1995; 371: 241-4

Incubation of human thrombocytes in the presence of [3H]palmitic acid leads to incorporation of this fatty acid into the alpha subunit of Gs as described [Linder et al., Proc. Natl. Acad. Sci. USA 90 (1993) 3675-3679; Degtyarev et al., Biochemistry 32 (1993) 8057-8061] but also into the catalyst of adenylyl cyclase which has not been recognized before. Treatment of labeled membranes with hydroxylamine released the label from both components. Label incorporated into the catalyst could be identified as [3H]palmitate. At the same time chemical deacylation caused partial loss of adenylyl cyclase activity.

The C2 catalytic domain of adenylyl cyclase contains the second metal ion (Mn2+) binding site.

Mitterauer T, Hohenegger M, Tang WJ, Nanoff C, Freissmuth M
Biochemistry. 1998; 37: 16183-91

Membrane-bound mammalian adenylyl cyclase isoforms contain two internally homologous cytoplasmic domains (C1 and C2). When expressed separately, C1 and C2 are catalytically inactive, but conversion of ATP to cAMP is observed if C1 and C2 are combined. By analogy with DNA polymerases, adenylyl cyclases are thought to require two divalent metal ions for nucleotide binding and phosphodiester formation; however, only one Mg2+ ion (liganded to C1) has been visualized in the recently solved crystal structure of a C1-C2 complex [Tesmer, J. J. G., Sunahara, R. K., Gilman, A. G., and Sprang, S. R. (1997) Science 278, 1907-1916]. Here, we have studied the binding of ATP to IIC2 (from type II adenylyl cyclase) using ATP analogues [2',3'-dialdehyde ATP (oATP), a quasi-irreversible inhibitor that is covalently incorporated via reduction of a Schiff base, the photoaffinity ligand 8-azido-ATP (8N3-ATP), and trinitrophenyl-ATP (TNP-ATP), a fluorescent analogue] and fluorescein isothiocyanate (FITC). [alpha-32P]oATP and 8N-[alpha-32P]ATP are specifically incorporated into IIC2. Labeling of IIC2 by [alpha-32P]oATP and by FITC is greatly enhanced by Mn2+ and to a much lesser extent by Mg2+. Similarly, TNP-ATP binds to IIC2 as determined by fluorescence enhancement, and this binding is promoted by Mn2+. Thus, a second metal ion binding site (preferring Mn2+) is contained within the C2 domain, and this finding highlights the analogy in the reaction catalyzed by DNA polymerases and adenylyl cyclases.

Interaction of vanadate and iodate oxyanions with adenylyl cyclase of ciliary processes.

Mittag TW, Guo WB, Taniguchi T
Biochem Pharmacol. 1993; 45: 1311-6

Vanadate (VO3-) was found to activate adenylyl cyclase (AC) in ocular ciliary process membrane. This response was additive to that of isoproterenol (ISO) and vasoactive intestinal peptide (VIP), but it was potentiative with forskolin (FSK) and also with Ca2+/calmodulin activation of AC activity. The potentiated response of FSK in the presence of VO3- was due to an increase in Vmax without a change in the apparent affinity of FSK or VO3-, and therefore differs from the potentiated response of activated G-protein (Gs) and FSK, where the affinity of FSK was increased by 1-2 orders of magnitude. Potentiation occurred at low Mg2+ and was not observed at free [Mg2+] > 3 mM. Iodate (IO3-) inhibited the FSK, ISO, and VO3- activations of AC in ciliary process membranes (IC50, 0.3 mM). In vivo topical treatment of the rabbit eye with 50 microL of 5% NaIO3 had no effect alone but completely blocked the intraocular pressure response to a 50-microL topical dose of 1% FSK and partially blocked the response to a 50-microL dose of 0.001% ISO. These findings indicate that some AC enzymes may have a binding site for oxyanions which can directly regulate enzyme activity.

Bicarbonate-activated adenylyl cyclase in fluid-transporting tissues.

Mittag TW, Guo WB, Kobayashi K
Am J Physiol. 1993; 264: 10604-10604

A bicarbonate-stimulated adenylyl cyclase (AC) activity was found in ocular ciliary processes, which secrete the aqueous humor of the eye. Other fluid-transporting tissues also showed HCO3(-)-stimulated AC activity. Relative to basal, the response to 10 mM NaHCO3 was greatest in the particulate fraction of bovine ciliary processes, followed by bovine corneal endothelium, bovine choroid plexus, and rat kidney (medulla and cortex). However, no activity was detectable in bovine retina, cerebral cortex, or cerebellum. The activity in ciliary processes was present only in the particulate fraction, was supported by Mg2+ or Mn2+, was independent of GTP, and was additive to the stimulatory G protein-dependent AC response (via beta-adrenergic receptor stimulation). The activation of AC was dose dependent up to 100 mM bicarbonate with a 50% effective concentration of 2-3 mM. The HCO3- response was unaffected by 1 mM methazolamide, a carbonic anhydrase inhibitor, but HSO3- was a partially selective inhibitor compared with its effect on forskolin-stimulated AC activities. The presence of membrane-bound bicarbonate-sensitive AC in fluid-transporting tissues suggests an autoregulatory mechanism for intracellular HCO3- concentration acting via adenosine 3',5'-cyclic monophosphate in the control of membrane ion transporters.

Partial amino acid sequence of calmodulin-independent bovine brain adenylate cyclase.

Mirzoeva SF, Dranitsyna SM, Chernova MN, Obukhov AN, Khramtsov NV, Lipkin VM, Ovchinnikov YuA
J Protein Chem. 1989; 8: 402-4

Yeast adenylyl cyclase assays.

Mintzer KA, Field J
Methods Enzymol. 1995; 255: 468-76

The stoichiometry of expression of protein components of the stimulatory adenylyl cyclase cascade and the regulation of information transfer.

Milligan G
Cell Signal. 1996; 8: 87-95

Quantitative analysis of the proteins which compromise the stimulatory arm of the adenylyl cyclase cascade indicate that the adenylyl cyclase catalytic component is usually the least highly expressed. The effects on both potency of agonist ligands and maximal output resulting from targetted alterations in expression levels of each element of this cascade are discussed.

Conformational basis for the activation of adenylate cyclase by adenosine.

Miles DL, Miles DW, Eyring H
Proc Natl Acad Sci U S A. 1977; 74: 2194-8

Molecular cloning of cDNAs and expression of mRNAs encoding alpha and beta subunits of soluble guanylyl cyclase from medaka fish Oryzias latipes.

Mikami T, Kusakabe T, Suzuki N
Eur J Biochem. 1998; 253: 42-8

Two cDNA clones encoding alpha and beta subunits of soluble guanylyl cyclase (GC) were isolated from medaka fish (Oryzias latipes) cDNA libraries and designated as OlGCS-alpha1 and OlGCS-beta1, respectively. The ORF of OlGCS-alpha1 encodes 678 amino acids and that of OlGCS-beta1 encodes 614 amino acids. Both subunits have a putative catalytic domain conserved with membrane GC and adenylyl cyclases. The amino acid sequences of OlGCS-beta1 and mammalian soluble GC subunits are highly conserved throughout the entire protein, suggesting that amino acid sequences outside the catalytic domain are important for the functioning of the beta subunit. On the contrary, amino acid sequences of alpha subunits outside the catalytic domain are fairly divergent between fish and mammals. Reverse transcription/PCR (RT/PCR) analysis showed that OlGCS-alpha1 and OlGCS-beta1 transcripts were abundant in the brain, eye, spleen and testis. RT/PCR analysis demonstrated that both transcripts were present in unfertilized eggs, were reduced immediately after fertilization, then increased again. In addition, our results suggest that expression patterns of OlGCS-alpha1 and OlGCS-beta1 genes are different from each other both during development and in adults.

Forskolin-stimulated platelet adenylyl cyclase activity is lower in persons with major depression.

Menninger JA, Tabakoff B
Biol Psychiatry. 1997; 42: 30-8

We investigated platelet adenylyl cyclase activity in 17 subjects with a history of major depression ("depressed subjects") and 20 controls. Forskolin was used to directly activate adenylyl cyclase, while guanine nucleotides (Gpp(NH)p) and fluoride ions were used to measure adenylyl cyclase activity modulated through the G proteins. Forskolin-stimulated adenylyl cyclase was significantly lower in the depressed subjects (p < 0.0005). There was a statistically significant difference in basal adenylyl cyclase activity between male depressed subjects and male controls. The basal adenylyl cyclase activity was also lower in female depressed subjects, but this difference did not reach statistical significance (p < 0.2). The adenylyl cyclase activity measured after stimulation with a guanine nucleotide or cesium fluoride did not differ between control and depressed male or female subjects. Severity of current depression and the current use of antidepressant medication were not related to the lower forskolin-stimulated enzyme activity in the depressed subjects. The difference in forskolin-stimulated adenylyl cyclase activity appears to reflect a qualitative difference in the adenylyl cyclase enzyme activity in persons with a history of major depression.

Fingerprinting of adenylyl cyclase activities during Dictyostelium development indicates a dominant role for adenylyl cyclase B in terminal differentiation.

Meima ME, Schaap P
Dev Biol. 1999; 212: 182-90

Activation of cAMP-dependent protein kinase (PKA) triggers terminal differentiation in Dictyostelium, without an obvious requirement for the G-protein-coupled adenylyl cyclase, ACA, or the osmosensory adenylyl cyclase, ACG. A third adenylyl cyclase, ACB, was recently detected in rapidly developing mutants. The specific characteristics of ACA, ACG, and ACB were used to determine their respective activities during development of wild-type cells. ACA was highly active during aggregation, with negligible activity in the slug stage. ACG activity was not present at significant levels until mature spores had formed. ACB activity increased strongly after slugs had formed with optimal activity at early fruiting body formation. The same high activity was observed in slugs of ACG null mutants and ACA null mutants that overexpress PKA (acaA/PKA), indicating that it was not due to either ACA or ACG. The detection of high adenylyl cyclase activity in acaA/PKA null mutants contradicts earlier conclusions (B. Wang and A. Kuspa, Science 277, 251-254, 1997) that these mutants can develop into fruiting bodies in the complete absence of cAMP. In contrast to slugs of null mutants for the intracellular cAMP-phosphodiesterase REGA, where both intact cells and lysates show ACB activity, wild-type slugs only show activity in lysates. This indicates that cAMP accumulation by ACB in living cells is controlled by REGA. Both REGA inhibition and PKA overexpression cause precocious terminal differentiation. The developmental regulation of ACB and its relationship to REGA suggest that ACB activates PKA and induces terminal differentiation.

Functional classification of cNMP-binding proteins and nucleotide cyclases with implications for novel regulatory pathways in Mycobacterium tuberculosis.

McCue LA, McDonough KA, Lawrence CE
Genome Res. 2000; 10: 204-19

We have analyzed the cyclic nucleotide (cNMP)-binding protein and nucleotide cyclase superfamilies using Bayesian computational methods of protein family identification and classification. In addition to the known cNMP-binding proteins (cNMP-dependent kinases, cNMP-gated channels, cAMP-guanine nucleotide exchange factors, and bacterial cAMP-dependent transcription factors), new functional groups of cNMP-binding proteins were identified, including putative ABC-transporter subunits, translocases, and esterases. Classification of the nucleotide cyclases revealed subtle differences in sequence conservation of the active site that distinguish the five classes of cyclases: the multicellular eukaryotic adenylyl cyclases, the eukaryotic receptor-type guanylyl cyclases, the eukaryotic soluble guanylyl cyclases, the unicellular eukaryotic and prokaryotic adenylyl cyclases, and the putative prokaryotic guanylyl cyclases. Phylogenetic distribution of the cNMP-binding proteins and cyclases was analyzed, with particular attention to the 22 complete archaeal and eubacterial genome sequences. Mycobacterium tuberculosis H37Rv and Synechocystis PCC6803 were each found to encode several more putative cNMP-binding proteins than other prokaryotes; many of these proteins are of unknown function. M. tuberculosis also encodes several more putative nucleotide cyclases than other prokaryotic species.

Inhibition of adenylyl cyclase by hormones: structural and functional correlates.

Mattera R, Toro MJ, Codina J, Birnbaumer L
Prog Clin Biol Res. 1987; 249: 43-64

Identification of novel guanylyl cyclases from chemosensory tissues of rat and cattle.

Matsuoka I, Mori T, Sato T, Sakai M, Kurihara K
Biochem Biophys Res Commun. 1995; 216: 242-8

A number of studies have shown that cGMP may play some roles in chemosensory transduction. To identify the structure of guanylyl cyclase in chemosensory tissues, cDNA fragments encoding guanylyl cyclase catalytic domain were amplified from rat and bovine olfactory and tongue epithelium using degenerate oligonucleotide primers and reverse transcription-polymerase chain reaction (RT-PCR). Three novel clones, two membrane type guanylyl cyclases (RAT GC-1, BOV GC-3) and one soluble type guanylyl cyclase (RAT GC-2) were identified. RAT GC-1 was distributed over various rat tissues in addition to these chemosensory organs. BOV GC-3 was similar to but distinct from recent cloned olfactory-specific guanylyl cyclase. RAT GC-2 was identified as rat homologue of alpha 2 subunit of the soluble guanylyl cyclase.

The role of Gs in activation of adenylate cyclase.

Martin BR, Farndale RW, Wong SK
Biochem Soc Trans. 1987; 15: 19-21

An adenylate cyclase from Saccharomyces cerevisiae that is stimulated by RAS proteins with effector mutations.

Marshall MS, Gibbs JB, Scolnick EM, Sigal IS
Mol Cell Biol. 1988; 8: 52-61

Conservative amino acid substitutions were introduced into the proposed effector regions of both mammalian Ha-ras (residues 32 to 40) and Saccharomyces cerevisiae RAS2 (residues 39 to 47) proteins. The RAS2[Ser 42] protein had reduced biological function in the yeast S. cerevisiae. A S. cerevisiae strain with a second-site suppressor mutation, SSR2-1, was isolated which could grow on nonfermentable carbon sources when the endogenous RAS2 protein was replaced by the RAS2[Ser 42] protein. The SSR2-1 mutation was mapped to the structural gene for adenylate cyclase (CYR1), and the gene containing SSR2-1 was cloned and sequenced. SSR2-1 corresponded to a point mutation that would create an amino acid substitution of a tyrosine residue for an aspartate residue at position 1547. The SSR2-1 gene encodes an adenylate cyclase that is dependent on ras proteins for activity, but is stimulated by Ha-ras and RAS2 mutant proteins that are unable to stimulate wild-type adenylate cyclase.

Sequence analysis of cDNA and genomic DNA, and mRNA expression of the medaka fish homolog of mammalian guanylyl cyclase C.

Mantoku T, Muramatsu R, Nakauchi M, Yamagami S, Kusakabe T, Suzuki N
J Biochem (Tokyo). 1999; 125: 476-86

We isolated the cDNA and genomic DNA encoding a membrane guanylyl cyclase of medaka fish (designated as OlGC6), and determined their complete nucleotide sequences. The open reading frame for OlGC6 cDNA predicted a protein of 1,075 amino acids. Phylogenetic analysis indicated that OlGC6 is a member of the enterotoxin/guanylin receptor family. We also determined the partial genomic structure of the gene of another membrane guanylyl cyclase of medaka fish, OlGC2, which is a member of the natriuretic peptide receptor family. The intron positions relative to the protein-coding sequence are highly conserved in the intracellular domains of OlGC6, OlGC2, mammalian GC-A, and GC-E. Despite their divergent primary structures, some intron positions also seem to be conserved in the extracellular domains of different membrane guanylyl cyclase genes. Northern blot analysis demonstrated that an OlGC6 transcript of 3.9 kb is only present in the intestine, while reverse transcription (RT)-PCR analysis demonstrated that the OlGC6 transcript is present in the kidney, spleen, liver, pancreas, gallbladder, ovary, testis, brain, and eye. RT-PCR also demonstrated that OlGC6 is only expressed zygotically and that transcripts are present from 1 day after fertilization, i.e. long before the intestinal tissues begin to develop.

Mice lacking the guanylyl cyclase C receptor are resistant to STa-induced intestinal secretion.

Mann EA, Jump ML, Wu J, Yee E, Giannella RA
Biochem Biophys Res Commun. 1997; 239: 463-6

Heat-stable enterotoxin (STa) is an important causative agent of diarrheal disease throughout the world. STa is known to bind specifically to receptors in the intestine, provoking intense intestinal secretion. Binding of STa, or of the mammalian endogenous ligands guanylin and uroguanylin, activates the guanylyl cyclase C receptor (GC-C); the resulting elevation of cGMP levels stimulates chloride secretion via CFTR. We have generated knockout mice which completely lack the GC-C receptor. These mice are viable and show no obvious alteration in intestinal fluidity. However, GC-C null mice are refractory to the secretory action of STa, proving that the GC-C receptor is necessary for the diarrheal response induced by STa.

[Effect of GM3 ganglioside on the adenyl and guanyl cyclase activity of cultured cells]

Mandel P, Yusufi A, Helwig JJ, Rebel G
C R Seances Soc Biol Fil. 1984; 178: 641-6

Addition of GM3 (10(-7) or 5.10(-5) M) did not affect the specific activity of adenyl and guanyl cyclase in confluent cultures of C6 glioma cells. Higher concentrations inhibit the cyclases.

Neuronal and glial cell cultures, a tool for investigation of ganglioside function.

Mandel P, Dreyfus H, Yusufi AN, Sarlieve L, Robert J, Neskovic N, Harth S, Rebel G
Adv Exp Med Biol. 1980; 125: 515-31

Guanylyl cyclases and signaling by cyclic GMP.

Lucas KA, Pitari GM, Kazerounian S, Ruiz-Stewart I, Park J, Schulz S, Chepenik KP, Waldman SA
Pharmacol Rev. 2000; 52: 375-414

Guanylyl cyclases are a family of enzymes that catalyze the conversion of GTP to cGMP. The family comprises both membrane-bound and soluble isoforms that are expressed in nearly all cell types. They are regulated by diverse extracellular agonists that include peptide hormones, bacterial toxins, and free radicals, as well as intracellular molecules, such as calcium and adenine nucleotides. Stimulation of guanylyl cyclases and the resultant accumulation of cGMP regulates complex signaling cascades through immediate downstream effectors, including cGMP-dependent protein kinases, cGMP-regulated phosphodiesterases, and cyclic nucleotide-gated ion channels. Guanylyl cyclases and cGMP-mediated signaling cascades play a central role in the regulation of diverse (patho)physiological processes, including vascular smooth muscle motility, intestinal fluid and electrolyte homeostasis, and retinal phototransduction. Topics addressed in this review include the structure and chromosomal localization of the genes for guanylyl cyclases, structure and function of the members of the guanylyl cyclase family, molecular mechanisms regulating enzymatic activity, and molecular sequences coupling ligand binding to catalytic activity. A brief overview is presented of the downstream events controlled by guanylyl cyclases, including the effectors that are regulated by cGMP and the role that guanylyl cyclases play in cell physiology and pathophysiology.

Sites for Galpha binding on the G protein beta subunit overlap with sites for regulation of phospholipase Cbeta and adenylyl cyclase.

Li Y, Sternweis PM, Charnecki S, Smith TF, Gilman AG, Neer EJ, Kozasa T
J Biol Chem. 1998; 273: 16265-72

Heterotrimeric G proteins, composed of alpha and betagamma subunits, forward signals from transmembrane receptors to intracellular effector enzymes and ion channels. Free betagamma activates downstream targets, but its action is terminated by association with GDP-liganded alpha subunits. Because alpha can inhibit activation of many effectors by betagamma, it is likely that the alpha subunit binding surfaces on betagamma overlap the surfaces necessary for effector activation. To test this hypothesis, we mutated residues on beta shown to contact alpha in the recently published crystal structures of the alphabetagamma heterotrimer (Wall, M. A., Coleman, D. E., Lee, E., Iniguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995) Cell 83, 1047-1058; Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319.). The alpha subunit binds to the flat, top surface of the toroidal beta subunit and also extends a helix along the side of the beta subunit at blade 1. We mutated four residues on the top surface of beta (Hbeta1[L117A], Hbeta1[D228R], Hbeta1[D246S], and Hbeta1[W332A]) and two residues on the side of beta that contacts alpha (Hbeta1[N88A/K89A]). Each of the mutant proteins was able to form beta gamma dimers, but they differed in their ability to bind alpha and to activate phospholipase C beta2 (PLCbeta2), PLCbeta3, and adenylyl cyclase II. Mutation of residues along the side of the torus at blade 1 diminish affinity for alpha but do not prevent activation of any of the effectors. Mutations on the alpha binding surface differentially affected PLCbeta2, PLCbeta3, and adenylyl cyclase II. Residues that affect PLCbeta and adenylyl cyclase II activity are found on opposite sides of the central tunnel, suggesting that PLC and adenylyl cyclase, like the alpha subunit, make many contacts on the top surface. None of the mutations affected the ability of betagamma to inhibit adenylyl cyclase I. We conclude that alpha, PLCbeta2, PLCbeta3, and adenylyl cyclase II share an interaction on the top surface of beta. The importance of individual residues is different for alpha binding and for effector activation and differs even between closely related isoforms of the same effector.

A guanylyl cyclase from Paramecium with 22 transmembrane spans. Expression of the catalytic domains and formation of chimeras with the catalytic domains of mammalian adenylyl cyclases.

Linder JU, Hoffmann T, Kurz U, Schultz JE
J Biol Chem. 2000; 275: 11235-40

Paramecium has a 280-kDa guanylyl cyclase. The N terminus resembles a P-type ATPase, and the C terminus is a guanylyl cyclase with the membrane topology of canonical mammalian adenylyl cyclases, yet with the cytosolic loops, C1 and C2, inverted compared with the mammalian order. We expressed in Escherichia coli the cytoplasmic domains of the protozoan guanylyl cyclase, independently and linked by a peptide, as soluble proteins. The His(6)-tagged proteins were enriched by affinity chromatography and analyzed by immunoblotting. Guanylyl cyclase activity was reconstituted upon mixing of the recombinant C1a- and C2-positioned domains and in a linked C1a-C2 construct. Adenylyl cyclase activity was minimal. The nucleotide substrate specificity was switched from GTP to ATP upon mutation of the substrate defining amino acids Glu(1681) and Ser(1748) in the C1-positioned domain to the adenylyl cyclase specific amino acids Lys and Asp. Using the C2 domains of mammalian adenylyl cyclases type II or IX and the C2-positioned domain from the Paramecium guanylyl cyclase we reconstituted a soluble, all C2 adenylyl cyclase. All enzymes containing protozoan domains were not affected by Galpha(s)/GTP or forskolin, and P site inhibitors were only slightly effective.

Guanylyl cyclases with the topology of mammalian adenylyl cyclases and an N-terminal P-type ATPase-like domain in Paramecium, Tetrahymena and Plasmodium.

Linder JU, Engel P, Reimer A, Kruger T, Plattner H, Schultz A, Schultz JE
EMBO J. 1999; 18: 4222-32

We cloned a guanylyl cyclase of 280 kDa from the ciliate Paramecium which has an N-terminus similar to that of a P-type ATPase and a C-terminus with a topology identical to mammalian adenylyl cyclases. Respective signature sequence motifs are conserved in both domains. The cytosolic catalytic C1a and C2a segments of the cyclase are inverted. Genes coding for topologically identical proteins with substantial sequence similarities have been cloned from Tetrahymena and were detected in sequences from Plasmodium deposited by the Malaria Genome Project. After 99 point mutations to convert the Paramecium TAA/TAG-Gln triplets to CAA/CAG, together with partial gene synthesis, the gene from Paramecium was heterologously expressed. In Sf9 cells, the holoenzyme is proteolytically processed into the two domains. Immunocytochemistry demonstrates expression of the protein in Paramecium and localizes it to cell surface membranes. The data provide a novel structural link between class III adenylyl and guanylyl cyclases and imply that the protozoan guanylyl cyclases evolved from an ancestral adenylyl cyclase independently of the mammalian guanylyl cyclase isoforms. Further, signal transmission in Ciliophora (Paramecium, Tetrahymena) and in the most important endoparasitic phylum Apicomplexa (Plasmodium) is, quite unexpectedly, closely related.

Myometrial adenylyl cyclase protein decreases on the last day of pregnancy in the rat.

Lindeman KS, Forrester DJ, Hirshman CA, Emala CW
Biol Reprod. 2000; 62: 1422-6

To determine whether gestation-related changes in responsiveness of the rat uterus to beta-adrenergic agonists are mediated at the level of adenylyl cyclase, we measured myometrial adenylyl cyclase activity and protein quantities during pregnancy and labor. In rat myometrial membranes, basal adenylyl cyclase activity increased from the nonpregnant state to mid (Days 12-14) and then late (Days 18-20) gestation and then decreased intrapartum (Day 22). Stimulated adenylyl cyclase activity, at the level of the beta-adrenergic receptor (isoproterenol, 10(-4) M), the G protein (GTP, 10(-5) M), or the adenylyl cyclase enzyme (MnCl(2), 20 mM), was similarly altered during gestation. Total adenylyl cyclase protein was quantified by [(3)H]forskolin binding assay in myometrial membranes from nonpregnant and pregnant (Day 14, Day 20, Day 21, and intrapartum Day 22) rats. Adenylyl cyclase protein increased progressively from nonpregnant rats to pregnant rats at mid (Day 14) and late (Day 20) gestation, but it decreased abruptly to nonpregnant levels on Day 21, the day before parturition, and remained at similar levels on Day 22 (intrapartum). The gestation-related increase in expression of myometrial adenylyl cyclase protein may facilitate uterine quiescence during pregnancy, and the abrupt decrease of adenylyl cyclase protein on the last day of pregnancy may be a contributing mechanism for the initiation of labor.

Identification of CRAC, a cytosolic regulator required for guanine nucleotide stimulation of adenylyl cyclase in Dictyostelium.

Lilly PJ, Devreotes PN
J Biol Chem. 1994; 269: 14123-9

As previously reported, guanine nucleotide regulation of adenylyl cyclase activity in the Dictyostelium mutant synag 7 can be restored in vitro by addition of a high-speed supernatant prepared from wild-type cells (Theibert, A., and Devreotes, P. N. (1986) J. Biol. Chem. 261, 15121-15125). We have designated this activity CRAC, for cytosolic regulator of adenylyl cyclase. Trypsinization of partially purified material demonstrated that this activity contains a protein. We report here a 50,000-fold purification of this protein using Q and S Sepharose fast flow and P11 cellulose chromatography, followed by sucrose gradient centrifugation and separation on SDS-polyacrylamide gel electrophoresis. Purification of wild-type and mutant supernatants in parallel allowed identification of an 88-kDa protein required for reconstituting activity. A polyclonal antibody was raised against this protein; it stains a band in unfractionated wild-type, but not mutant, supernatants. Immunoblots of fractions from each purification step show that activity and the immunostaining band cochromatograph. We have determined a short N-terminal sequence of the 88-kDa CRAC polypeptide, which matches a portion of the deduced N terminus of a gene, dagA, isolated from a mutant similar to synag 7. Expression of the dagA cDNA in synag 7 cells restores both the 88 kDa band and CRAC activity.

Identification of functional domains of adenylyl cyclase using in vivo chimeras.

Levin LR, Reed RR
J Biol Chem. 1995; 270: 7573-9

Adenylyl cyclase, the effector molecule of the cAMP signaling pathway, is composed of a family of isoforms that differ in their modes of regulation. Many of these modulatory interactions are dependent upon well characterized molecules from various second messenger pathways; however, very little is known about their mechanisms or sites of action on adenylyl cyclase. Chimeras were produced by a novel in vivo mechanism between two differentially modulated adenylyl cyclases to identify their regulatory domains. The basal activity of the type I adenylyl cyclase (AC1) is activated by calcium/calmodulin, inhibited by G protein beta gamma subunits, and insensitive to protein kinase C regulation. In contrast, type II adenylyl cyclase (AC2) is insensitive to calcium/calmodulin regulation and is activated by G protein beta gamma subunits as well as by activated protein kinase C. Expression and biochemical characterization of chimeras between AC1 and AC2 identified a single specific domain of AC1 responsible for calmodulin binding and a small, well defined region near the C terminus of AC2 required for protein kinase C activation.

The Drosophila learning and memory gene rutabaga encodes a Ca2+/Calmodulin-responsive adenylyl cyclase.

Levin LR, Han PL, Hwang PM, Feinstein PG, Davis RL, Reed RR
Cell. 1992; 68: 479-89

Four putative adenylyl cyclase genes from Drosophila melanogaster were identified by virtue of their extensive sequence homology with mammalian cyclases. One corresponds to the learning and memory gene rutabaga and is most similar to the mammalian brain Ca2+/calmodulin (CaM)-responsive cyclase. In a mammalian expression system, rutabaga cyclase activity was stimulated approximately 5-fold by the presence of Ca2+/CaM. A point mutation, identified at this locus in rut1 mutant flies, resulted in loss of detectable adenylyl cyclase activity. New P element insertion-induced rutabaga mutations mapped to within 200 nucleotides of the 5' end of the rutabaga cDNA. These data confirm the identity of the rutabaga locus as the structural gene for the Ca2+/CaM-responsive adenylyl cyclase and show that the inactivation of this cyclase leads to a learning and memory defect.

Hypothyroidism alters the effect of GTP on adenylyl cyclase in forebrain and hindbrain synaptosomal membranes from 15-day-old rats.

Leung FC, Saggerson ED, Clark JB
Int J Dev Neurosci. 1996; 14: 701-5

The effect of GTP concentration of forskolin-stimulated adenylyl cyclase activity was examined in synaptosomal membranes from 15-day-old rats that were hypothyroid owing to administration of propylthiouracil and a low-iodine diet to the mothers during pregnancy and suckling. In membranes from the forebrain hypothyroidism abolished the overall stimulatory effect of GTP, which was seen in the euthyroid case. In membranes from the hindbrain hypothyroidism had the opposite effect in that there was an enhancement of an overall stimulatory effect of GTP. It is suggested that these findings reflect changes during early development of the brain in the expression of various G-proteins and/or the expression of different isoforms of adenylyl cyclase.

A novel Ras-interacting protein required for chemotaxis and cyclic adenosine monophosphate signal relay in Dictyostelium.

Lee S, Parent CA, Insall R, Firtel RA
Mol Biol Cell. 1999; 10: 2829-45

We have identified a novel Ras-interacting protein from Dictyostelium, RIP3, whose function is required for both chemotaxis and the synthesis and relay of the cyclic AMP (cAMP) chemoattractant signal. rip3 null cells are unable to aggregate and lack receptor activation of adenylyl cyclase but are able, in response to cAMP, to induce aggregation-stage, postaggregative, and cell-type-specific gene expression in suspension culture. In addition, rip3 null cells are unable to properly polarize in a cAMP gradient and chemotaxis is highly impaired. We demonstrate that cAMP stimulation of guanylyl cyclase, which is required for chemotaxis, is reduced approximately 60% in rip3 null cells. This reduced activation of guanylyl cyclase may account, in part, for the defect in chemotaxis. When cells are pulsed with cAMP for 5 h to mimic the endogenous cAMP oscillations that occur in wild-type strains, the cells will form aggregates, most of which, however, arrest at the mound stage. Unlike the response seen in wild-type strains, the rip3 null cell aggregates that form under these experimental conditions are very small, which is probably due to the rip3 null cell chemotaxis defect. Many of the phenotypes of the rip3 null cell, including the inability to activate adenylyl cyclase in response to cAMP and defects in chemotaxis, are very similar to those of strains carrying a disruption of the gene encoding the putative Ras exchange factor AleA. We demonstrate that aleA null cells also exhibit a defect in cAMP-mediated activation of guanylyl cyclase similar to that of rip3 null cells. A double-knockout mutant (rip3/aleA null cells) exhibits a further reduction in receptor activation of guanylyl cyclase, and these cells display almost no cell polarization or movement in cAMP gradients. As RIP3 preferentially interacts with an activated form of the Dictyostelium Ras protein RasG, which itself is important for cell movement, we propose that RIP3 and AleA are components of a Ras-regulated pathway involved in integrating chemotaxis and signal relay pathways that are essential for aggregation.

Forskolin: a specific stimulator of adenylyl cyclase or a diterpene with multiple sites of action?

Laurenza A, Sutkowski EM, Seamon KB
Trends Pharmacol Sci. 1989; 10: 442-7

Forskolin, a naturally occurring diterpene, directly stimulates adenylyl cyclase and has been used extensively to increase cAMP and to elicit cAMP-dependent physiological responses. More recently, forskolin has been shown to inhibit a number of membrane transport proteins and channel proteins through a mechanism that does not involve the production of cAMP. Many of these channel proteins are predicted to have similar topographies in the membrane bilayer and it is tempting to speculate that forskolin may be binding at structurally homologous sites. Kenneth Seamon and colleagues discuss the cAMP-independent effects of forskolin and the structural similarity between forskolin and other physiologically important substances such as hexoses and steroids with respect to potential forskolin binding sites.

The kinase homology domain of retinal guanylyl cyclases 1 and 2 specifies the affinity and cooperativity of interaction with guanylyl cyclase activating protein-2.

Laura RP, Hurley JB
Biochemistry. 1998; 37: 11264-71

RetGC-1 and RetGC-2 are photoreceptor membrane guanylyl cyclases that are regulated by the Ca2+-binding protein, GCAP-2. We used a protease protection assay to localize regions of the intracellular domains of RetGCs important for the interaction with GCAP-2 and found that GCAP-2 reduces the access of trypsin to a site in the kinase homology domain (KHD) of RetGC-1. The protective effect of GCAP-2 is independent of Ca2+. We also found that RetGC-2 and GCAP-2 interact cooperatively with high affinity, but RetGC-1 and GCAP-2 interact noncooperatively with low affinity. By analyzing RetGC-1/RetGC-2 chimeras we demonstrated that the affinity and cooperativity of the interaction with GCAP-2 is dictated by the structure of the KHD. These findings suggest that GCAP-2 interacts constituitively with the KHDs of RetGC-1 and RetGC-2 and that cGMP synthesis is controlled by Ca2+-dependent conformational changes in the RetGC/GCAP complex.

The membrane guanylyl cyclase, retinal guanylyl cyclase-1, is activated through its intracellular domain.

Laura RP, Dizhoor AM, Hurley JB
J Biol Chem. 1996; 271: 11646-51

Retinal guanylyl cyclase-1 (RetGC-1) is a membrane guanylyl cyclase found in photoreceptor outer segments. It consists of an apparent extracellular domain (ECD) linked by a single transmembrane segment to an intracellular domain (ICD). Guanylyl cyclase activating protein-2 (GCAP-2) is a Ca2+-binding protein that activates RetGC-1 in a Ca2+-sensitive manner. To establish whether GCAP-2 stimulates RetGC-1 through the ECD or ICD, we made deletion mutants lacking either the ECD or both the ECD and transmembrane domains (TMD) of RetGC-1. Recombinant wild type RetGC-1 and both deletion mutants were expressed in HEK 293 cells, and their sensitivities to GCAP-2, Ca2+, and ATP were compared. Our data demonstrate that both deletion mutants are regulated similarly to wild type RetGC-1 with indistinguishable EC50 values for Ca2+ and similar K1/2 values for activation by GCAP-2. This shows that GCAP-2 functions through the ICD of RetGC-1 and that removal of the ECD and TMD do not significantly alter regulation by these factors. Our data also show that ATP potentiates stimulation of guanylyl cyclase activity by GCAP-2 and that neither the ECD nor the TMD of RetGC-1 participate in its regulation by ATP.

Regions in vertebrate photoreceptor guanylyl cyclase ROS-GC1 involved in Ca(2+)-dependent regulation by guanylyl cyclase-activating protein GCAP-1.

Lange C, Duda T, Beyermann M, Sharma RK, Koch KW
FEBS Lett. 1999; 460: 27-31

The membrane bound guanylyl cyclase (GC) photoreceptor membrane GC1 (ROS-GCI) of photoreceptor cells synthesizes cGMP, the intracellular transmitter of vertebrate phototransduction. The activity of ROS-GCI is controlled by small Ca(2+)-binding proteins, named GC-activating proteins (GCAPs). We identified and characterized two short regulatory regions (M445-L456 and L503-1522) in the juxtamembrane domain (JMD) of ROS-GC1 by peptide competition and mutagenesis studies. Both regions are critical for the activation of ROS-GCI by GCAP-1.

The fission yeast git5 gene encodes a Gbeta subunit required for glucose-triggered adenylate cyclase activation.

Landry S, Pettit MT, Apolinario E, Hoffman CS
Genetics. 2000; 154: 1463-71

Fission yeast adenylate cyclase is activated by the gpa2 Galpha subunit of a heterotrimeric guanine-nucleotide binding protein (G protein). We show that the git5 gene, also required for this activation, encodes a Gbeta subunit. In contrast to another study, we show that git5 is not a negative regulator of the gpa1 Galpha involved in the pheromone response pathway. While 43% identical to mammalian Gbeta's, the git5 protein lacks the amino-terminal coiled-coil found in other Gbeta subunits, yet the gene possesses some of the coding capacity for this structure 5' to its ORF. Although both gpa2 (Galpha) and git5 (Gbeta) are required for adenylate cyclase activation, only gpa2 is needed to maintain basal cAMP levels. Strains bearing a git5 disruption are derepressed for fbp1 transcription and sexual development even while growing in a glucose-rich environment, although fbp1 derepression is half that observed in gpa2 deletion strains. Multicopy gpa2 partially suppresses the loss of git5, while the converse is not true. These data suggest that Gbeta is required for activation of adenylate cyclase either by promoting the activation of Galpha or by independently activating adenylate cyclase subsequent to Galpha stimulation as seen in type II mammalian adenylate cyclase activation.

The N terminus domain of type VI adenylyl cyclase mediates its inhibition by protein kinase C.

Lai HL, Lin TH, Kao YY, Lin WJ, Hwang MJ, Chern Y
Mol Pharmacol. 1999; 56: 644-50

Previous results from our laboratory have shown that phosphorylation of type VI adenylyl cyclase (ACVI) by protein kinase C (PKC) caused suppression of adenylyl cyclase activity. In the present study, we investigated the role of the N terminus cytosolic domain of ACVI in this PKC-mediated inhibition of ACVI. Removal of amino acids 1 to 86 of ACVI or mutation of Ser(10) (a potential PKC phosphorylation site) into alanine significantly relieved the PKC-mediated inhibition and markedly reduced the PKC-evoked protein phosphorylation. PKC also effectively phosphorylated a recombinant N terminus cytosolic domain (amino acids 1-160) protein of ACVI and a synthetic peptide representing Ser(10). In addition, the amino acids 1 to 86 truncated mutant exhibited kinetic properties similar to those of the wild type. Taken together, these data demonstrate that the highly variable N terminus cytoplasmic domain of ACVI is a regulatory domain with a critical role in PKC-mediated suppression, which is a hallmark of this adenylyl cyclase isozyme. In addition, Ser(10) was found to serve as an acceptor for the PKC-mediated phosphorylating transfer of ACVI.

Regulation of guanylyl cyclase by a cGMP-binding protein during chemotaxis in Dictyostelium discoideum.

Kuwayama H, Van Haastert PJ
J Biol Chem. 1996; 271: 23718-24

Chemoattractants transiently activate guanylyl cyclase in Dictyostelium discoideum cells. Mutant analysis demonstrates that the produced cGMP plays an essential role in chemotactic signal transduction, controlling the actomyosin-dependent motive force. Guanylyl cyclase activity is associated with the particulate fraction of a cell homogenate. The addition of the cytosol stimulates guanylyl cyclase activity, whereas the cytosol plus ATP/Mg2+ inhibits enzyme activity. We have analyzed the regulation of guanylyl cyclase in chemotactic mutants and present evidence that a cGMP-binding protein mediates both stimulation and ATP-dependent inhibition of guanylyl cyclase. Upon chromatography of cytosolic proteins, cGMP binding activity co-elutes with both guanylyl cyclase-stimulating and ATP-dependent-inhibiting activities. In addition, ATP-dependent inhibition of guanylyl cyclase activity is enhanced by the cGMP analogue 8-Br-cGMP, suggesting that a cGMP-binding protein regulates guanylyl cyclase activity. Mutant KI-4 has an aberrant cGMP binding activity with very low Kd and shows a very small chemoattractant-mediated cGMP response; the cytosol from this mutant does not stimulate guanylyl cyclase. In contrast to KI-4, the aberrant cGMP binding activity of mutant KI-7 has a very high Kd and chemoattractants induce a prolonged cGMP response. The cytosol of this mutant stimulates guanylyl cyclase activity, but ATP does not inhibit the enzyme. Thus, two previously isolated chemotactic mutants are defective in the activation and inhibition of guanylyl cyclase, respectively. The positive and negative regulation of guanylyl cyclase by its product cGMP may well explain how cells process the temporospatial information of chemotactic signals, which is necessary for sensing the direction of the chemoattractant.

Protection against osmotic stress by cGMP-mediated myosin phosphorylation.

Kuwayama H, Ecke M, Gerisch G, Van Haastert PJ
Science. 1996; 271: 207-9

Conventional myosin functions universally as a generator of motive force in eukaryotic cells. Analysis of mutants of the microorganism Dictyostelium discoideum revealed that myosin also provides resistance against high external osmolarities. An osmo-induced increase of intracellular guanosine 3',5'-monophosphate was shown to mediate phosphorylation of three threonine residues on the myosin tail, which caused a relocalization of myosin required to resist osmotic stress. This redistribution of myosin allowed cells to adopt a spherical shape and may provide physical strength to withstand extensive cell shrinkage in high osmolarities.

Expression pattern of adenylyl cyclase isoforms in the inner ear of the rat by RT-PCR and immunochemical localization of calcineurin in the organ of Corti.

Kumagami H, Beitz E, Wild K, Zenner HP, Ruppersberg JP, Schultz JE
Hear Res. 1999; 132: 69-75

Most studies concerning adenylyl cyclases in the inner ear were carried out before the advent of molecular biology. In a PCR approach using cDNAs of six inner ear tissues (stria vascularis, endolymphatic sac, organ of Corti, vestibulum, cochlear and vestibular nerve) we found tissue specific expression of adenylyl cyclase isoforms. Adenylyl cyclases types 2 and 4 are predominant in the fluid controlling tissues, i.e. in the stria vascularis and endolymphatic sac. In the organ of Corti and vestibulum the Ca2+-modulated isoforms types 1, 6 and 9 were expressed. The regulation of adenylyl cyclase 9, which is the major isoform expressed in the organ of Corti, proceeds via the Ca2+-activated protein phosphatase 2B (calcineurin, PPP3). PCR with specific primers for calcineurin demonstrated its abundant expression in the organ of Corti. Using a monoclonal antibody we localized calcineurin immunochemically to the cochlear nerve, the nerve fibers and the inner hair cells. In the cochlear and vestibular nerves a characteristic neuronal expression pattern of adenylyl cyclase isoforms was observed, i.e. adenylyl cyclases types 2, 3 and 8. The functional consequences of the adenylyl cyclase expression pattern in the inner ear are discussed in conjunction with its unique sensory performance.

Inhibition of adenylyl and guanylyl cyclase isoforms by the antiviral drug foscarnet.

Kudlacek O, Mitterauer T, Nanoff C, Hohenegger M, Tang WJ, Freissmuth M, Kleuss C
J Biol Chem. 2001; 276: 3010-6

The pyrophosphate (PP(i)) analog foscarnet inhibits viral DNA-polymerases and is used to treat cytomegalovirus and human immunodeficiency vius infections. Nucleotide cyclases and DNA-polymerases catalyze analogous reactions, i.e. a phosphodiester bond formation, and have similar topologies in their active sites. Inhibition by foscarnet of adenylyl cyclase isoforms was therefore tested with (i) purified catalytic domains C1 and C2 of types I and VII (IC1 and VIIC1) and of type II (IIC2) and (ii) membrane-bound holoenzymes (from mammalian tissues and types I, II, and V heterologously expressed in Sf9 cell membranes). Foscarnet was more potent than PP(i) in suppressing forskolin-stimulated catalysis by both, IC1/IIC2 and VIIC1/IIC2. Stimulation of VIIC1/IIC2 by Galpha(s) relieved the inhibition by foscarnet but not that by PP(i). The IC(50) of foscarnet on membrane-bound adenylyl cyclases also depended on their mode of regulation. These findings predict that receptor-dependent cAMP formation is sensitive to inhibition by foscarnet in some, but not all, cells. This was verified with two cell lines; foscarnet blocked cAMP accumulation after A(2A)-adenosine receptor stimulation in PC12 but not in HEK-A(2A) cells. Foscarnet also inhibited soluble and, to a lesser extent, particulate guanylyl cylase. Thus, foscarnet interferes with the generation of cyclic nucleotides, an effect which may give rise to clinical side effects. The extent of inhibition varies with the enzyme isoform and with the regulatory input.

Mapping sites in guanylyl cyclase activating protein-1 required for regulation of photoreceptor membrane guanylyl cyclases.

Krylov DM, Niemi GA, Dizhoor AM, Hurley JB
J Biol Chem. 1999; 274: 10833-9

Guanylyl cyclase activating protein (GCAP)-1 regulates photoreceptor membrane guanylyl cyclase, RetGC, in a Ca2+-sensitive manner. It contains four Ca2+-binding motifs, EF-hands, three of which are capable of binding Ca2+. GCAP-1 activates RetGC in low Ca2+ and inhibits it in high Ca2+. In this study we used deletion and substitution analysis to identify regions of GCAP-1 sequence that are specifically required for inhibition and activation. A COOH-terminal sequence within Met157 to Arg182 is required for activation but not for inhibition of RetGC. We localized one essential stretch to 5 residues from Arg178 to Arg182. Another sequence essential for activation is within the N-terminal residues Trp21 to Thr27. The region between EF-hands 1 and 3 of GCAP-1 also contains elements needed for activation of RetGC. Finally, we found that inhibition of RetGC requires the first 9 amino-terminal residues of GCAP-1, but none of the residues from Gln33 to the COOH-terminal Gly205 are specifically required for inhibition. The ability of GCAP-1 mutants to regulate RetGC was tested on total guanylyl cyclase activity present in rod outer segments. In addition, the key mutants were also shown to produce similar effects on recombinant bovine outer segment cyclases GC1 and GC2.

Adenylyl cyclase amino acid sequence: possible channel- or transporter-like structure.

Krupinski J, Coussen F, Bakalyar HA, Tang WJ, Feinstein PG, Orth K, Slaughter C, Reed RR, Gilman AG
Science. 1989; 244: 1558-64

Complementary DNA's that encode an adenylyl cyclase were isolated from a bovine brain library. Most of the deduced amino acid sequence of 1134 residues is divisible into two alternating sets of hydrophobic and hydrophilic domains. Each of the two large hydrophobic domains appears to contain six transmembrane spans. Each of the two large hydrophilic domains contains a sequence that is homologous to a single cytoplasmic domain of several guanylyl cyclases; these sequences may represent nucleotide binding sites. An unexpected topographical resemblance between adenylyl cyclase and various plasma membrane channels and transporters was observed. This structural complexity suggests possible, unappreciated functions for this important enzyme.

Molecular diversity of the adenylyl cyclases.

Krupinski J, Cali JJ
Adv Second Messenger Phosphoprotein Res. 1998; 32: 53-79

The adenylyl cyclase family.

Krupinski J
Mol Cell Biochem. 1991; 104: 73-9

Hormone-sensitive adenylyl cyclase is a model system for the study of receptor-mediated signal transduction. It is comprised of three types of components: 1) receptors for hormones that regulate cyclic AMP (cAMP) synthesis, 2) regulatory GTP binding proteins (G proteins), and 3) the family of enzymes, the adenylyl cyclases. Concentrations of cAMP are altered by at least 35 different stimulatory or inhibitory hormones and neurotransmitters. Other signalling pathways may also influence cAMP production through regulation of particular adenylyl cyclase subtypes. The second messenger, cAMP propagates the hormone signal through the effects of cAMP-dependent protein kinase. While structural information on the adenylyl cyclases is limited, a cDNA clone for a calmodulin-sensitive form of bovine brain adenylyl cyclase has been isolated. The amino acid sequence encoded by the Type I cDNA is approximately 40% identical to those specified by three other adenylyl cyclase cDNAs that have been cloned subsequently. This degree of structural variation implies that there must be functional differences between the adenylyl cyclases.

The guanylin and uroguanylin peptide hormones and their receptors.

Krause WJ, London RM, Freeman RH, Forte LR
Acta Anat (Basel). 1997; 160: 213-31

Guanylin and uroguanylin are newly discovered, related peptides that activate common guanylyl cyclase signaling molecules and via 3', 5'-guanosine cyclic monophosphate regulate the activity of a variety of tissues and organs. Additionally, the message for both peptides is expressed in a variety of tissues and organs, including the intestinal tract and kidney, and thus may serve as part of a functional endocrine axis linking these two major organ systems in fluid/volume homeostasis. This manuscript reviews the discovery and nature of the guanylin and uroguanylin peptides, their actions on the intestinal mucosa and kidney, the distribution and molecular biology of the guanylyl cyclase C receptor, and explores the future directions of this rapidly developing, expanding field of inquiry.

Guanylyl cyclase receptors and guanylin-like peptides in reptilian intestine.

Krause WJ, Freeman RH, Eber SL, Hamra FK, Currie MG, Forte LR
Gen Comp Endocrinol. 1997; 107: 229-39

Receptors for guanylin and uroguanylin were identified on the mucosal surface of enterocytes lining the intestine of the bobtail skink (Tiliqua rugosa), king's skink (Egernia kingii), and knight anole (Anolis equestris) by receptor autoradiography using 125I-ST (Escherichia coli heat-stable enterotoxin) as the radioligand. Specific, high-affinity binding of 125I-ST to receptors was found on the microvillus border of enterocytes and little or no specific binding of 125I-ST was observed in other strata comprising the gut wall. The American alligator (Alligator mississippensis) also exhibited receptor binding, but unlike the other three species had relatively high levels of apparent nonspecific binding. A comparison of intestinal cGMP accumulation responses between the American alligator and the knight anole demonstrated a greater magnitude of cGMP responses to ST and guanylin in vitro in the knight anole relative to the tissue cGMP accumulation responses of alligators. Treatment with ST resulted in markedly greater tissue cGMP accumulation responses in both species compared to treatment with guanylin. To complete a paracrine signaling pathway in reptilian intestine, guanylin-like peptides that stimulated cGMP accumulation in human T84 intestinal cells were isolated from the intestinal mucosa of alligators. We conclude that functional receptor-guanylyl cyclases and one or more endogenous guanylin/uroguanylin-like peptides occur in the intestinal tract of reptiles as well as in the intestines of mammals and birds. Thus, higher vertebrates have a conserved signaling pathway that regulates intestinal function through the first-messenger peptides, guanylin and/or uroguanylin, and the intracellular second messenger, cGMP.

G protein hyperactivation of the Caenorhabditis elegans adenylyl cyclase SGS-1 induces neuronal degeneration.

Korswagen HC, van der Linden AM, Plasterk RH
EMBO J. 1998; 17: 5059-65

Expression of a constitutively activated version of the heterotrimeric G protein alpha-subunit Galphas results in the swelling and vacuolization of a specific subset of ventral nerve cord motoneurons of Caenorhabditis elegans. A second site modifier (sgs-1) that completely suppresses this neuronal degeneration has been isolated. sgs-1 was cloned and was shown to encode an adenylyl cyclase which is most similar to mammalian adenylyl cyclase type IX. Mutations in sgs-1 change residues that are conserved among different adenylyl cyclases. These mutations are located in the two catalytic domains and in the first multiple transmembrane spanning region of the predicted protein. An sgs-1 reporter construct shows a general neuronal expression pattern, demonstrating that sgs-1 is expressed in the neurons that are susceptible to activated Galphas-induced cell death. A second C.elegans adenylyl cyclase gene (acy-2) was analyzed as well. In contrast to sgs-1, acy-2 shows a restricted expression pattern and loss of acy-2 function results in early larval lethality. These results suggest that SGS-1 is a target of Galphas signaling in motoneurons, whereas an interaction of Galphas with ACY-2, probably in the canal-associated neurons, is required for viability.

A new type soluble guanylyl cyclase, which contains a kinase-like domain: its structure and expression.

Kojima M, Hisaki K, Matsuo H, Kangawa K
Biochem Biophys Res Commun. 1995; 217: 993-1000

A new type soluble guanylyl cyclase, designated as ksGC (kinase-like domain containing soluble guanylyl cyclase), was identified from a kidney cDNA library. By Northern blot analysis, expression of this novel guanylyl cyclase mRNA was found in lung, kidney and skeletal muscle. Although ksGC is a soluble type guanylyl cyclase, it is not a member of soluble alpha or beta guanylyl cyclases. The ksGC contains in its N-terminal region a protein kinase-like domain, which is usually a characteristic feature only of particulate type guanylyl cyclases. This unique feature of ksGC suggests a novel pathway of cGMP synthesis.

Cloning and functional expression of the rat alpha(2) subunit of soluble guanylyl cyclase.

Koglin M, Behrends S
Biochim Biophys Acta. 2000; 1494: 286-9

Nitric oxide-sensitive guanylyl cyclase is a heterodimeric enzyme consisting of one alpha and one beta subunit. Here, we clone the first alpha(2) subunit ortholog and functionally express the cDNA in Sf-9 cells. Our data indicate a high degree of conservation of the primary sequence and functional activity of the rat alpha(2) subunit.

Sequence homologies between guanylyl cyclases and structural analogies to other signal-transducing proteins.

Koesling D, Schultz G, Bohme E
FEBS Lett. 1991; 280: 301-6

The cyclic GMP-forming enzyme guanylyl cyclase exists in cytosolic and in membrane-bound forms differing in structure and regulations. Determination of the primary structures of the guanylyl cyclases revealed that the cytosolic enzyme form consists of two similar subunits and that membrane-bound guanylyl cyclases represent enzyme forms in which the catalytic part is located in an intracellular, C-terminal domain and is regulated by an extracellular, N-terminal receptor domain. A domain of 250 amino acids conserved in all guanylyl cyclases appears to be required for the formation of cyclic nucleotide, as this homologous domain is also found in the cytosolic regions of the adenylyl cyclase. The general structures of guanylyl cyclases shows similarities with other signal transducing enzymes such as protein-tyrosine phosphatases and protein-tyrosine kinases, which also exist in cytosolic and receptor-linked forms.

The primary structure of the 70 kDa subunit of bovine soluble guanylate cyclase.

Koesling D, Herz J, Gausepohl H, Niroomand F, Hinsch KD, Mulsch A, Bohme E, Schultz G, Frank R
FEBS Lett. 1988; 239: 29-34

The primary structure of the 70 kDa subunit of soluble bovine guanylate cyclase, which catalyzes the formation of cyclic GMP from GTP, has been determined. The alignment of six different clones out of two bovine libraries yielded a total of 3.1 kb with a coding region of 1857 bases. The open reading frame encodes a protein of 619 amino acids and a molecular mass of 70.5 kDa. Antibodies raised against a synthetic peptide, which corresponded to the C-terminus of the deduced sequence precipitated guanylate cyclase activity from guanylate cyclase-enriched preparations.

The primary structure of the larger subunit of soluble guanylyl cyclase from bovine lung. Homology between the two subunits of the enzyme.

Koesling D, Harteneck C, Humbert P, Bosserhoff A, Frank R, Schultz G, Bohme E
FEBS Lett. 1990; 266: 128-32

The primary structure of the larger subunit of the soluble guanylyl cyclase from bovine lung, which catalyzes the formation of cyclic GMP from GTP, has been determined. Two clones, isolated from two bovine libraries yielded a total of 3261 bp with a coding region of 2073 bp. The open reading frame encodes a protein of 691 amino acids and a molecular mass of 77,500. The deduced amino acid sequence reveals regions which are, to a large extent, homologous to the sequence of the smaller subunit of the enzyme as well as to the sequences of other gyanylyl and adenylyl to a large extent, homologous to the sequence of the smaller subunit of the enzyme as well as to the sequences of other gyanylyl and adenylyl cyclases.

Soluble guanylyl cyclase: structure and regulation.

Koesling D, Friebe A
Rev Physiol Biochem Pharmacol. 1999; 135: 41-65

Guanylyl cyclases, a growing family of signal-transducing enzymes.

Koesling D, Bohme E, Schultz G
FASEB J. 1991; 5: 2785-91

Guanylyl cyclases, which catalyze the formation of the intracellular signal molecule cyclic GMP from GTP, display structural features similar to other signal-transducing enzymes such as protein tyrosine-kinases and protein tyrosine-phosphatases. So far, three isoforms of mammalian membrane-bound guanylyl cyclases (GC-A, GC-B, GC-C), which are stimulated by either natriuretic peptides (GC-A, GC-B) or by the enterotoxin of Escherichia coli (GC-C), have been identified. These proteins belong to the group of receptor-linked enzymes, with different NH2-terminal extracellular receptor domains coupled to a common intracellular catalytic domain. In contrast to the membrane-bound enzymes, the heme-containing soluble guanylyl cyclase is stimulated by NO and NO-containing compounds and consists of two subunits (alpha 1 and beta 1). Both subunits contain the putative catalytic domain, which is conserved in the membrane-bound guanylyl cyclases and is found twice in adenylyl cyclases. Coexpression of the alpha 1- and beta 1-subunit is required to yield a catalytically active enzyme. Recently, another subunit of soluble guanylyl cyclase was identified and designated beta 2, revealing heterogeneity among the subunits of soluble guanylyl cyclase. Thus, different enzyme subunits may be expressed in a tissue-specific manner, leading to the assembly of various heterodimeric enzyme forms. The implications concerning the physiological regulation of soluble guanylyl cyclase are not known, but different mechanisms of soluble enzyme activation may be due to heterogeneity among the subunits of soluble guanylyl cyclase.

Studying the structure and regulation of soluble guanylyl cyclase.

Koesling D
Methods. 1999; 19: 485-93

Soluble guanylyl cyclase acts as the receptor for the signaling molecule nitric oxide. The enzyme consists of two different subunits. Each subunit shows the cyclase catalytic domain, which is also conserved in the membrane-bound guanylyl cyclases and the adenylyl cyclases. The N-terminal regions of the subunits are responsible for binding of the prosthetic heme group of the enzyme, which is required for the stimulatory effect of nitric oxide (NO). The five-coordinated ferrous heme displays a histidine as the axial ligand; activation of soluble guanylyl cyclase by NO is initiated by binding of NO to the heme iron and proceeds via breaking of the histidine-to-iron bond. Recently, a novel pharmacological and possibly physiological principle of guanylyl cyclase sensitization was demonstrated. The substance YC-1 has been shown to activate the enzyme independent of NO, to potentiate the effect of submaximally effective NO concentrations, and to turn carbon monoxide into an effective activator of soluble guanylyl cyclase.

Modulators of soluble guanylyl cyclase.

Koesling D
Naunyn Schmiedebergs Arch Pharmacol. 1998; 358: 123-6

Particulate guanylyl cyclases: multiple mechanisms of activation.

Kobialka M, Gorczyca WA
Acta Biochim Pol. 2000; 47: 517-28

Cyclic GMP (cGMP), a key messenger in several signal transduction pathways, is synthesized from GTP by a family of enzymes termed guanylyl cyclases, which are found in two forms: cytosolic (soluble) and membrane-bound (particulate). The past decade has brought significant progress in understanding the molecular mechanisms that underlie the regulation of particulate guanylyl cyclases and new members of their family have been identified. It has become more evident that the basic mechanism of catalysis of guanylyl cyclases is analogous to that recognized in adenylyl cyclases. Here we review the known basic mechanisms that contribute to the regulation of particulate guanylyl cyclases.

The expression of a novel Ca2+/CaM stimulated adenylyl cyclase activity in the neuroblastoma cell line Lan-1 is regulated by cell density.

Kluxen FW, Hulster A, Droste M, Pfeuffer T
Brain Res. 1996; 740: 169-74

In the human neuroblastoma cell line Lan-1, the mRNA encoding the Ca2+/calmodulin (CaM) sensitive adenylyl cyclase type-1 (AC-1) was detected by reverse transcription-polymerase chain reaction (RT-PCR) as well as by Northern blotting. However, neither Ca2+/CaM stimulated AC activity was found nor could AC-1 type protein be detected by a specific antibody (anti-1Cl). In contrast, when cells were grown to high cell density, Ca2+/CaM stimulated AC-activity could be indeed found in membranes. The large increase in activity was paralleled by the appearance of a 110 kDa protein detected by the monoclonal AC antibody BBC-2. At the same time a 150 kDa adenylyl cyclase species present in growing cells was absent. The 110 kDa protein co-migrated with bovine AC-1 and was slightly larger than the human AC-1. Unexpectedly, however, the antibody anti-1CI was not able to precipitate the newly induced Lan-1 AC. In addition, no increase in type-1 AC mRNA could be detected either by PCR or by Northern blotting. Treatment of Lan-1 cells with 10 microM retinoic acid for 7 days caused growth arrest and morphological differentiation of the cells, yet the induction of the Ca2+/CaM-stimulated AC activity was much lower than in the dense grown control cultures. It is concluded that the Ca2+/CaM-activated AC of M(r) 110 kDa in Lan-1 cells is not related to the previously known Ca2+/CaM stimulated AC isoforms, and might thus represent a novel AC.

Gsalpha contains an unidentified covalent modification that increases its affinity for adenylyl cyclase.

Kleuss C, Gilman AG
Proc Natl Acad Sci U S A. 1997; 94: 6116-20

Many G protein alpha subunits are dually acylated with myristate and palmitate or are palmitoylated on more than one cysteine residue near their N termini. The Galpha protein that activates adenylyl cyclase, alphas, is not myristoylated but can be reversibly palmitoylated. It appears that alphas contains another, as-yet-unidentified covalent modification that decreases its apparent dissociation constant for adenylyl cyclase from 50 nM to <0. 5 nM. This modification is at or near the N terminus of the protein and is hydrophobic. Palmitoylation of native alphas does not account for its high affinity for adenylyl cyclase.

Physiological regulation of blood pressure and kidney function by guanylyl cyclase isoforms.

Kishimoto I, Garbers DL
Curr Opin Nephrol Hypertens. 1997; 6: 58-63

Guanylyl cyclase isoforms can serve as receptors for various molecules including atrial natriuretic peptide, brain natriuretic peptide, C-type natriuretic peptide and nitric oxide. It has been speculated these signaling pathways are important regulators of cardiovascular and renal physiology. Within the past year, gene targeting in particular has strengthened this speculation, suggesting critical roles of guanylyl cyclase receptors in both the chronic and acute regulation of blood pressure.

Characterization of adenylyl cyclase in goldfish brain.

Kirkham DM, Henley JM
Biochem Pharmacol. 1993; 46: 1559-63

The rate of production of cAMP by the adenylyl cyclase enzyme from goldfish brain was linear with time and with protein concentration. In agreement with mammalian adenylyl cyclase systems the enzyme is divalent cation dependent, being activated in the presence of either Mg2+ or Mn2+. Forskolin also stimulated the rate of reaction in a dose-dependent manner with a half-maximal effect of 1 microM. The activated enzyme was inhibited by high concentrations of Ca2+ but was independent of Na+ concentration. The presence of guanine nucleotide binding proteins (G-proteins) was demonstrated by the fact that both NaF and guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) stimulated the basal rate. In addition, the p[NH]ppG dose-response curve of the forskolin-stimulated enzyme was biphasic, similar to that observed for other systems. At low concentrations of p[NH]ppG a small inhibition was observed while higher concentrations produced a stimulation. These data suggest that the goldfish brain adenylyl cyclase enzyme complex includes both stimulatory and inhibitory G-proteins in addition to the catalytic unit. A series of known and putative goldfish neurotransmitter substances failed to either stimulate or inhibit the adenylyl cyclase activity. The endogenous neurotransmitters which interact with this second messenger system remain to be determined.

Identification and partial purification of an endogenous inhibitor of soluble guanylyl cyclase from bovine lung.

Kim TD, Burstyn JN
J Biol Chem. 1994; 269: 15540-5

An endogenous inhibitor of soluble guanylyl cyclase from bovine lung has been partially purified by use of anion exchange, hydrophobic interaction, and gel filtration chromatography. This inhibitor is a protein with a molecular weight of about 149,000 which was estimated from its elution behavior, versus that of a series of standards, on a Sephacryl S-300-HR column. Its activity was measured by comparison of the level of cGMP production from soluble guanylyl cyclase in the presence and absence of the inhibitor protein. Soluble guanylyl cyclase is inhibited by this protein when either Mg2+ or Mn2+ is used as a cofactor. The insensitivity of its inhibitory activity to both isobutylmethylxanthine and the presence of cGMP demonstrates that this new protein is not a phosphodiesterase. This new protein inhibits both activated and unactivated soluble guanylyl cyclase. Because the inhibition was found to be noncompetitive with respect to the substrate, GTP, it appears that this inhibitor may be an allosteric regulator of soluble guanylyl cyclase.

Phosphorylation of chemoattractant receptors is not essential for chemotaxis or termination of G-protein-mediated responses.

Kim JY, Soede RD, Schaap P, Valkema R, Borleis JA, Van Haastert PJ, Devreotes PN, Hereld D
J Biol Chem. 1997; 272: 27313-8

In several G-protein-coupled signaling systems, ligand-induced receptor phosphorylation by specific kinases is suggested to lead to desensitization via mechanisms including receptor/G-protein uncoupling, receptor internalization, and receptor down-regulation. We report here that elimination of phosphorylation of a chemoattractant receptor of Dictyostelium, either by site-directed substitution of the serines or by truncation of the C-terminal cytoplasmic domain, completely prevented agonist-induced loss of ligand binding but did not impair the adaptation of several receptor-mediated responses including the activation of adenylyl and guanylyl cyclases and actin polymerization. In addition, the phosphorylation-deficient receptors were capable of mediating chemotaxis, aggregation, and differentiation. We propose that for chemoattractant receptors agonist-induced phosphorylation regulates surface binding activity but other phosphorylation-independent mechanisms mediate response adaptation.

Genetic and biochemical analysis of the adenylyl cyclase of Schizosaccharomyces pombe.

Kawamukai M, Ferguson K, Wigler M, Young D
Cell Regul. 1991; 2: 155-64

The adenylyl cyclase gene, cyr1, of Schizosaccharomyces pombe has been cloned. We have begun an analysis of the function and regulation of adenylyl cyclase by disrupting this gene and by over-expressing all or parts of this gene in various strains. cyr1- strains are viable and contain no measurable cyclic AMP. They conjugate and sporulate under conditions that normally inhibit wild-type strains. Strains containing the cyr1 coding sequences transcribed from the strong adh1 promoter contain greatly elevated adenylyl cyclase activity, as measured in vitro, but only modestly elevated cAMP levels. Such strains conjugate and sporulate less frequently than wild-type cells upon nutrient limitation. Strains which carry the wild-type cyr1 gene but that also express high levels of the amino terminal domain of adenylyl cyclase behave much like cyr1-strains, suggesting that the amino terminal domain can bind a positive regulator. A protein that copurifies with the adenylyl cyclase of S. pombe cross-reacts to antiserum raised against the S. cerevisiae adenylyl cyclase-associated regulatory protein, CAP.

Isolation of a cotton CAP gene: a homologue of adenylyl cyclase-associated protein highly expressed during fiber elongation.

Kawai M, Aotsuka S, Uchimiya H
Plant Cell Physiol. 1998; 39: 1380-3

The cDNA encoding CAP (adenylyl cyclase-associated protein) was isolated from a cotton (Gossypium hirsutum) fiber cDNA library. The cDNA (GhCAP) contained an open reading frame that encoded 471 amino acid residues. RNA blot analysis showed that the cotton CAP gene was expressed mainly in young fibers.

Soluble adenylyl cyclase from Spodoptera frugiperda (Sf9) cells. Purification and biochemical characterization.

Kawabe Ji, Toya Y, Schwencke C, Oka N, Ebina T, Ishikawa Y
J Biol Chem. 1996; 271: 20132-7

An insect ovarian cell, Spodoptera frugiperda (Sf9), has been widely used to express recombinant proteins, including adenylyl cyclase, as a host cell in the baculovirus expression system. We report the presence and characterization of a soluble adenylyl cyclase (sAC) distinct from a membrane-bound form of adenylyl cyclase (mAC) that is also present in Sf9 cells. sAC was purified 3,500-fold to near homogeneity; a single band at 25 kDa on SDS-polyacrylamide gel electrophoresis correlated well with adenylyl cyclase catalytic activity. The purified enzyme had a catalytic activity of 0.1 micromol/min.mg and the Km of 0.55 mM for the substrate ATP. In contrast to mAC, sAC was heat-stable. Enzymatic activity of sAC was not stimulated by forskolin and was inhibited by salts at high concentrations. sAC utilized both manganese- and magnesium-ATP as substrate. Di- or triphosphate-containing nucleotides, such as GTP and GDP, as well as pyrophosphate, noncompetitively inhibited sAC. Our data suggest that the physical and biochemical characteristics of sAC are different from those of mAC in Sf9 cells as well as from those of other known forms of adenylyl cyclase in animal cells; sAC in Sf9 cells may constitute a new member of adenylyl cyclase found in animals.

A novel peptide inhibitor of adenylyl cyclase (AC). A peptide from type V AC directly inhibits AC catalytic activity.

Kawabe J, Ebina T, Ismail S, Kitchen DB, Homcy CJ, Ishikawa Y
J Biol Chem. 1994; 269: 24906-11

Peptides derived from various regions of type V adenylyl cyclase (AC) were studied to determine their effect on AC catalytic activity. Out of 10 examined, only one peptide, peptide 2 (Lys425-Cys444), significantly inhibited both basal and stimulated (forskolin or GTP gamma S (guanosine 5'-O-thiotriphosphate)) type V AC catalytic activity overexpressed in CMT cells. The sequence of this peptide was taken from the first cytoplasmic domain of type V AC, which has a high sequence homology to other AC isoforms. Competition studies performed between the peptide and the substrate ATP showed that the inhibition was noncompetitive. Mutation or truncation of the peptide designed to destroy its secondary structure totally negated the inhibitory effect. This peptide also inhibited the catalytic activity of purified types II and V AC, as well as that of various cells, including S49 cyc- cells. Our data indicate that the peptide directly interacts with AC to inhibit catalytic activity; this provides new information regarding regions of the enzyme involved in its catalytic activation.

In vivo generation of an adenylylcyclase isoform with a half-molecule motif.

Katsushika S, Kawabe J, Homcy CJ, Ishikawa Y
J Biol Chem. 1993; 268: 2273-6

A truncated form of adenylylcyclase (type V-alpha) has been cloned from a cardiac cDNA library. It constitutes a half-molecule of type V adenylylcyclase diverging at the end of the first cytoplasmic loop. Northern blotting study has revealed the presence of such a mRNA species (approximately 3.5 kilobases in size) in the heart. Genomic sequence analysis has revealed that type V-alpha is generated via usage of a polyadenylation signal located within an intronic sequence of type V adenylylcyclase gene. When type V-alpha is co-expressed with an artificially generated half-molecule constituting the latter half of type V adenylylcyclase, the catalytic activity in transfected cell membranes is significantly higher than that of controls. However, when either alone is overexpressed, no significant increase in catalytic activity results. These results indicate that a half-molecule of adenylylcyclase, i.e. a protein containing six-transmembrane spans followed by a single cytoplasmic domain, can be generated in vivo, but catalytic activity is lacking unless heterodimerization can occur. This finding identifies another potential mechanism for generating diversity within this enzyme family.

Cloning and characterization of a sixth adenylyl cyclase isoform: types V and VI constitute a subgroup within the mammalian adenylyl cyclase family.

Katsushika S, Chen L, Kawabe J, Nilakantan R, Halnon NJ, Homcy CJ, Ishikawa Y
Proc Natl Acad Sci U S A. 1992; 89: 8774-8

A sixth member of the mammalian adenylyl cyclase family has been isolated from a canine cardiac cDNA library. This isoform is more highly homologous to type V than to the other adenylyl cyclase types; sequence similarity is apparent even in the transmembrane regions where the greatest divergence among the types exists. Type VI mRNA expression is most abundant in heart and brain; however, unlike type V, a low level of expression is also observed in a variety of other tissues examined. Type VI adenylyl cyclase can be stimulated by NaF, guanosine 5'-[gamma-thio]triphosphate, and forskolin but not by Ca2+/calmodulin, whereas it is inhibited by adenosine and its analogues. Comparison of both their structural and biochemical properties suggests that types V and VI constitute a distinct subgroup of the mammalian adenylyl cyclase family.

The Spirulina platensis adenylate cyclase gene, cyaC, encodes a novel signal transduction protein.

Kasahara M, Yashiro K, Sakamoto T, Ohmori M
Plant Cell Physiol. 1997; 38: 828-36

A cyaC gene encoding an adenylate cyclase of the filamentous cyanobacterium Spirulina platensis was sequenced. The predicted amino acid sequence of the C-terminal region of cyaC is similar to the catalytic domains of adenylate cyclases in other cyanobacteria and eukaryotes. The sequences of other regions are similar to those of proteins consisting of the bacterial two-component signal transduction system: the sensory kinase and the response regulator. The predicted gene product of cyaC contains, from the N-terminal end, a receiver domain of the response regulator protein (R1), a domain similar to the ETR1 of Arabidopsis thaliana, a transmitter domain of the sensory kinase protein, a receiver domain of the response regulator protein (R2), and a catalytic domain of adenylate cyclase. The cyaC gene was expressed as an affinity-tagged protein in Escherichia coli, and the recombinant protein was purified. The purified protein had adenylate cyclase activity which was activated by Mu2+. The results of Western blotting using an anti-CyaC antiserum and the S.platensis cell extract confirmed that cyaC gene is expressed in S. platensis.

CyaG, a novel cyanobacterial adenylyl cyclase and a possible ancestor of mammalian guanylyl cyclases.

Kasahara M, Unno T, Yashiro K, Ohmori M
J Biol Chem. 2001; 276: 10564-9

A novel gene encoding an adenylyl cyclase, designated cyaG, was identified in the filamentous cyanobacterium Spirulina platensis. The predicted amino acid sequence of the C-terminal region of cyaG was similar to the catalytic domains of Class III adenylyl and guanylyl cyclases. The N-terminal region next to the catalytic domain of CyaG was similar to the dimerization domain, which is highly conserved among guanylyl cyclases. As a whole, CyaG is more closely related to guanylyl cyclases than to adenylyl cyclases in its primary structure. The catalytic domain of CyaG was expressed in Escherichia coli and partially purified. CyaG showed adenylyl cyclase (but not guanylyl cyclase) activity. By site-directed mutagenesis of three amino acid residues (Lys(533), Ile(603), and Asp(605)) within the purine ring recognition site of CyaG to Glu, Arg, and Cys, respectively, CyaG was transformed to a guanylyl cyclase that produced cGMP instead of cAMP. Thus having properties of both cyclases, CyaG may therefore represent a critical position in the evolution of Class III adenylyl and guanylyl cyclases.

Activation of a cyanobacterial adenylate cyclase, CyaC, by autophosphorylation and a subsequent phosphotransfer reaction.

Kasahara M, Ohmori M
J Biol Chem. 1999; 274: 15167-72

The CyaC protein, a cyanobacterial adenylate cyclase, has a unique primary structure composed of the catalytic domain of adenylate cyclase and the conserved domains of bacterial two-component regulatory systems, one transmitter domain and two receiver domains. In the present work, CyaC was produced in Escherichia coli as a histidine-tagged recombinant protein and purified to homogeneity. CyaC showed ability to autophosphorylate in vitro with the gamma-phosphate of [gamma-32P]ATP. CyaC derivatives were constructed by site-directed mutagenesis in which the highly conserved phosphorylation sites in the transmitter domain (His572) and receiver domains (Asp60 or Asp895) were replaced by glutamine and alanine residues, respectively. After autophosphorylation of the CyaC derivatives, the chemical stabilities of the phosphoryl groups bound to the derivatives were determined. It was found that His572 is the initial phosphorylation site and that the phosphoryl group once bound to His572 is transferred to Asp895. The enzyme activities of the CyaC derivatives defective in His572 or Asp895 were considerably reduced. Asp895 is phosphorylated by acetyl [32P]phosphate, a small phosphoryl molecule, but Asp60 is not. Acetyl phosphate stimulates adenylate cyclase activity only when Asp895 is intact. These results suggest that the phosphorylation of Asp895 is essential for the activation of adenylate cyclase and that Asp60 functions differently from Asp895 in regulating the enzyme activity.

Neuropeptides, adenylyl cyclase, and memory storage.

Kandel E, Abel T
Science. 1995; 268: 825-6

Analysis of effects of corticotropin, forskolin and fluoride on activity of adenylate cyclase of bovine adrenal cortex.

Juska A, de Foresta B
Biochim Biophys Acta. 1995; 1236: 289-98

A mathematical model relating the activity of adenylate cyclase (AC) with concentrations of stimulators, equilibrium dissociation constants, specific activity and efficacies of AC depending on the states of its binding sites has been developed and used for analysis of the data on activation of AC of bovine adrenal cortex plasma membranes presented in (De Foresta et al. (1987) FEBS Lett. 216, 107-112). Equilibrium dissociation constants. chi h and chi l, corresponding to high- and low-affinity forskolin-binding sites were estimated to be 0.37 and 17 microM: these constants characterize forskolin's potency more adequately than does ED50, the concentration eliciting half-asymptotic activity of AC. Corticotropin does not affect the affinity of AC for forskolin whereas fluoride increases this affinity, thus augmenting forskolin's potency. Hormone receptor of adenylate cyclase of bovine adrenal cortex has been suggested to have two or more binding sites for corticotropin. Some unidentified factor(s) may be responsible for the differences found in adenylate cyclase activity in different experiments carried out under similar conditions. The model applied for the analysis may be thought to be the best means for the moment to relate dose-response dependencies with what is known or can be hypothesized about the mechanisms underlying activation of adenylate cyclase.

Analysis of effects of adrenaline and collagen on the activity of prostaglandin E1-stimulated human platelet adenylyl cyclase.

Juska A
Blood Coagul Fibrinolysis. 1996; 7: 199-201

A mathematical model relating the activity of adenylyl cyclase (AC) with concentrations of inhibitors, equilibrium dissociation constants, specific activity and efficacies of AC depending on the states of binding sites of the receptors was used for analysis of the data on inhibition of PGE1-stimulated AC of human platelet membranes (Farndale et al. Biochem J 1992; 282; 25-32). The equilibrium dissociation constant, x2 characterizing the affinity of adrenaline for its receptor, was estimated to be 0.14 microM; this constant is a better characteristic of adrenaline's potency than IC50, the concentration corresponding to half-asymptotic inhibition of AC. Collagen does not affect the affinity of adrenaline for its receptor. G protein involved in inhibition of AC activity by collagen is different from Gi2 mediating inhibition by alpha 2-adrenergic agonists. The model applied for the analysis may be thought to be the best means presently to relate dose-response dependencies to what is known or can be hypothesized about the mechanisms underlying inhibition of AC activity.

Enzymatic preparation of 32P-labeled beta-L-2',3',-dd-5'ATP and its use as a high-affinity, conformation-specific ligand for labeling adenylyl cyclases.

Johnson RA, Shoshani I, Dessauer C, Gosselin G
Nucleosides Nucleotides. 1999; 18: 839-42

An enzymatic method was developed for the preparation of unlabeled and [beta-32P]-labeled beta-L-2',3'-dd-5'ATP from the monophosphate with near quantitative yields. beta-L-2',3'-dd-5'ATP was a competitive and potent inhibitor of adenylyl cyclases (IC5 approximately 30 nM). Upon uv-irradiation beta-L-2',3'-dd-[beta-32P]-5'ATP directly crosslinked to a chimeric construct of this enzyme. Data suggest that this is a pre-transition state inhibitor and contrasts with the equipotent 2',5'-dd-3'ATP, a post-transition state, noncompetitive inhibitor.

Preparation and use of "P"-site-targeted affinity ligands for adenylyl cyclases.

Johnson RA, Shoshani I
Methods Enzymol. 1994; 238: 56-71

Selection of gbeta subunits with point mutations that fail to activate specific signaling pathways in vivo: dissecting cellular responses mediated by a heterotrimeric G protein in Dictyostelium discoideum.

Jin T, Amzel M, Devreotes PN, Wu L
Mol Biol Cell. 1998; 9: 2949-61

In Dictyostelium discoideum, a unique Gbeta subunit is required for a G protein-coupled receptor system that mediates a variety of cellular responses. Binding of cAMP to cAR1, the receptor linked to the G protein G2, triggers a cascade of responses, including activation of adenylyl cyclase, gene induction, actin polymerization, and chemotaxis. Null mutations of the cAR1, Galpha2, and Gbeta genes completely impair all these responses. To dissect specificity in Gbetagamma signaling to downstream effectors in living cells, we screened a randomly mutagenized library of Gbeta genes and isolated Gbeta alleles that lacked the capacity to activate some effectors but retained the ability to regulate others. These mutant Gbeta subunits were able to link cAR1 to G2, to support gene expression, and to mediate cAMP-induced actin polymerization, and some were able to mediate to chemotaxis toward cAMP. None was able to activate adenylyl cyclase, and some did not support chemotaxis. Thus, we separated in vivo functions of Gbetagamma by making point mutations on Gbeta. Using the structure of the heterotrimeric G protein displayed in the computer program CHAIN, we examined the positions and the molecular interactions of the amino acids substituted in each of the mutant Gbetas and analyzed the possible effects of each replacement. We identified several residues that are crucial for activation of the adenylyl cyclase. These residues formed an area that overlaps but is not identical to regions where bovine Gtbetagamma interacts with its regulators, Galpha and phosducin.

Adenylate cyclase--properties, activity and methodology.

Jaworska-Adamu J, Cybulska R
Folia Morphol (Warsz). 1991; 50: 1-12

Effects of the phospholipid environment in the plasma membrane on receptor interaction with the adenylyl cyclase complex of intact cells.

Jansson C, Harmala AS, Toivola DM, Slotte JP
Biochim Biophys Acta. 1993; 1145: 311-9

In this study we have examined the effects of variations of the plasma membrane phospholipid and cholesterol content on the metabolic functions of the adenylyl cyclase complex in intact cells. Exposure of cells to 0.1 U/ml of sphingomyelinase led to the degradation of 75, 55 and 40% of the cellular total sphingomyelin mass in human skin fibroblasts (HSF), Chinese hamster lung fibroblasts (CHLF) and rat liver hepatocytes (RLH), respectively. Degradation of sphingomyelin in native cells led in turn to a reduction (within 60 min) of the plasma membrane cholesterol content (by 25, 15 and 10%, respectively). This manipulation of the plasma membrane lipid content did not affect the forskolin or prostaglandin E1-induced activation of adenylyl cyclase (as measured from the conversion of [3H]adenine via [3H]ATP to [3H]cAMP). These manipulations did, however, increase the basal rate of [3H]cAMP formation in rat liver hepatocytes (but not in the fibroblast cell types). With Chinese hamster lung fibroblasts, transfected to express an alpha 2-adrenergic receptor, it was observed that the alpha 2-adrenergic receptor-induced inhibition of adenylyl cyclase activity was slightly (but significantly) diminished in sphingomyelin and cholesterol-depleted cells. With isolated rat liver hepatocytes it was observed that the glucagon (receptor) mediated activation of adenylyl cyclase was also reduced in sphingomyelinase-treated cells. In another set of experiments, CHLF and RLH cells were exposed for 2 h to vesicles prepared from dilauroylphosphatidylcholine, to increase the lateral packing density in the outer leaflet of the plasma membrane. In such treated cells, the receptor-coupling to adenylyl cyclase was markedly reduced both in CHLF (the alpha 2-adrenergic receptor) and RLH (the glucagon-receptor) cells. We conclude that the direct activation of adenylyl cyclase (i.e., by forskolin) is not markedly affected by manipulations outer leaflet phospholipid composition (either reduction of sphingomyelin or increase of phosphatidylcholine), whereas receptor-coupled events clearly are.

Molecular and functional diversity of mammalian Gs-stimulated adenylyl cyclases.

Iyengar R
FASEB J. 1993; 7: 768-75

Receptor-regulated adenylyl cyclase in mammalian systems is among the best-studied of the cell surface signaling pathways that utilize G-proteins as transducers. In addition to the multiplicity of receptors and Gs proteins that function in this pathway, recent studies have shown that substantial molecular diversity exists in the effector as well. Full-length cDNAs encoding six different G-protein-regulated adenylyl cyclases have been isolated, and partial sequences identifying two more are known. These eight mammalian adenylyl cyclases can be grouped into five distinct families. The different types share some common properties such as stimulation by Gs and the diterpene forskolin. They show very distinct patterns of regulation by the beta gamma-subunits of G-proteins and protein kinases such as protein kinase C. The different types also appear to be localized in a tissue-specific manner. This diversity of regulatory features indicates that the effector can play an important role in determining the routing of signals to the cAMP pathway. The tissue and cell type-specific localization of the individual forms suggests that effectors such as adenylyl cyclase could be potential targets for a new generation of cell and tissue-specific drugs.

Multiple families of Gs-regulated adenylyl cyclases.

Iyengar R
Adv Second Messenger Phosphoprotein Res. 1993; 28: 27-36

[Structure and regulation of adenylyl cyclase]

Itoh H
Tanpakushitsu Kakusan Koso. 1997; 42: 371-8

The adenylyl cyclases as integrators of transmembrane signal transduction.

Ishikawa Y, Homcy CJ
Circ Res. 1997; 80: 297-304

Immunodetection of adenylyl cyclase protein in tissues.

Ishikawa Y, Grant BS, Okumura S, Schwencke C, Yamamoto M
Mol Cell Endocrinol. 2000; 162: 107-12

Immunodetection of the adenylyl cyclase isoforms has been difficult in tissues because of its low quantity of protein expression. We have developed a one-step cellular protein purification method that enables a simple immunodetection of the adenylyl cyclase isoforms. The type I isoform was detected exclusively in the brain. The type III isoform was detected in the brain and lungs. Further, the protein expression of type III adenylyl cyclase in lungs changed ontogenically and was the lowest in neonates. Thus, the comparison of the amount of certain adenylyl cyclase isoforms protein in each tissue is now feasible using our method.

Cloning and characterization of a Drosophila adenylyl cyclase homologous to mammalian type IX.

Iourgenko V, Kliot B, Cann MJ, Levin LR
FEBS Lett. 1997; 413: 104-8

A novel Drosophila adenylyl cyclase (AC) was identified by PCR using degenerate primers specific for the known metazoan ACs. The full-length cDNA predicts a protein displaying significant sequence homology with mammalian Type IX AC (AC9). The abundance and size of the message for the Drosophila AC9 homolog (DAC9) changes through development. Biochemical analysis of DAC9 confirms it encodes a functional enzyme which can be activated by forskolin or G protein. Together with the Drosophila Type I AC homolog encoded by the learning and memory gene, rutabaga, the molecular identification of DAC9 demonstrates there is a family of Drosophila AC isoforms reflecting at least part of the diversity of mammalian AC isoforms.

Differential activation of yeast adenylyl cyclase by Ras1 and Ras2 depends on the conserved N terminus.

Hurwitz N, Segal M, Marbach I, Levitzki A
Proc Natl Acad Sci U S A. 1995; 92: 11009-13

Although both Ras1 and Ras2 activate adenylyl cyclase in yeast, a number of differences can be observed regarding their function in the cAMP pathway. To explore the relative contribution of conserved and variable domains in determining these differences, chimeric RAS1-RAS2 or RAS2-RAS1 genes were constructed by swapping the sequences encoding the variable C-terminal domains. These constructs were expressed in a cdc25ts ras1 ras2 strain. Biochemical data show that the difference in efficacy of adenylyl cyclase activation between the two Ras proteins resides in the highly conserved N-terminal domain. This finding is supported by the observation that Ras2 delta, in which the C-terminal domain of Ras2 has been deleted, is a more potent activator of the yeast adenylyl cyclase than Ras1 delta, in which the C-terminal domain of Ras1 has been deleted. These observations suggest that amino acid residues other than the highly conserved residues of the effector domain within the N terminus may determine the efficiency of functional interaction with adenylyl cyclase. Similar levels of intracellular cAMP were found in Ras1, Ras1-Ras2, Ras1 delta, Ras2, and Ras2-Ras1 strains throughout the growth curve. This was found to result from the higher expression of Ras1 and Ras1-Ras2, which compensate for their lower efficacy in activating adenylyl cyclase. These results suggest that the difference between the Ras1 and the Ras2 phenotype is not due to their different efficacy in activating the cAMP pathway and that the divergent C-terminal domains are responsible for these differences, through interaction with other regulatory elements.

The adenylyl and guanylyl cyclase superfamily.

Hurley JH
Curr Opin Struct Biol. 1998; 8: 770-7

New structures solved in 1997 revealed that the adenylyl cyclase core consists of a pair of catalytic domains arranged in a wreath. Homologous catalytic domains are arranged in diverse adenylyl and guanylyl cyclases as symmetric homodimers or pseudosymmetric heterodimers. The kinship of the adenylyl and guanylyl cyclases has been confirmed by the structure-based interconversion of their nucleotide specificities. Catalysis is activated when two metal-binding aspartate residues on one domain are juxtaposed with a key aspargine-arginine pair on the other. Allosteric activators of mammalian adenylyl cyclase, forskolin and the stimulatory G protein alpha subunit, promote the catalytically optimal juxtaposition of the two domains.

Heterologous expression and assays for photoreceptor guanylyl cyclases and guanylyl cyclase activating proteins.

Hurley JB, Dizhoor AM
Methods Enzymol. 2000; 315: 708-17

Organization of G proteins and adenylyl cyclase at the plasma membrane.

Huang C, Hepler JR, Chen LT, Gilman AG, Anderson RG, Mumby SM
Mol Biol Cell. 1997; 8: 2365-78

There is mounting evidence for the organization and compartmentation of signaling molecules at the plasma membrane. We find that hormone-sensitive adenylyl cyclase activity is enriched in a subset of regulatory G protein-containing fractions of the plasma membrane. These subfractions resemble, in low buoyant density, structures of the plasma membrane termed caveolae. Immunofluorescence experiments revealed a punctate pattern of G protein alpha and beta subunits, consistent with concentration of these proteins at distinct sites on the plasma membrane. Partial coincidence of localization of G protein alpha subunits with caveolin (a marker for caveolae) was observed by double immunofluorescence. Results of immunogold electron microscopy suggest that some G protein is associated with invaginated caveolae, but most of the protein resides in irregular structures of the plasma membrane that could not be identified morphologically. Because regulated adenylyl cyclase activity is present in low-density subfractions of plasma membrane from a cell type (S49 lymphoma) that does not express caveolin, this protein is not required for organization of the adenylyl cyclase system. The data suggest that hormone-sensitive adenylyl cyclase systems are localized in a specialized subdomain of the plasma membrane that may optimize the efficiency and fidelity of signal transduction.

New glucagon analogues with conformational restrictions and altered amphiphilicity: effects on binding, adenylate cyclase and glycogenolytic activities.

Hruby VJ, Gysin B, Trivedi D, Johnson DG
Life Sci. 1993; 52: 845-55

In an effort to obtain highly potent glucagon antagonists, we have investigated glucagon (1) structure-function relationships utilizing the following design principles: (1) structural changes known to lead to partial agonist activities; (2) conformational restrictions; (3) changes in the conformational probabilities of the primary sequence; and (4) increased amphiphilicity. In this report we present the total synthesis, purification, receptor binding, adenylate cyclase activity, in vivo glycogenolytic activity and CD spectrum of the following four glucagon analogues: [Ahx17,18]glucagon (2), [D-Phe4,Tyr5, 3,5-diiodo-Tyr10,Arg12,Lys17,18,Glu21]glucagon (3), [Asp9,Lys12,Lys17,18,Glu21]glucagon 4, and [Glu15,Lys17,18]glucagon 5. Compound 2 binds exclusively to the high affinity receptor and compound 3 was a highly potent antagonist with respect to adenylate cyclase activity. Analog 4 showed distinct biphasic binding (IC50 5.6 nM and 630 nM), with only the low affinity binding leading to adenylate cyclase activity. Furthermore in analogue 5 receptor binding and adenylate cyclase activity were dissociated by a factor of 5. The results are consistent with a multistep binding mechanism in which glucagon interacts first nonspecifically with the anisotropic interphase of the cell membrane, followed by a conformational transition which occurs in the sequences 10-14 and 15-18 when the membrane bound peptide binds to its receptor.

Nonclassical cannabinoid analgetics inhibit adenylate cyclase: development of a cannabinoid receptor model.

Howlett AC, Johnson MR, Melvin LS, Milne GM
Mol Pharmacol. 1988; 33: 297-302

Extensive structure-activity relationship studies have demonstrated that specific requirements within the cannabinoid structure are necessary to produce potent analgesia. A three-point association between the agonist and the receptor mediating analgesia consists of: 1) the C ring hydroxyl, 2) the phenolic A ring hydroxyl, and 3) the A ring alkyl hydrophobic side chain. Potent tricyclic and bicyclic structures were synthesized as "nonclassical" cannabinoid analgetics that conform to this agonist-receptor three-point interaction model. At the cellular level, centrally active cannabinoid drugs inhibit adenylate cyclase activity in a neuroblastoma cell line. The structure-activity relationship profile for inhibition of adenylate cyclase in vitro was consistent with this same three-point association of agonists with the receptor. A correlation exists between the potency of drugs to produce analgesia in vivo and to inhibit adenylate cyclase in vitro. Enantio- and stereoselectivity were exhibited by the nonclassical cannabinoid compounds for both the analgetic response and the ability to inhibit adenylate cyclase. The magnitude of the enantioselective response was equal for both the biochemical and physiological endpoints. Based on the parallels in structure-activity relationships and the enantioselective effects, it is postulated that the receptor that is associated with the regulation of adenylate cyclase in vitro may be the same receptor as that mediating analgesia in vivo. A conceptualization of the cannabinoid analgetic receptor is presented.

The amino terminus of Galpha(z) is required for receptor recognition, whereas its alpha4/beta6 loop is essential for inhibition of adenylyl cyclase.

Ho MK, Wong YH
Mol Pharmacol. 2000; 58: 993-1000

G(z) couples to most of the known G(i)-linked receptors and its alpha subunit (Galpha(z)) inhibits adenylyl cyclases as efficiently as Galpha(i) subtypes. A series of chimeric Galpha subunits with different portions of Galpha(z) and Galpha(t1) (a regulator of cGMP phosphodiesterase) were constructed to study the essential structural elements of Galpha(z) that determine receptor coupling and effector interaction. The receptor-mediated functions of the chimeras were assessed in two aspects: 1) stimulation of type 2 adenylyl cyclase through the release of betagamma subunits from the chimeras, and 2) inhibition of isoproterenol-stimulated adenylyl cyclase by the chimeric Galpha subunits. The results suggested that the presence of both termini of Galpha(z) were critical for coupling to delta-opioid receptor, with the N-terminal region being more important. Moreover, a stretch of amino acids (295-319) corresponding to the alpha4/beta6 loop was identified as one of the adenylyl cyclase inhibitory domains of Galpha(z).

Two thromboxane A2 receptor isoforms in human platelets. Opposite coupling to adenylyl cyclase with different sensitivity to Arg60 to Leu mutation.

Hirata T, Ushikubi F, Kakizuka A, Okuma M, Narumiya S
J Clin Invest. 1996; 97: 949-56

Thromboxane A2 (TXA2) receptor is a key molecule in hemostasis as its abnormality leads to bleeding disorders. Two isoforms of the human TXA2 receptor have been cloned; one from placenta and the other from endothelium, here referred to as TXR alpha and TXR beta, respectively. These isoforms differ only in their carboxyl-terminal tails. We report that both isoforms are present in human platelets. The two isoforms expressed in cultured cells show similar ligand binding characteristics and phospholipase C (PLC) activation but oppositely regulate adenylyl cyclase activity; TXR alpha activates adenylyl cyclase, while TXR beta inhibits it. The Arg60 to Leu mutant of TXR alpha, which has been shown to impair PLC activation (Hirata, T., A. Kakizuka, F. Ushikubi, I. Fuse, M. Okuma, and S. Narumiya. 1994. J. Clin. Invest. 94: 1662-1667), also impairs adenylyl cyclase stimulation, whereas that of TXR beta retains its activity to inhibit adenylyl cyclase. These findings suggest that the pathway linked to adenylyl cyclase inhibition might be involved in some of the TXA2-induced platelet responses such as shape change and phospholipase A2 activation which remain unaffected in the patients with this mutation.

[Structure and activity of heat-stable enterotoxin]

Hidaka Y
Seikagaku. 1996; 68: 1448-52

The characterization of a novel human adenylyl cyclase which is present in brain and other tissues.

Hellevuo K, Yoshimura M, Mons N, Hoffman PL, Cooper DM, Tabakoff B
J Biol Chem. 1995; 270: 11581-9

We characterized a human cDNA clone which encodes a novel adenylyl cyclase. Data from Southern and Northern blot analysis, and analysis of sequence similarity with a recently cloned mouse adenylyl cyclase (10), indicated that the human adenylyl cyclase was a species variant of type VII adenylyl cyclase. The sequence of the novel human adenylyl cyclase indicated it was a member of the type II adenylyl cyclase family, and we compared the regulatory characteristics of the novel human enzyme with those of type II adenylyl cyclase. The human type VII and rat type II adenylyl cyclases, expressed in human embryonic kidney 293 cells, were activated by prostaglandin E1 (PGE1), but only type VII was activated by isoproterenol. The stimulation of type VII adenylyl cyclase by PGE1 and isoproterenol was attenuated by pretreatment of the cells with staurosporine. Phorbol 12,13-dibutyrate synergistically enhanced the stimulation of both type VII and type II enzyme activity by PGE1 and by the constitutively active Gs mutant Gs (Q227L). The human type VII adenylyl cyclase activity was unresponsive to capacitatively induced changes in intracellular Ca2+. The functional characteristics of human type VII adenylyl cyclase resemble those of the rat type II enzyme, but the enzymes may respond differently to in vivo phosphorylation conditions. While the mRNA for adenylyl cyclase type II was found in several brain areas, the message for type VII adenylyl cyclase was localized primarily to the cerebellar granule cell layer.

Human adenylyl cyclase type 7 contains polymorphic repeats in the 3' untranslated region: investigations of association with alcoholism.

Hellevuo K, Welborn R, Menninger JA, Tabakoff B
Am J Med Genet. 1997; 74: 95-8

Platelet adenylyl cyclase activity has been proposed as a trait marker for alcoholism [Tabakoff et al. (1988): N Engl J Med 318:134-13;9; Parsian et al. (1996): Alcohol Clin Exp Res 20:745-751]. Human adenylyl cyclase type 7 (ADCY7) is a member of the adenylyl cyclase gene family, and it may be the major form of adenylyl cyclase expressed in human platelets. The published cDNA sequence of ADCY7 indicated the presence of potentially polymorphic regions in the 3' untranslated region of ADCY7. PCR techniques combined with fluorescently labeled primers were used to amplify two separate tetranucleotide repeat regions [(AACA)n] in the 3' untranslated region of ADCY7 from the genomic DNA of 62 unrelated individuals. The upstream (AACA)4-repeat was not polymorphic. Five different genotypes were found in the downstream (AACA)5-7 tetranucleotide repeat region. We also tested the association of the tetranucleotide polymorphism to alcohol dependence. When 30 alcoholic and 17 control individuals were compared, no difference was found in the ADCY7 tetranucleotide polymorphism between alcohol-dependent and control groups. Nevertheless, to our knowledge these are the first polymorphisms reported in an adenylyl cyclase gene. Adenylyl cyclases are important receptor-G protein-coupled effectors and are involved in numerous neuronal functions in the central nervous system. Whether variations in ADCY7 and possible variations in other members of this gene family are underlying other psychiatric disorders remains to be studied.

Adenylyl cyclases: mRNA and characteristics of enzyme activity in three areas of brain.

Hellevuo K, Hoffman PL, Tabakoff B
J Neurochem. 1996; 67: 177-85

RNase protection assays were used in a comparative analysis of the quantities of mRNA for five "calcium-sensitive" (types I, III, V, VI, and VIII) adenylyl cyclases and one "calcium-insensitive" (type II) adenylyl cyclase in mouse cerebral cortex, cerebellum, and nucleus accumbens. The mRNA levels for type V adenylyl cyclase were dominant in the nucleus accumbens. Type V adenylyl cyclase mRNA was also found in the cerebral cortex and at low levels in the cerebellum. Type I adenylyl cyclase mRNA was the major form in the cerebellum with 15-50-fold higher levels compared with other adenylyl cyclase mRNAs. Type I adenylyl cyclase mRNA was also the most prominent adenylyl cyclase mRNA in the cerebral cortex, although the mRNA levels of other adenylyl cyclase forms were more comparable to those of the type I enzyme in this brain area. The mRNA levels for adenylyl cyclase types II, III, VI, and VIII were intermediate to low depending on the brain area. Cell membranes from the nucleus accumbens demonstrated adenylyl cyclase activity that was synergistically activated by concomitant addition of GTP and forskolin to assay mixtures, reflecting a characteristic of type V adenylyl cyclase protein. Calcium?calmodulin stimulated adenylyl cyclase activity in membranes from all three brain areas. However, synergistic activation of adenylyl cyclase activity by GTP and calcium/calmodulin was noted only with cortical membranes, and this characteristic may reflect the presence of type VIII adenylyl cyclase mRNA in the cortex. Although mRNA for type VIII adenylyl cyclase was almost equivalent in the cortex and cerebellum, the lack of a synergistic effect of GTP plus calcium/calmodulin on the cerebellar enzyme activity may be a result of the significant dominance of type I adenylyl cyclase mRNA (and protein) in the cerebellum. In general, the mRNA levels for the various adenylyl cyclases were predictive of the regulatory characteristics of adenylyl cyclase activity in membranes of the brain areas studied.

Isolation and characterization of constitutively active mutants of mammalian adenylyl cyclase.

Hatley ME, Benton BK, Xu J, Manfredi JP, Gilman AG, Sunahara RK
J Biol Chem. 2000; 275: 38626-32

A genetic screen in Saccharomyces cerevisiae identified mutations in mammalian adenylyl cyclase that activate the enzyme in the absence of G(s)alpha. Thirteen of these mutant proteins were characterized biochemically in an assay system that depends on a mixture of the two cytosolic domains (C(1) and C(2)) of mammalian adenylyl cyclases. Three mutations, I1010M, K1014N, and P1015Q located in the beta4-beta5 loop of the C(2) domain of type II adenylyl cyclase, increase enzymatic activity in the absence of activators. The K1014N mutation displays both increased maximal activity and apparent affinity for the C(1) domain of type V adenylyl cyclase in the absence of activators of the enzyme. The increased affinity of the mutant C(2) domain of adenylyl cyclase for the wild type C(1) domain was exploited to isolate a complex containing VC(1), IIC(2), and G(s)alpha-guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) in the absence of forskolin and a complex of VC(1), IIC(2), forskolin, and P-site inhibitor in the absence of G(s)alpha-GTPgammaS. The isolation of these complexes should facilitate solution of crystal structures of low activity states of adenylyl cyclase and thus determination of the mechanism of activation of the enzyme by forskolin and G(s)alpha.

Determination of the binding site on the extracellular domain of guanylyl cyclase C to heat-stable enterotoxin.

Hasegawa M, Hidaka Y, Matsumoto Y, Sanni T, Shimonishi Y
J Biol Chem. 1999; 274: 31713-8

Guanylyl cyclase C, one of the family of membrane-bound guanylyl cyclases, consists of an extracellular domain and an intracellular domain, which are connected by a single transmembrane polypeptide. The extracellular domain binds unique small polypeptides with high specificity, which include the endogenous peptide hormones, guanylin and uroguanylin, as well as an exogenous enterotoxigenic peptide, heat-stable enterotoxin, secreted by pathogenic Escherichia coli. Information on this specific binding is propagated into the intracellular domain, followed by the synthesis of cGMP, a second messenger that regulates a variety of intracellular physiological processes. This study reports the design of a photoaffinity labeled analog of heat-stable enterotoxin (biotinyl-(AC(5))(2)-[Gly(4), Pap(11)]STp(4-17)), which incorporates a Pap residue (p-azidophenylalanine) at position 11 and a biotin moiety at the N terminus, and the use of this analog to determine the ligand-binding region of the extracellular domain of guanylyl cyclase C. The endoproteinase Lys-C digestion of the extracellular domain, which was covalently labeled by this ligand, and mass spectrometric analyses of the digest revealed that the ligand specifically binds to the region (residue 387 to residue 393) of guanylyl cyclase C. This region is localized close to the transmembrane portion of guanylyl cyclase C on the external cellular surface. This result was further confirmed by characterization of site-directed mutants of guanylyl cyclase C in which each amino acid residue was substituted by an Ala residue instead of residues normally located in the region. This experiment provides the first direct demonstration of the ligand-binding site of guanylyl cyclase C and will contribute toward an understanding of the receptor recognition of a ligand and the modeling of the interaction of the receptor and its ligand at the molecular level.

Adenylyl cyclase activity in clonally derived human myoblast cultures: evidence for myoblast heterogeneity.

Hardiman O, Sweeney DJ, Keenan AK
Neuromuscul Disord. 1996; 6: 283-91

In vitro myogenesis recapitulates the programme of myogenesis in vivo. During the process of muscle differentiation, cAMP plays an important role in the control of gene expression and in the integration of metabolic functions. cAMP generation may be affected by drugs or hormones that interact with the membrane-bound enzyme adenylyl cyclase, including adrenergic agents and glucocorticoids. In this study, adenylyl cyclase activity was evaluated in membranes prepared from human clonally derived muscle cultures. In control cultures, there was considerable inter-clonal variation in basal, sodium-fluoride and forskolin-stimulated adenylyl cyclase activity. Cultures differed in their response to steroids: adenylyl cyclase activity was markedly enhanced in some clones, and was significantly inhibited in other clones. Pre-treatment of cultures with pertussis toxin indicated that the effects of steroids are mediated in part by modulation of G-protein activity. These findings indicate a substantial heterogeneity among myoblast clones with respect to the modulating effect of steroids on adenylyl cyclase activity. This observation may account for the conflicting reports of steroid effects on muscle in vitro, and may be of relevance to the understanding of possible transmembrane signalling alterations in muscle disease.

Adenylyl cyclases: structure, regulation and function in an enzyme superfamily.

Hanoune J, Pouille Y, Tzavara E, Shen T, Lipskaya L, Miyamoto N, Suzuki Y, Defer N
Mol Cell Endocrinol. 1997; 128: 179-94

Analysis and prediction of functional sub-types from protein sequence alignments.

Hannenhalli SS, Russell RB
J Mol Biol. 2000; 303: 61-76

The increasing number and diversity of protein sequence families requires new methods to define and predict details regarding function. Here, we present a method for analysis and prediction of functional sub-types from multiple protein sequence alignments. Given an alignment and set of proteins grouped into sub-types according to some definition of function, such as enzymatic specificity, the method identifies positions that are indicative of functional differences by comparison of sub-type specific sequence profiles, and analysis of positional entropy in the alignment. Alignment positions with significantly high positional relative entropy correlate with those known to be involved in defining sub-types for nucleotidyl cyclases, protein kinases, lactate/malate dehydrogenases and trypsin-like serine proteases. We highlight new positions for these proteins that suggest additional experiments to elucidate the basis of specificity. The method is also able to predict sub-type for unclassified sequences. We assess several variations on a prediction method, and compare them to simple sequence comparisons. For assessment, we remove close homologues to the sequence for which a prediction is to be made (by a sequence identity above a threshold). This simulates situations where a protein is known to belong to a protein family, but is not a close relative of another protein of known sub-type. Considering the four families above, and a sequence identity threshold of 30 %, our best method gives an accuracy of 96 % compared to 80 % obtained for sequence similarity and 74 % for BLAST. We describe the derivation of a set of sub-type groupings derived from an automated parsing of alignments from PFAM and the SWISSPROT database, and use this to perform a large-scale assessment. The best method gives an average accuracy of 94 % compared to 68 % for sequence similarity and 79 % for BLAST. We discuss implications for experimental design, genome annotation and the prediction of protein function and protein intra-residue distances.

Genetic selection in Saccharomyces of mutant mammalian adenylyl cyclases with elevated basal activities.

Haney SA, Xu J, Lee SY, Ma CL, Duzic E, Broach JR, Manfredi JP
Mol Genet Genomics. 2001; 265: 1120-8

We show that co-expression of rat Galphas together with type I, II, IV, or VI mammalian adenylyl cyclase (AC) can suppress the growth defect of cyr1 strains of Saccharomyces cerevisiae, which lack a functional endogenous AC. Complemention of cvr1 is not observed in the absence of Galphas, indicating that the mammalian ACs retain their normal regulatory behavior in yeast. Selection for Galphas-independent growth of (cyr1 strains expressing type IV AC yielded several ACIV mutants with enhanced basal activity, each of which had a single amino acid substitution in the conserved C1a or C2a region of the protein. Expression of two of the mutant ACs in HEK293 cells resulted in increased levels of cAMP and elevated adenylyl cyclase activity. Further selection for reverting mutations in one of these constitutively active AC mutants yielded three independent intragenic suppressor mutations. The distribution of the activating and suppressor mutations throughout both C1a and C2a is consistent with a model in which the enhanced basal activity results from an increase in the affinity between C1a and C2a. These results demonstrate the utility of Saccharomyces as a tool for the identification of informative mutant forms of mammalian ACs.

Cloning, chromosomal mapping, and regulatory properties of the human type 9 adenylyl cyclase (ADCY9).

Hacker BM, Tomlinson JE, Wayman GA, Sultana R, Chan G, Villacres E, Disteche C, Storm DR
Genomics. 1998; 50: 97-104

The type 9 adenylyl cyclase (AC9) is a widely distributed adenylyl cyclase that was originally cloned from a mouse cDNA library. Here we report the cloning, chromosomal mapping, and regulatory properties of human AC9 (HGMW-approved symbol ADCY9). Although the human AC9 sequence shows 86% homology with mouse AC9, divergence at the C2a/C2b junction results in an alternative C2b amino acid sequence. In situ hybridization localized the human AC9 gene to both human and mouse chromosomes 16. AC9 mRNA is present in all tissues examined, with the highest levels found in skeletal muscle, heart, and brain. To characterize the regulatory properties of human AC9 in vivo, the enzyme was expressed in HEK-293 cells. Human AC9 is stimulated by beta-adrenergic receptor activation but is insensitive to forskolin, Ca2+ and somatostatin. In contrast to mouse AC9, the activity of human AC9 is unaffected by inhibitors of calcineurin. These data emphasize the importance of determining the regulatory properties of human adenylyl cyclases.

Expression and purification of soluble, active heterodimeric guanylyl cyclase from baculovirus.

Gupta G, Kim J, Yang L, Sturley SL, Danziger RS
Protein Expr Purif. 1997; 10: 325-30

A method for expression and purification of active cytosolic heterodimeric histidine (His)-tagged guanylyl cyclase of the alpha 1/beta 1 isoform has been developed using recombinant baculovirus-transfected insect cells. Confirmation of expression of active cyclase was obtained by both Western analysis and enzymatic activity. A His tag on the COOH-terminus of the alpha 1 and beta 1 subunits allowed rapid purification of the heterodimeric form of guanylyl cyclase in a single affinity step using a nickel column. A second gel-filtration step was applied to reconstitute into the complex heme, a required cofactor. This was confirmed spectroscopically by absorbance in the Soret region. Like enzyme purified from tissue, the activity of recombinant guanylyl cyclase was increased by protoporphyrin IX and inhibited by both Zn- and Sn-protoporphyrin. The method described here should provide a general approach for the expression and purification of alternate forms of cytosolic guanylyl cyclase and facilitate mechanistic and structural studies of this important family of enzymes. Furthermore, the procedure demonstrates the utility of the His-tag system to purify multimeric proteins.

The beta2 subunit inhibits stimulation of the alpha1/beta1 form of soluble guanylyl cyclase by nitric oxide. Potential relevance to regulation of blood pressure.

Gupta G, Azam M, Yang L, Danziger RS
J Clin Invest. 1997; 100: 1488-92

Cytosolic guanylyl cyclases (GTP pyrophosphate-lyase [cyclizing; EC 4.6.1.2]), primary receptors for nitric oxide (NO) generated by NO synthases, are obligate heterodimers consisting of an alpha and a beta subunit. The alpha1/beta1 form of guanylyl cyclase has the greatest activity and is considered the universal form. An isomer of the beta1 subunit, i.e., beta2, has been detected in the liver and kidney, however, its role is not known. In this study, we investigated the function of beta2. Immunoprecipitation experiments showed that the beta2 subunit forms a heterodimer with the alpha1 subunit. NO-stimulated cGMP formation in COS 7 cells cotransfected with the alpha1 and beta2 subunits was approximately 1/3 of that when alpha1 and beta1 subunits were cotransfected. The beta2 subunit inhibited NO-stimulated activity of the alpha1/beta1 form of guanylyl cyclase and NO-stimulated cGMP formation in cultured smooth muscle cells. Our results provide the first evidence that the beta2 subunit can regulate NO sensitivity of the alpha1/beta1 form of guanylyl cyclase. Northern analysis for guanylyl cyclase subunits was performed on RNA from kidneys of Dahl salt-sensitive rats, which have been shown to have decreased renal sensitivity to NO. Compared to the Dahl salt-resistant rat, message for beta2 was increased, beta1 was decreased, and alpha1 was unchanged. These results suggest a molecular basis for decreased renal guanylyl cyclase activity, i.e. , an increase in the alpha1/beta2 heterodimer, and decrease in the alpha1/beta1 heterodimer.

Adenylyl cyclase Rv1625c of Mycobacterium tuberculosis: a progenitor of mammalian adenylyl cyclases.

Guo YL, Seebacher T, Kurz U, Linder JU, Schultz JE
EMBO J. 2001; 20: 3667-75

The gene Rv1625c from Mycobacterium tuberculosis encodes a membrane-anchored adenylyl cyclase corresponding to exactly one-half of a mammalian adenylyl cyclase. An engineered, soluble form of Rv1625c was expressed in Escherichia coli. It formed a homodimeric cyclase with two catalytic centers. Amino acid mutations predicted to affect catalysis resulted in inactive monomers. A single catalytic center with wild-type activity could be reconstituted from mutated monomers in stringent analogy to the mammalian heterodimeric cyclase structure. The proposed existence of supramolecular adenylyl cyclase complexes was established by reconstitution from peptide-linked, mutation-inactivated homodimers resulting in pseudo-trimeric and -tetrameric complexes. The mycobacterial holoenzyme was expressed successfully in E.coli and mammalian HEK293 cells, i.e. its membrane targeting sequence was compatible with the bacterial and eukaryotic machinery for processing and membrane insertion. The membrane-anchored mycobacterial cyclase expressed in E.coli was purified to homogeneity as a first step toward the complete structural elucidation of this important protein. As the closest progenitor of the mammalian adenylyl cyclase family to date, the mycobacterial cyclase probably was spread by horizontal gene transfer.

A systematic study of effects of non-ionic detergens on solubilization and activity of pig kidney adenylate cyclase.

Guillon G, Roy C, Jard S
Eur J Biochem. 1978; 92: 341-8

Persistent interactions between the two transmembrane clusters dictate the targeting and functional assembly of adenylyl cyclase.

Gu C, Sorkin A, Cooper DM
Curr Biol. 2001; 11: 185-90

Adenylyl cyclases possess complex structures like those of the ATP binding cassette (ABC) transporter family, which includes the cystic fibrosis transmembrane regulator, the P-glycoprotein, and ATP-sensitive K(+) channels [1-4]. These structures comprise a cytosolic N terminus followed by two tandem six-transmembrane cassettes, each associated with a highly homologous (ATP binding) cytosolic loop [5-8]. The catalytic domains, which are located in the two large cytoplasmic loops, are highly conserved and well studied. The crystal structure of these domains has even been described recently [9, 10]. However, nothing is known of the function or organization of the 12 transmembrane segments. In the present study we adopted a range of strategies including live-cell fluorescence resonance energy transfer (FRET) microscopy, coimmunoprecipitation, and functional assays of various truncated and substituted, fluorescently-tagged molecules to analyze the trafficking and activity of this molecule. When expressed as individual peptides, the two transmembrane domains - largely independently of any cytosolic region - formed a tight complex that was delivered to the plasma membrane. This cooperation between the two intact transmembrane domains was essential and sufficient to target the enzyme to the plasma membrane of the cell. The extracellular loop between the ninth and tenth transmembrane segments, which contains an N-glycosylation site, was also necessary. Furthermore, the interaction between the two transmembrane clusters played a critical role in bringing together the cytosolic catalytic domains to express functional adenylyl cyclase activity in the intact cell.

Calmodulin-binding sites on adenylyl cyclase type VIII.

Gu C, Cooper DM
J Biol Chem. 1999; 274: 8012-21

Ca2+ stimulation of adenylyl cyclase type VIII (ACVIII) occurs through loosely bound calmodulin. However, where calmodulin binds in ACVIII and how the binding activates this cyclase have not yet been investigated. We have located two putative calmodulin-binding sites in ACVIII. One site is located at the N terminus as revealed by overlay assays; the other is located at the C terminus, as indicated by mutagenesis studies. Both of these calmodulin-binding sites were confirmed by synthetic peptide studies. The N-terminal site has the typical motif of a Ca2+-dependent calmodulin-binding domain, which is defined by a characteristic pattern of hydrophobic amino acids, basic and aromatic amino acids, and a tendency to form amphipathic alpha-helix structures. Functional, mutagenesis studies suggest that this binding makes a minor contribution to the Ca2+ stimulation of ACVIII activity, although it might be involved in calmodulin trapping by ACVIII. The primary structure of the C-terminal site resembles another calmodulin-binding motif, the so-called IQ motif, which is commonly Ca2+-independent. Mutagenesis and functional assays indicate that this latter site is a calcium-dependent calmodulin-binding site, which is largely responsible for the Ca2+ stimulation of ACVIII. Removal of this latter calmodulin-binding region from ACVIII results in a hyperactivated enzyme state and a loss of Ca2+ sensitivity. Thus, Ca2+/calmodulin regulation of ACVIII may be through a disinhibitory mechanism, as is the case for a number of other targets of Ca2+/calmodulin.

Identification of common and distinct residues involved in the interaction of alphai2 and alphas with adenylyl cyclase.

Grishina G, Berlot CH
J Biol Chem. 1997; 272: 20619-26

The G protein alpha subunits, alphas and alphai2, have stimulatory and inhibitory effects, respectively, on a common effector protein, adenylyl cyclase. These effects require a GTP-dependent conformational change that involves three alpha subunit regions (Switches I-III). alphas residues in three adjacent loops, including Switch II, specify activation of adenylyl cyclase. The adenylyl cyclase-specifying region of alphai2 is located within a 78-residue segment that includes two of these loops but none of the conformational switch regions. We have used an alanine-scanning mutagenesis approach within Switches I-III and the 78-residue segment of alphai2 to identify residues required for inhibition of adenylyl cyclase. We found a cluster of conserved residues in Switch II in which substitutions cause major losses in the abilities of both alphai2 and alphas to modulate adenylyl cyclase activity but do not affect alpha subunit expression or the GTP-induced conformational change. We also found two regions within the 78-residue segment of alphai2 in which substitutions reduce the ability of alphai2 to inhibit adenylyl cyclase, one of which corresponds to an effector-activating region of alphas. Thus, both alphai2 and alphas interact with adenylyl cyclase using: 1) conserved Switch II residues that communicate the conformational state of the alpha subunit and 2) divergent residues that specify particular effectors and the nature of their modulation.

Cyclase enzymes. Deciphering the mosaic.

Green NM
Nature. 1989; 340: 679-80

Corpora allata of the larval tobacco hornworm contain a calcium/calmodulin-sensitive adenylyl cyclase.

Granger NA, Allen LG, Sturgis SL, Combest W, Ebersohl R
Arch Insect Biochem Physiol. 1995; 30: 149-64

An assay was developed with which to study basic characteristics of an adenylyl cyclase in the corpora allata (CA) of the tobacco hornworm, Manduca sexta. The assay used glands collected and frozen at -80 degrees C, to circumvent the problem of tissue availability. With this protocol for storage of tissue, less than 25% of the enzyme activity in fresh tissue was lost. Substances such as sodium fluoride (NaF) and Gpp(NH)p (a non-hydrolyzable GTP analog), which typically stimulate the adenylyl cyclases in other insect tissues, increased enzyme activity several-fold. There was a progressive decrease in the capacity of the CA adenylyl cyclase to be stimulated by NaF during the fifth stadium, suggesting a possible developmental change in the capacity of the associated G protein to be stimulated by NaF. The calcium/calmodulin (CaM) dependence of adenylyl cyclase activity was also investigated. The results demonstrated that addition of up to 10(-4) M calcium to assays of enzyme activity in whole gland homogenates of both larval (day 0) and prepupal (day 6) CA resulted in only a slight increase in the activity of the enzyme over basal rates in the presence of the calcium chelator EGTA. However, addition of as little as 5 microM CaM in the presence of 10(-4) to 10(-3) M calcium increased adenylyl cyclase activity three-to five-fold. A similar stimulation was obtained with washed membrane preparations of day 0 and day 6 glands, but required a substantially higher concentration of CaM. Results demonstrated that the CA possess a calcium/CaM-dependent adenylyl cyclase from day 0 through day 6.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of guanylyl cyclase in purified myelin.

Grabow M, Chakraborty G, Ledeen RW
Neurochem Res. 1996; 21: 457-62

This study was undertaken to characterize the enzymatic properties of the particulate guanylyl cyclase previously shown to be present at a high level of activity in purified rat brain myelin. Significant activation was achieved by both Lubrol-PX and Triton X-100, the latter being somewhat more effective. A pH optimum of 7.8 was observed, compared to 7.4 for microsomes. Employing 1.2 mM GTP with 1% Triton X-100, linearity of response was observed up to 60 min and approximately 1.2 mg of myelin protein. Kinetic analysis revealed Km values of 0.258mM and 0.486mM for myelin and microsomes, respectively, similar values being obtained by Lineweaver-Burke analysis or Direct Linear Plot. Vmax values were 20 and 266 pmol/mg protein/min for myelin and microsomes, respectively. Washing of the myelin with 0.5 M NaCl or 0.1% Na taurocholate did not remove a significant amount of guanylyl cyclase activity, indicating the enzyme to be intrinsic to the myelin sheath.

Cellular localization of the Mn2+-dependent adenylyl cyclase (AC) in rat testis.

Gordeladze JO, Purvis K, Clausen OP, Rommerts FF, Hansson V
Int J Androl. 1981; 4: 172-82

In the present study we have examined the exact cellular localization of the Mn2+-dependent adenylyl cyclase (AC) in the rat testis. Several pieces of evidence indicate that this testis specific enzyme is exclusively localized to haploid cells in the testis: firstly, the appearance of Mn2+-dependent adenylyl cyclase coincides in time with the occurrence of haploid cells (IC) in the rat testis. Secondly, tests of tfm rats and mice, in which little or no haploid cells are found, exhibit negligible Mn2+-dependent AC activity. Finally, separation of various testicular cells by BSA gradient sedimentation (Staput fractionation), revealed that this enzyme activity follows the location of haploid cells; the highest specific AC activities being found in the fractions containing elongated spermatids. Some activity found in the primary spermatocyte fraction (4C cells) can to a large extent be explained by the contamination of haploid cells in this fraction.

Mn2+-dependent adenylyl cyclase (AC) in rat testis: kinetic properties and optimalization of assay conditions.

Gordeladze JO, Hansson V
Int J Androl. 1980; 3: 539-52

The aim of the present study was to investigate some kinetic properties of the soluble Mn2+-dependent adenylyl cyclase (AC) in rat testis and to examine the viability of this enzyme under various assay conditions. 1. Constant AC activity was found during the first 35 min of incubation, and AC activity was proportional to protein concentrations in the range tested (10-45 micrograms/tube). 2. The enzyme activity was constant for at least 24 h at 4 degrees C and for at least 2 h at 35 degrees C. At higher temperatures (above 45 degrees C) the AC activity rapidly declines. 3. The enzyme displays a pH optimum in the physiological range (7.0-7.4) and increasing ionic strength (0-0.4 m NaCl/KCl) caused only a marginal decrease in enzyme activity. 5. The soluble Mn2+-dependent AC in rat testis could not be stimulated by hormones (hCG, FSH, prostaglandins), guanyl-nucleotides (GMP-P(NH)P, 2-4 x 10(-5) M) or F-. Ca2+ (0.1-5 mM) caused a dose-dependent stimulation of AC activity; the most pronounced effect was seen at low Mn2+ concentrations. 6. The Km for MnATP2- was estimated to 3.0 mM and the Km for Mn2+ to 7.2 mM. Increasing concentrations of Mn2+ did not alter the affinity for MnATP2- and neither did varying levels of ATP affect the affinity for Mn2+.

[Guanylyl cyclases]

Gorczyca WA
Postepy Hig Med Dosw. 1999; 53: 209-22

Guanylyl cyclases constitute still growing family of enzymes, which catalyze synthesis of cGMP and can be activated in several ways. This review describes activation mechanisms for different forms of guanylyl cyclases. Possible signalling pathways mediated by cGMP are also indicated.

Structural and functional characterization of a second subfamily member of the calcium-modulated bovine rod outer segment membrane guanylate cyclase, ROS-GC2.

Goraczniak R, Duda T, Sharma RK
Biochem Biophys Res Commun. 1997; 234: 666-70

A native bovine calcium-modulated rod outer segment membrane guanylate cyclase (ROS-GC) has been cloned and reconstituted to show its linkage consistent to the process of phototransduction. In the present study, a second form of the membrane guanylate cyclase has been cloned from the bovine retina. This cyclase shares a high sequence identity with ROS-GC, is specifically expressed in the bovine retina, and, like ROS-GC, is modulated in low Ca2+ by a calmodulin-like Ca2+-binding protein, termed GCAP2. For this reason, this cyclase has now been named ROS-GC2 and the previously described ROS-GC as ROS-GC1. The tail end of ROS-GC2 contains a stretch of five amino acids, a structural feature unique to itself. These findings support the existence of a calcium-modulated subfamily of ROS-GC and indicate that ROS-GC2 embodies a five amino acid signature element at its tail end.

Cloning and expression of an adenylyl cyclase localized to the corpus striatum.

Glatt CE, Snyder SH
Nature. 1993; 361: 536-8

The neurotransmitter dopamine acts through various receptor subtypes that are largely associated with enhancement or inhibition of adenylyl cyclases. These dopamine-sensitive adenylyl cyclases are highly concentrated in the corpus stratum and associated limbic structures of the brain, where their levels exceed by orders of magnitude those in other areas of the brain. Here we use in situ hybridization to show that messenger RNA for three of these adenylyl cyclases is not found in the corpus striatum. We have isolated and expressed a complementary DNA encoding new adenylyl cyclase whose selective concentration in the corpus striatum indicates that it may be responsible for the synaptic actions of dopamine.

Nobel Lecture. G proteins and regulation of adenylyl cyclase.

Gilman AG
Biosci Rep. 1995; 15: 65-97

Transmembrane signaling, G proteins, and adenylyl cyclase.

Gilman AG
Harvey Lect. 1989; 85: 153-72

Mammalian adenylyl cyclase family members are randomly located on different chromosomes.

Gaudin C, Homcy CJ, Ishikawa Y
Hum Genet. 1994; 94: 527-9

Molecular cloning studies have elucidated the presence of multiple isoforms of mammalian adenylyl cyclase. So far, six different isoforms (I to VI) have been fully characterized. Comparison of their structural and biochemical characteristics suggests that the mammalian adenylyl cyclase family can be classified into four subfamilies: type I, type III, type II/IV, and type V/VI. We have determined the chromosomal localization of these genes. Type I gene was assigned to chromosome 7, type III to chromosome 2, types II and IV to chromosomes 5 and 14, and types V and VI to chromosomes 3 and 12. Our results indicate that the different adenylyl cyclase isoforms, even within the same subfamily, are distributed randomly in the genome, in contrast to the chromosomal organization of other components within the same signaling pathway, such as catecholamine receptors and G proteins.

Guanylate cyclase receptor family.

Garbers DL
Recent Prog Horm Res. 1990; 46: 85-96

The plasma membrane forms of guanylate cyclase contain a highly conserved catalytic domain, which is also conserved in the soluble form of the enzyme and in mammalian adenylate cyclase. A protein kinase-like domain lies to the amino-terminal side of the catalytic domain and appears to be required for signaling via cGMP; it might also signal, itself, through phosphotransferase activity. This domain is present in the growth factor receptors, but appears not to be a component of other guanylate cyclases or adenylate cyclases. A single transmembrane domain then separates the cyclase catalytic and protein kinase-like domains from the putative ligand-binding domain. At least two plasma membrane forms of gunaylate cyclase (i.e., GC-A and GC-B) have now been identified, and their ligand specificities appear to be distinctly different. The tissue/cellular distribution of this family of receptors is now of potential importance, since specific agonists might differentially regulate physiological processes via the secondary messenger, cGMP, dependent on cellular distribution of the receptors.

Interruption of specific guanylyl cyclase signaling pathways.

Gao Z, Yuen PS, Garbers DL
Adv Second Messenger Phosphoprotein Res. 1997; 31: 183-90

Cloning and expression of a widely distributed (type IV) adenylyl cyclase.

Gao BN, Gilman AG
Proc Natl Acad Sci U S A. 1991; 88: 10178-82

We have cloned and expressed a cDNA that encodes a widely distributed form of mammalian adenylyl cyclase (EC 4.6.1.1). Although those adenylyl cyclases described previously have a rather narrow tissue distribution, this enzyme (type IV) is apparently synthesized in a variety of peripheral tissues and in the central nervous system. The protein resembles the other adenylyl cyclases in its proposed structure. It most resembles the type II adenylyl cyclase described in the preceding paper [Feinstein, P. G., Schrader, K. A., Bakalyar, H. A., Tang, W.-J., Krupinski, J., Gilman, A. G. & Reed, R. R. (1991) Proc. Natl. Acad. Sci. USA 88, 10173-10177] in its amino acid sequence, lack of response to calmodulin, and synergistic activation by a combination of the Gs alpha subunit and the G-protein beta gamma subunit complex.

A receptor guanylyl cyclase expressed specifically in olfactory sensory neurons.

Fulle HJ, Vassar R, Foster DC, Yang RB, Axel R, Garbers DL
Proc Natl Acad Sci U S A. 1995; 92: 3571-5

We have cloned an additional member (GC-D) of the membrane receptor guanylyl cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] family that is specifically expressed in a subpopulation of olfactory sensory neurons. The extracellular, putative ligand-binding domain of the olfactory cyclase is similar in primary structure to two guanylyl cyclases expressed in the retina but diverges considerably from other known guanylyl cyclases. The expression of GC-D RNA is restricted to a small, randomly dispersed population of neurons that is within a single topographic zone in the olfactory neuroepithelium and resembles the pattern of the more diverse seven-transmembrane-domain odorant receptors. These observations suggest that GC-D may function directly in odor recognition or in modulating the sensitivity of a subpopulation of sensory neurons to specific odors.

Guanylyl cyclases: a family of receptor-linked enzymes.

Fulle HJ, Garbers DL
Cell Biochem Funct. 1994; 12: 157-65

Functions of conserved cysteines of soluble guanylyl cyclase.

Friebe A, Wedel B, Harteneck C, Foerster J, Schultz G, Koesling D
Biochemistry. 1997; 36: 1194-8

Soluble guanylyl cyclase (sGC), a heme-containing heterodimeric enzyme, is stimulated by NO and catalyzes the formation of the intracellular signaling molecule cGMP. Cysteine residues of sGC have been considered to be important as they were thought to play a significant role in the regulation of the enzyme. The aim of this study was to investigate the possible function of conserved cysteine residues of sGC. Fifteen conserved cysteine residues on sGC were point-mutated to serine, using site-directed mutagenesis. All of the resulting recombinant enzymes were able to synthesize cGMP. Mutation of two cysteines located in the N-terminal, putative heme-binding region of the beta1 subunit yielded proteins that were insensitive to NO. Spectrophotometric analysis of the NO-insensitive mutants purified from Sf9 cells revealed a loss of the prosthetic heme group. Both mutants could be reconstituted with heme and, as a consequence, NO sensitivity of the mutants was restored. Our data show that mutation of two cysteines of the beta1 subunit (Cys-78 and Cys-214) reduces the affinity of sGC for heme. Mutation of the corresponding cysteines on the alpha1 subunit did not alter NO responsiveness, indicating that heme-binding is mainly a feature of the N-terminal domain of the beta1 subunit.

Inactivation of adenylate cyclase by phenylglyoxal and other dicarbonyls. Evidence for existence of essential arginyl residues.

Franks DJ, Tunnicliff G, Ngo TT
Biochim Biophys Acta. 1980; 611: 358-62

Rat brain adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) activity was markedly reduced when the enzyme was preincubated at 23 degrees C for periods up to 30 min with phenylglyoxyal (2.5-20 mM), an agent that binds specifically to arinyl residues. The kinetics indicated the interaction of phenylgloxal with one arginyl residue was responsible for the inactivation. Protection of inactivation of the enzyme by phenylglyoxal was attained in the presence of ATP and to a lesser extent by ADP and 5'-AMP but not by cyclic-AMP or Mg2+. In addition, 2,3-butanedione and 1,2-hexanedione, compounds that also react with arginyl residues, each inactivated adenylate cyclase to varying degrees. Furthermore, this inactivation was enhanced in the presence of borate ions. These observations strongly suggest that brain adenylate cyclase possesses essential arginyl residues.

Guanylin peptides: cyclic GMP signaling mechanisms.

Forte LR, Freeman RH, Krause WJ, London RM
Braz J Med Biol Res. 1999; 32: 1329-36

Guanylate cyclases (GC) serve in two different signaling pathways involving cytosolic and membrane enzymes. Membrane GCs are receptors for guanylin and atriopeptin peptides, two families of cGMP-regulating peptides. Three subclasses of guanylin peptides contain one intramolecular disulfide (lymphoguanylin), two disulfides (guanylin and uroguanylin) and three disulfides (E. coli stable toxin, ST). The peptides activate membrane receptor-GCs and regulate intestinal Cl- and HCO3- secretion via cGMP in target enterocytes. Uroguanylin and ST also elicit diuretic and natriuretic responses in the kidney. GC-C is an intestinal receptor-GC for guanylin and uroguanylin, but GC-C may not be involved in renal cGMP pathways. A novel receptor-GC expressed in the opossum kidney (OK-GC) has been identified by molecular cloning. OK-GC cDNAs encode receptor-GCs in renal tubules that are activated by guanylins. Lymphoguanylin is highly expressed in the kidney and heart where it may influence cGMP pathways. Guanylin and uroguanylin are highly expressed in intestinal mucosa to regulate intestinal salt and water transport via paracrine actions on GC-C. Uroguanylin and guanylin are also secreted from intestinal mucosa into plasma where uroguanylin serves as an intestinal natriuretic hormone to influence body Na+ homeostasis by endocrine mechanisms. Thus, guanylin peptides control salt and water transport in the kidney and intestine mediated by cGMP via membrane receptors with intrinsic guanylate cyclase activity.

[A molecular hybrid having gonadotropic activity in frogs composed of the alpha subunit of bovine luteinizing hormone and a subunit of a gonadotropin of a teleost fish]

Fontaine MM
C R Acad Sci Hebd Seances Acad Sci D. 1976; 282: 97-100

The alpha subunit of bovine LH and the subunit SU II of carp gonadotropin are able to associate. The hybrid is about one hundred times less active than c-GTH or b-LH, respectively on goldfish and rat ovarian adenyl cyclase. However it is more efficient than both horomones on an Amphibian (Rana esculenta), on spermiation in vivo as well as on ovarian adenyl cyclase in vitro.

MDL 12330A inhibits the non-neuronal adenylyl cyclase from the freshwater snail Planorbarius corneus, but the neuronal enzyme is activated by this compound.

Ferretti ME, Sonetti D, Pareschi MC, Buzzi M, Colamussi ML, Biondi C
Neurosci Lett. 1996; 207: 191-4

N-(Cis-2-phenyl-cyclopentyl)azacyclotridecan-2-imine-hydrochloride (MDL 12330A), considered an inhibitor of adenylyl cyclase, has been tested on the enzyme activity of neuronal and non-neuronal tissues from the freshwater snail Planorbarius corneus. The drug dose-dependently activates the basal as well as agonist-stimulated adenylyl cyclase in the ganglionic preparations, while it exerts an inhibitory effect on the enzyme present in the non-nervous tissues examined. SQ 22536 and forskolin, respectively an inhibitor and activator of adenylyl cyclase, behave as generally reported both in central and peripheral tissues of the snail. This is, to our knowledge, the first report of a stimulatory action of MDL 12330A on an adenylyl cyclase system.

Molecular cloning and characterization of a Ca2+/calmodulin-insensitive adenylyl cyclase from rat brain.

Feinstein PG, Schrader KA, Bakalyar HA, Tang WJ, Krupinski J, Gilman AG, Reed RR
Proc Natl Acad Sci U S A. 1991; 88: 10173-7

Biochemical, immunological, and molecular cloning studies have suggested the existence of multiple forms of adenylyl cyclase (EC 4.6.1.1). An adenylyl cyclase cDNA clone (type II) was isolated from a rat brain library and found to encode a protein of 1090 amino acids that was homologous to but distinct from the previously described Ca2+/calmodulin-stimulated adenylyl cyclase from bovine brain. Expression of the type II cDNA in an insect cell line resulted in an increased level of adenylyl cyclase activity that was insensitive to Ca2+/calmodulin. Addition of activated Gs alpha protein to type II-containing membranes increased enzyme activity. The mRNA encoding the type II protein was expressed at high levels in brain tissue and at low levels in olfactory epithelium and lung. The existence of multiple adenylyl cyclase enzymes may provide for complex and distinct modes of biochemical regulation of cAMP levels in the brain.

Activity trends of adenylate and guanylate cyclases and cAMP and cGMP levels during the first embryonic stages of Bufo bufo.

Farnesi RM, Secca T, Tei S, Vagnetti D, Santarella B, Roscani C
Comp Biochem Physiol Comp Physiol. 1993; 105: 319-22

1. During the first stages of embryonic development of Bufo bufo, the levels of cAMP and cGMP showed interesting opposite trends analogous to the trends of their respective enzymes. 2. In the late segmentation, the high increase of guanylate cyclase specific activity and the corresponding rise of embryonic level of cGMP, could indicate the involvement of this nucleotide in cellular proliferation. 3. During the following stage of the gastrulation, the increase of adenylate cyclase specific activity, coupled to the loss of guanylate cyclase specific activity, could suggest the importance of cAMP in the phenomena of differentiation induction. 4. Furthermore, the cytochemical investigation of adenylate and guanylate cyclase localization seems to confirm the prominent role of cAMP during the differentiation phases.

Regulation of adenylate cyclase by adenosine.

Fain JN, Malbon CC
Mol Cell Biochem. 1979; 25: 143-69

Synthesis and biological activities of arginine-vasopressin analogues with reactive groups.

Fahrenholz F, Thierauch KH, Crause P
Hoppe Seylers Z Physiol Chem. 1980; 361: 153-67

The synthesis and biological activities of arginine-vasopressin analogues are described, where p-azido-L-phenylalanine [Phe(pN3)] or p-(bromoacetylamino)-L-phenylalanine [Phe-(pNHCOCH2Br)] replace Tyr2 or Phe3. The hormone analogues are prepared via precursors containing p-aminophenylalanine [Phe(pNH2)] in position 2 or 3. During peptide synthesis the p-amino group of [Phe(pNH2)] is protected by the tert-butyloxycarbonyl or the benzyloxycarbonyl group, the side chains of cysteine and arginine by the acetamidomethyl residue and the tosyl group, respectively. The amino and guanidino protecting groups are removed from the nonapeptides by trifluoromethanesulfonic acid yielding the S-protected derivatives which are cyclized by means of iodine. The ring closure by disulfide formation is confirmed by Edman degradation, CD and 1H-NMR spectroscopy. Modification at the p- and alpha-amino groups result in [Phe(pN3)2]-vasopressin, [Phe(pNHCOCH2Br)2]vasopressin, Nalpha-dansyl-[Phe(pN3)2]vasopressin, [Phe2,Phe-(pN3)3]vasopressin and [Phe2,Phe(pNHCOCH2-Br)3]vasopressin. The analogues modified only in position 2, [Phe(pN3)2]vasopressin stimulate the adenylate cyclase derived from bovine kidney inner medulla to similar maximal velocities as arginine vasopressin and show high apparent affinities for enzyme activation. The Nalpha-dansyl derivative and the analogues with reactive groups in position 3 have reduced maximal velocities and apparent affinities for vasopressin-sensitive adenylate cyclase. These results suggest that especially the derivatives with reactive groups in position 2 are useful for the labelling of vasopressin receptors in plasma membranes and for studies of covalent hormone-receptor complexes.

Adenylyl cyclase messenger ribonucleic acid in myometrium: splice variant of type IV.

Emala CW, Kumasaka D, Hirshman CA, Lindeman KS
Biol Reprod. 1998; 59: 169-75

Regulation of uterine tone during pregnancy and parturition involves G protein-coupled receptors that modulate cellular levels of cAMP. Cyclic AMP is synthesized by a family of at least nine adenylyl cyclase enzymes that have unique tissue distributions, basal activities, and modes of regulation. Little is known regarding the expression of adenylyl cyclase isoforms in myometrium. Reverse transcription-polymerase chain reaction (PCR) was performed using RNA extracted from freshly dissected rat uteri and a cell line of homogeneous rat myometrial cells. Using PCR primers specific for adenylyl cyclases I-IX, mRNA encoding adenylyl cyclases II-IX were identified in both fresh and cultured myometrial cells. A splice variant of adenylyl cyclase type IV was also identified in RNA from both sources. These data show that multiple isoforms of adenylyl cyclases are expressed at the mRNA level in myometrial cells and suggest that the regulation of cellular levels of cAMP likely involves a complex integration of cellular second messengers acting upon multiple isoforms of adenylyl cyclases.

Assay of adenylyl cyclase activity by quantitative mass spectrometry.

el-Shazley H, Newton RP, Harris FM, Brenton AG, Walton TJ, Restall CJ, Bayliss M, Wilkins A, Games DE, Langridge JI
Biochem Soc Trans. 1996; 24: 474-474

Localization of soluble guanylyl cyclase alpha-subunit in identified insect neurons.

Elphick MR, Jones IW
Brain Res. 1998; 800: 174-9

The distribution of soluble guanylyl cyclase in the brain of the locust Schistocerca gregaria was analysed using antisera to a highly conserved region (X-peptide) of the Drosophila soluble guanylyl cyclase alpha-subunit (SGCalpha). Analysis of locust brain and locust eye homogenates in Western blots using X-peptide antisera revealed specific staining of a approximately 65 kDa band, which is similar to the expected molecular mass for a SGCalpha-subunit. SGCalpha-immunoreactivity was localized in identified neuronal components of several sensory systems including photoreceptors of the compound eyes and ocelli, large ocellar interneurons, antennal mechanosensory neurons and olfactory interneurons. These neurons are putative targets for the gas nitric oxide which activates guanylyl cyclase activity in the locust brain.

Nucleoside phosphorothioates.

Eckstein F
Annu Rev Biochem. 1985; 54: 367-402

Isoform-specific regulation of adenylyl cyclase by oxidized catecholamines.

Ebina T, Toya Y, Oka N, Schwencke C, Kawabe J, Ishikawa Y
J Mol Cell Cardiol. 1997; 29: 1247-54

Both epinephrine and manganese are known to stimulate cAMP production in cardiac homogenates. When added together, however, they inhibited adenylyl cyclase catalytic activity. Type V adenylyl cyclase, the major isoform in the heart, was also inhibited when an increasing concentration of epinephrine was added in the presence of manganese. Inhibition was not dependent on the condition of stimulation or preparation of the enzyme. However, this inhibition was abolished in the presence of anti-oxidant. Other catecholamines, including dopamine and isoproterenol, as well as adrenochrome, an oxidized product of epinephrine, similarly inhibited the activity of this enzyme. Kinetic analyses revealed that the K(m) for the substrate ATP was unchanged, but the V(max) was significantly decreased. In contrast, type II adenylyl cyclase, a non-cardiac isoform, was resistant to such inhibition by adrenochrome and was somewhat stimulated by it. Thus, catecholamines, when oxidized, directly interacted with adenylyl cyclase in an isoform-specific manner in the absence of G proteins. Our findings suggest that adenylyl cyclase isoforms have different sensitivity to various stresses, including oxidative stress.

Isoform-dependent activation of adenylyl cyclase by proteolysis.

Ebina T, Toya Y, Oka N, Kawabe J, Schwencke C, Ishikawa Y
FEBS Lett. 1997; 401: 223-6

Recent findings have suggested that the cellular proteolytic system plays a major role in the regulation of various intra- and extra-cellular signaling. It was previously shown that proteolytic treatment of adenylyl cyclase leads to the activation of this enzyme. We demonstrate that this activation occurs in an adenylyl cyclase isoform-dependent manner. The type II isoform was strongly activated (approximately 500%), the type III isoform was modestly activated (approximately 30%),and the type V isoform was inhibited by trypsin. Activation of type II adenylyl cyclase occurred in trypsin dose- and time-dependent manners and was blocked by a trypsin inhibitor in a dose-dependent manner. Other proteases, such as thrombin and plasminogen, similarly activated the type II isoform, but not the others. Our data suggest that proteolytic activation is an isoform- and thus cell type-dependent mechanism of altering adenylyl cyclase catalytic activity.

Regulation of adenylyl cyclase isoforms by N-alkanols.

Ebina T, Toya Y, Kawabe J, Ishikawa Y
J Cell Biochem. 1997; 66: 450-6

We examined the effect of n-alkanols on adenylyl cyclase isoforms (types II and V) overexpressed in insect cells. Ethanol stimulated the type II isoform but not the type V isoform. Ethanol stimulated type II adenylyl cyclase greater than GTP gamma S, and the treatment of the membrane with GDP beta S or cholera toxin did not affect this stimulation. Other n-alkanols inhibited type V adenylyl cyclase activity in proportion to their lipophilic potency. In contrast, type II adenylyl cyclase was stimulated by weakly lipophilic n-alkanols and inhibited by strongly lipophilic n-alkanols. When solubilized membranes and purified preparations were used, all the n-alkanols inhibited type II adenylyl cyclase. Our data suggest that n-alkanols regulated adenylyl cyclase isoform-dependently. Stimulation of the type II isoform was independent from the interaction with Gs alpha but required the presence of an intact membrane structure. Our study may provide another step to understanding how membrane protein subtypes are differentially regulated by n-alkanols.

Conformation-dependent activation of type II adenylyl cyclase by protein kinase C.

Ebina T, Kawabe J, Katada T, Ohno S, Homcy CJ, Ishikawa Y
J Cell Biochem. 1997; 64: 492-8

Phorbol ester treatment enhanced the catalytic activity of type II adenylyl cyclase overexpressed in insect cells. In cells coexpressing type II adenylyl cyclase and protein kinase C-alpha, type II adenylyl cyclase catalytic activity was higher even in the absence of phorbol ester treatment; phorbol ester treatment further and markedly enhanced type II adenylyl cyclase catalytic activity. However, this enhancement, either by phorbol ester treatment or by coexpression of protein kinase C-alpha, was lost following membrane solubilization with detergents. This attenuation was unaffected by phosphatase inhibitor or salts. In contrast, membrane solubilization did not affect forskolin-stimulated type II adenylyl cyclase catalytic activity. Purified type II adenylyl cyclase was stimulated by forskolin and Gs alpha, but not by protein kinase C-alpha. Therefore, a specific mammalian protein kinase C isoenzyme can activate type II adenylyl cyclase, but the mechanism clearly differs from that underlying either Gs alpha- or forskolin-mediated stimulation.

Gs alpha meets its target--shedding light on a key signal transduction event.

Dumas JJ, Lambright DG
Structure. 1998; 6: 407-11

The recently determined crystal structure of Gs alpha bound to a catalytically active form of adenylyl cyclase reveals the location of the enzyme's active site and provides the first view of heterotrimeric G protein alpha subunit activating a downstream effector. Comparison with the structure of a catalytically inactive form of adenylyl cyclase suggests a plausible allosteric mechanism whereby the synergistic activators Gs alpha and forskolin stimulate the activity of adenylyl cyclase.

Oxidation of critical cysteine residues of type I adenylyl cyclase by o-iodosobenzoate or nitric oxide reversibly inhibits stimulation by calcium and calmodulin.

Duhe RJ, Nielsen MD, Dittman AH, Villacres EC, Choi EJ, Storm DR
J Biol Chem. 1994; 269: 7290-6

The calmodulin binding domain of the type I adenylyl cyclase has recently been identified as an amino acid sequence (residues 495-522) that contains 2 cysteine residues. Therefore, we examined the effect of several sulfhydryl reagents on the calmodulin sensitivity of the enzyme. Treatment of membranes containing the type I adenylyl cyclase with N-ethylmaleimide rapidly inhibited basal, calcium/calmodulin-stimulated, and forskolin-stimulated adenylyl cyclase activity. When the enzyme was treated with limiting amounts of o-iodosobenzoate, which oxidizes vicinal sulfhydryls to disulfides, stimulation by Ca2+ and calmodulin was eliminated at concentrations which did not affect basal adenylyl cyclase activity. Calmodulin stimulation of the enzyme was restored by treatment with dithiothreitol or glutathione which reduce disulfides to free thiols. NO and sodium nitroprusside also reversible inhibited calmodulin stimulation of the enzyme. We propose that the loss in calmodulin sensitivity caused by these reagents may be due to the oxidation one or more sets of vicinal thiols present in the enzyme.

An extended conformation of calmodulin induces interactions between the structural domains of adenylyl cyclase from Bacillus anthracis to promote catalysis.

Drum CL, Yan SZ, Sarac R, Mabuchi Y, Beckingham K, Bohm A, Grabarek Z, Tang WJ
J Biol Chem. 2000; 275: 36334-40

The edema factor exotoxin produced by Bacillus anthracis is an adenylyl cyclase that is activated by calmodulin (CaM) at resting state calcium concentrations in infected cells. A C-terminal 60-kDa fragment corresponding to the catalytic domain of edema factor (EF3) was cloned, overexpressed in Escherichia coli, and purified. The N-terminal 43-kDa domain (EF3-N) of EF3, the sole domain of edema factor homologous to adenylyl cyclases from Bordetella pertussis and Pseudomonas aeruginosa, is highly resistant to protease digestion. The C-terminal 160-amino acid domain (EF3-C) of EF3 is sensitive to proteolysis in the absence of CaM. The addition of CaM protects EF3-C from being digested by proteases. EF3-N and EF3-C were expressed separately, and both fragments were required to reconstitute full CaM-sensitive enzyme activity. Fluorescence resonance energy transfer experiments using a double-labeled CaM molecule were performed and indicated that CaM adopts an extended conformation upon binding to EF3. This contrasts sharply with the compact conformation adopted by CaM upon binding myosin light chain kinase and CaM-dependent protein kinase type II. Mutations in each of the four calcium binding sites of CaM were examined for their effect on EF3 activation. Sites 3 and 4 were found critical for the activation, and neither the N- nor the C-terminal domain of CaM alone was capable of activating EF3. A genetic screen probing loss-of-function mutations of EF3 and site-directed mutations based on the homology of the edema factor family revealed a conserved pair of aspartate residues and an arginine that are important for catalysis. Similar residues are essential for di-metal-mediated catalysis in mammalian adenylyl cyclases and a family of DNA polymerases and nucleotidyltransferases. This suggests that edema factor may utilize a similar catalytic mechanism.

Localisation of an ATP-binding site on adenylyl cyclase type I after chemical and enzymatic fragmentation.

Droste M, Mollner S, Pfeuffer T
FEBS Lett. 1996; 391: 209-14

Photolabeling of partially purified bovine brain adenylyl cyclase (AC I) with [gamma 32P]8-N3-ATP led to incorporation of 32P into the 115 kDa catalyst. Further treatment with N-chlorosuccinimide, which cleaves proteins at tryptophan residues, yielded a 14 kDa 32P-labeled fragment. The latter was immunoprecipitated by antibody BBC1, recognizing the extreme C-terminus of AC I, but not by antibody BBC2, recognizing a more remote epitope. Further fragmentation of photolabeled AC I by the proteases Glu-C and Asp-N yielded 32P-labeled peptides corresponding to 2.9 kDa and 5.6 kDa fragments, which were not recognized by any of these antibodies. This narrows the ATP binding site down to a 25 amino acid sequence containing a general motif G(X0-7)KG(X0-4)L/M(X5-7)S/T present in all eukaryotic adenylyl cyclases so far cloned, but also in a variety of bacterial adenylyl cyclases (Peterkofsky et al. (1993) Progr. Nucleic Acids Res. Mol. Biol. 44, 31-65).

The family of guanylyl cyclase receptors and their ligands.

Drewett JG, Garbers DL
Endocr Rev. 1994; 15: 135-62

Direct photoaffinity labeling of individual cytosolic domains of adenylyl cyclase by [32P]2'-deoxy-3'-AMP and [alpha-32P]5'-ATP.

Doronin S, Dessauer C, Johnson RA
J Biol Chem. 1998; 273: 32416-20

The susceptibility of purines to form a covalent attachment with proteins upon exposure to UV irradiation was applied to adenylyl cyclase by use of [32P]2'-d-3'-AMP, a dead-end inhibitor that binds to the post-transition configuration of the enzyme. [32P]2'-d-3'-AMP was synthesized enzymatically. It and [alpha-32P]5'-ATP were used for direct photocross-linking to individually expressed cytosolic domains of adenylyl cyclase. Both the C1 domain of the type V isozyme (VC1) and the C2 domain of the type II isozyme (IIC2) were labeled, whether alone or combined, upon photolysis of [32P]2'-d-3'-AMP in the presence of acetone. Labeling of VC1 and IIC2 was greatly enhanced in the presence of PPi, was almost completely suppressed by 50 microM 2',5'-dideoxy-3'-ATP, the most potent reported P-site inhibitor of adenylyl cyclases, but was partially suppressed by 1 mM 3'-IMP, a ligand that does not inhibit the enzyme via the P-site. Neither 3':5'-cAMP nor 5'-ATP had a major effect on labeling by [32P]2'-d-3'-AMP. Direct cross-linking of VC1 with [alpha-32P]5'-ATP was substantially suppressed by 2', 5'-dideoxy-3'-ATP and partially suppressed by 2'-d-3'-AMP, whereas cross-linking of IIC2 was less affected by the 3'-triphosphate. The data imply that either cytosolic domain can interact directly with either substrate or P-site ligand and that subunit interaction modifies the susceptibility of each domain to UV-induced covalent modification by either [alpha-32P]5'-ATP or [32P]2'-d-3'-AMP.

Effects of adenylate cyclase activation on electrical resistance of scala media.

Doi K, Mori N, Matsunaga T
Acta Otolaryngol Suppl. 1993; 501: 76-9

Electrical resistance of scala media at the second turn of the guinea pig cochlea was measured while cochlear adenylate cyclase was activated by perilymphatic perfusion with 2 x 10(-4) M forskolin. The electrical resistance decreased from the pre-forskolin value (6.36 +/- 0.27 k omega to 5.22 +/- 0.33 k omega, n = 5), while the endocochlear potential (EP) increased from the pre-forskolin value (70.2 +/- 2.1 mV to 81.2 +/- 2.8 mV, n = 5). The time course of the electrical resistance change was more rapid than that of the EP change but almost identical to the time course of the change in the Cl- activity of the endolymph. The results suggest that the forskolin-induced decrease in the electrical resistance of the perilymph-endolymph barrier (presumably Reissner's membrane), and the consequent increase in Cl- flux into the endolymph may trigger EP elevation.

Regulation of photoreceptor membrane guanylyl cyclases by guanylyl cyclase activator proteins.

Dizhoor AM, Hurley JB
Methods. 1999; 19: 521-31

Guanylyl cyclase (GC) plays a central role in the responses of vertebrate rod and cone photoreceptors to light. cGMP is an internal messenger molecule of vertebrate phototransduction. Light stimulates hydrolysis of cGMP, causing the closure of cGMP-dependent cation channels in the plasma membranes of photoreceptor outer segments. Light also lowers the concentration of intracellular free Ca(2+) and by doing so it stimulates resynthesis of cGMP by guanylyl cyclase. The guanylyl cyclases that couple Ca(2+) to cGMP synthesis in photoreceptors are members of a family of transmembrane guanylyl cyclases that includes atrial natriuretic peptide receptors and the heat-stable enterotoxin receptor. The photoreceptor membrane guanylyl cyclases, RetGC-1 and RetGC-2 (also referred to as GC-E and GC-F), are regulated intracellularly by two Ca(2+)-binding proteins, GCAP-1 and GCAP-2. GCAPs bind Ca(2+) at three functional EF-hand structures. Several lines of biochemical evidence suggest that guanylyl cyclase activator proteins (GCAPs) bind constitutively to an intracellular domain of RetGCs. In the absence of Ca(2+) GCAP stimulates and in the presence of Ca(2+) it inhibits cyclase activity. Proper functioning of RetGC and GCAP is necessary not only for normal photoresponses but also for photoreceptor viability since mutations in RetGC and in GCAP cause photoreceptor degeneration.

Inactivation of EF-hands makes GCAP-2 (p24) a constitutive activator of photoreceptor guanylyl cyclase by preventing a Ca2+-induced "activator-to-inhibitor" transition.

Dizhoor AM, Hurley JB
J Biol Chem. 1996; 271: 19346-50

Guanylyl cyclase activator proteins GCAP-1 and GCAP-2 (Dizhoor et al. , 1995, Gorczyca et al., 1995) are members of a recently identified subclass of EF-hand type Ca2+-binding proteins that respond to Ca2+ differently than any other known members of the EF-hand superfamily. GCAPs acquire an activating conformation only in their Ca2+-free form. Free Ca2+ concentrations corresponding to levels in dark-adapted vertebrate photoreceptors inhibit the ability of GCAPs to activate photoreceptor guanylyl cyclases (RetGCs). We studied the effects of mutations that block binding of Ca2+ to the EF-hands of GCAP-2. Unlike other EF-hand proteins, which fail to activate their target when their EF-hands are inactivated by mutations, GCAP-2 with any single EF-hand inactivated remains active and is 3-6 times less sensitive to the inhibitory effect of Ca2+. Inactivation of any two or all three EF-hands produces active forms of GCAP-2 that are insensitive to inhibition by physiological intracellular concentrations of Ca2+. Unexpectedly we also found that activation of RetGCs by a Ca2+-insensitive mutant is inhibited by Ca2+-loaded wild type GCAP-2. We propose the following. 1) GCAP-2 can exist in two extreme functional forms: an apo form that activates RetGCs and a Ca2+-loaded form that blocks activation of RetGCs. 2) All three EF-hands of GCAP-2 contribute to the inhibitory effect of Ca2+. 3) Inactivation of two or three EF-hands is sufficient to shift the "activator-inhibitor" transition outside the physiological range of intracellular free Ca2+.

Sensitization of olfactory guanylyl cyclase to a specific imprinted odorant in coho salmon.

Dittman AH, Quinn TP, Nevitt GA, Hacker B, Storm DR
Neuron. 1997; 19: 381-9

The role of cGMP in olfactory signaling is not fully understood, but it is believed to play a modulatory role in intracellular signaling in vertebrate olfactory receptor neurons (ORNs). Here, we present evidence that cGMP in ORNs may play an important role in recognition of biologically relevant odors and olfactory learning. Specifically, we investigated the cellular mechanisms underlying olfactory imprinting in salmon. Salmon learn odors associated with their natal site as juveniles and later use these odors to guide their homing migration. This imprinting is believed to involve sensitization of the peripheral olfactory system to specific homestream odorants. We imprinted juvenile salmon to the odorant beta-phenylethyl alcohol (PEA) and examined the sensitivity of olfactory adenylyl and guanylyl cyclases to PEA during development. Stimulation of guanylyl cyclase activity by PEA was significantly greater in olfactory cilia isolated from PEA-imprinted salmon compared with PEA-naive fish only at the time of the homing migration, 2 years after PEA exposure. These results suggest that sensitization of olfactory guanylyl cyclase may play an important role in olfactory imprinting by salmon.

Mutations of aromatic residues in the first transmembrane helix impair signalling by the secretin receptor.

Di Paolo E, Petry H, Moguilevsky N, Bollen A, De Neef P, Waelbroeck M, Robberecht P
Receptors Channels. 1999; 6: 309-15

The secretin amino terminal residues are essential for high affinity binding to the cognate receptor and for the subsequent activation of adenylate cyclase. It has been already established that two basic residues of the receptor TM 2 are involved in the interaction with aspartate 3 of the ligand. The present work investigated the hypothesis that two conserved tyrosine residues of the TM 1 (Tyrosines 124 and 128) could also participate to the positioning of the amino terminus of the ligand. Tyrosines 124 and 128 were mutated into alanine and histidine residues, and the properties of the mutant receptors, expressed in CHO cells, were compared with those of the wild-type receptor. Mutation of tyrosine 124 to Ala or His decreased the affinity of the receptor for secretin, [Glu3]secretin, [Asn3]secretin and the secretin fragment 2-27, and reduced the intrinsic activity of [Asn3]secretin. Mutation of tyrosine 128 to Ala, but not to His reduced 50-fold secretin and [Asn3]secretin affinity but only 3-fold that of [Glu3]secretin. Secretin and [Glu3]Sn were equipotent in that mutant receptor. These results suggested that tyrosine 128 of the secretin receptor interacted directly with the [Asp3] residue of secretin and thus that the amino terminal domain of secretin interacts with amino acids buried in both the TM 1 and TM 2 helices.

Characterization and localization of adenylate and guanylate cyclases.

De Vries GW, Ferrendelli JA
Methods Enzymol. 1982; 81: 517-22

Identification of a Gialpha binding site on type V adenylyl cyclase.

Dessauer CW, Tesmer JJ, Sprang SR, Gilman AG
J Biol Chem. 1998; 273: 25831-9

The stimulatory G protein alpha subunit Gsalpha binds within a cleft in adenylyl cyclase formed by the alpha1-alpha2 and alpha3-beta4 loops of the C2 domain. The pseudosymmetry of the C1 and C2 domains of adenylyl cyclase suggests that the homologous inhibitory alpha subunit Gialpha could bind to the analogous cleft within C1. We demonstrate that myristoylated guanosine 5'-3-O-(thio)triphosphate-Gialpha1 forms a stable complex with the C1 (but not the C2) domain of type V adenylyl cyclase. Mutagenesis of the membrane-bound enzyme identified residues whose alteration either increased or substantially decreased the IC50 for inhibition by Gialpha1. These mutations suggest binding of Gialpha within the cleft formed by the alpha2 and alpha3 helices of C1, analogous to the Gsalpha binding site in C2. Adenylyl cyclase activity reconstituted by mixture of the C1 and C2 domains of type V adenylyl cyclase was also inhibited by Gialpha. The C1b domain of the type V enzyme contributed to affinity for Gialpha, but the source of C2 had little effect. Mutations in this soluble system faithfully reflected the phenotypes observed with the membrane-bound enzyme. The pseudosymmetrical structure of adenylyl cyclase permits bidirectional regulation of activity by homologous G protein alpha subunits.

The interactions of adenylate cyclases with P-site inhibitors.

Dessauer CW, Tesmer JJ, Sprang SR, Gilman AG
Trends Pharmacol Sci. 1999; 20: 205-10

Recent kinetic, binding and crystallographic studies using P-site inhibitors of mammalian adenylate bases provide new insights into the catalytic mechanism of these highly regulated enzymes. Here, Carmen Dessauer and colleagues discuss the conformational states of adenylate cyclase, the structural determinants of inhibitor binding and the potential uses of these inhibitors as pharmacological agents.

Interactions of forskolin and ATP with the cytosolic domains of mammalian adenylyl cyclase.

Dessauer CW, Scully TT, Gilman AG
J Biol Chem. 1997; 272: 22272-7

Fragments of the two cytoplasmic domains of mammalian adenylyl cyclases can be synthesized independently (and abundantly) as soluble proteins; Gsalpha- and forskolin-stimulated enzymatic activity is restored upon their mixture. We have utilized this system to characterize the interactions of adenylyl cyclase with forskolin and its substrate, ATP. In the presence of Gsalpha, adenylyl cyclase is activated in response to occupation of only one forskolin-binding site. A single binding site for forskolin was identified by equilibrium dialysis; its Kd (0.1 microM) corresponds to the EC50 for enzyme activation. The affinity of forskolin for adenylyl cyclase is greatly reduced in the absence of Gsalpha ( approximately 40 microM). Binding of forskolin to the individual cytoplasmic domains of the enzyme was not detected. A single binding site for the ATP analog, alpha,beta-methylene ATP (Ap(CH2)pp), was also detected by equilibrium dialysis. Such binding was not observed with the individual domains. Binding of Ap(CH2)pp was unaffected by P-site inhibitors of adenylyl cyclase. A modified P-loop sequence located near the carboxyl terminus of adenylyl cyclase has been implicated in ATP binding. Mutation of the conserved, non-glycine residues within this region caused no significant changes in the Km for ATP or the Ki for Ap(CH2)pp. It thus seems unlikely that this region is part of the active site. However, a mutation in the C1 domain (E518A) causes a 10-fold decrease in the binding affinity for Ap(CH2)pp. This residue and the active site of the enzyme may lie at the interface between the two cytosolic domains.

Visualizing signal transduction: receptors, G-proteins, and adenylate cyclases.

Dessauer CW, Posner BA, Gilman AG
Clin Sci (Colch). 1996; 91: 527-37

1. The first glimpses of heterotrimeric G-proteins came with the discoveries of the ubiquitous adenylate cyclase activator, Gs, and the specialized retinal cGMP phosphodiesterase activator, Gi or transducin. The model that evolved for regulation of adenylate cyclase activity by G-proteins soon proved to be a general paradigm for a large number of signalling pathways. Although many different G-proteins interact with a diverse array of receptors and effectors, each is composed of a guaninenucleotide-binding alpha-subunit and a tightly associated complex of a beta- and a gamma-subunit. 2. Receptors catalyse the activation of G-proteins by promoting exchange of GDP for GTP, while G-proteins catalyse their own deactivation as a result of their intrinsic GTPase activity. Crystallographic analysis has described several of the various conformational states that G-proteins undergo as they are activated and deactivated and has provided great insight into the kinetic models of G-protein-mediated signal transduction. 3. The regulation of adenylate cyclase has proven to be intriguing and complex. Gsx activates all forms of mammalian adenylate cyclase; other G-proteins (Gi, Go and Gz) inhibit certain isoforms of the enzyme. The discovery of new isoforms of adenylate cyclase has revealed synergistic and conditional mechanisms of regulation. These include activation or inhibition by the G-protein beta gamma-subunit complex, activation by Ca(2+)-calmodulin, and phosphorylation by protein kinases. The large number of receptors, G-proteins and adenylate cyclases provides a complex signalling network that integrates and interprets a multitude of convergent inputs.

Purification and characterization of a soluble form of mammalian adenylyl cyclase.

Dessauer CW, Gilman AG
J Biol Chem. 1996; 271: 16967-74

An engineered, soluble form of mammalian adenylyl cyclase has been expressed in Escherichia coli and purified by three chromatographic steps. The enzyme utilizes one molecule of ATP to synthesize one molecule of cyclic AMP and pyrophosphate at a maximal specific activity of 12.8 micromol/min/mg, corresponding to a turnover number of 720 min-1. Although devoid of membrane spans, the enzyme displays all of the regulatory properties that are common to mammalian adenylyl cyclases. It is activated synergistically by Gsalpha and forskolin and is inhibited by adenosine (P-site) analogs with kinetic patterns that are identical to those displayed by the native enzymes. The purified enzyme is also inhibited directly by the G protein betagamma subunit complex. After adenovirus-mediated expression in adenylyl cyclase-deficient HC-1 cells, the enzyme can be stimulated synergistically by Gs-coupled receptors and forskolin.

The catalytic mechanism of mammalian adenylyl cyclase. Equilibrium binding and kinetic analysis of P-site inhibition.

Dessauer CW, Gilman AG
J Biol Chem. 1997; 272: 27787-95

The mechanism of P-site inhibition of adenylyl cyclase has been probed by equilibrium binding measurements using 2'-[3H]deoxyadenosine, a P-site inhibitor, and by kinetic analysis of both the forward and reverse reactions (i.e. cyclic AMP and ATP synthesis, respectively). There is one binding site for 2'-deoxyadenosine per C1/C2 heterodimer; the Kd is 40 +/- 3 microM. Binding is observed only in the presence of one of the products of the adenylyl cyclase reaction, pyrophosphate (PPi). A substrate analog, Ap(CH2)pp (alpha,beta-methylene adenosine 5'-triphosphate), and cyclic AMP compete for the P-site in the presence of PPi, but P-site analogs do not compete for substrate binding (in the absence of PPi). Kinetic analysis indicates that release of products from the enzyme is random. These facts permit formulation of a model for the adenylyl cyclase reaction, for which we provide substantial kinetic support. We propose that P-site analogs act as dead-end inhibitors of product release, stabilizing an enzyme-product (E-PPi) complex by binding at the active site. Although product release is random, cyclic AMP dissociates from the enzyme preferentially. Release of PPi is slow and partially rate-limiting.

Cytoplasmic domains mediate the ligand-induced affinity shift of guanylyl cyclase C.

Deshmane SP, Parkinson SJ, Crupper SS, Robertson DC, Schulz S, Waldman SA
Biochemistry. 1997; 36: 12921-9

Guanylyl cyclase C (GCC), the receptor for the Escherichia coli heat-stable enterotoxin (ST), exhibits multiple binding affinities, including high (RH) and low (RL) affinity sites and a ligand-induced conversion of low-affinity sites from a higher (RL1) to a lower (RL2) affinity state. Occupancy of the lowest affinity state of low-affinity sites is coupled to ligand-induced catalytic activation. In the present studies, ligand binding and catalytic activation properties of a series of intracellular deletion mutants of GCC were examined to identify the structural domains underlying expression of high- and low-affinity sites and the ligand-induced shift in low-affinity sites. These studies demonstrated that the cytoplasmic domains of GCC are not required, but extracellular and transmembrane domains are sufficient, for expression of high-affinity binding sites. In addition, the cytoplasmic juxtamembrane and kinase homology domains are required for expression of the ligand-induced affinity shift in low-affinity sites. Of significance, this shift in affinity was insensitive to adenine nucleotides, in contrast to other members of the receptor guanylyl cyclase family, such as guanylyl cyclase A (GCA). Also, the juxtamembrane and kinase homology domains are critical for coupling ST-receptor binding and guanylyl cyclase catalytic activation. Indeed, deletion of those domains from GCC results in a constitutively inhibited enzyme, suggesting that they function as positive effectors of ligand activation, in contrast to GCA and GCB, in which the kinase homology domain represses basal catalytic activity. These data suggest that the mechanisms regulating different members of the receptor guanylyl cyclase family are overlapping but not identical.

2',5'-Dideoxyadenosine 3'-polyphosphates are potent inhibitors of adenylyl cyclases.

Desaubry L, Shoshani I, Johnson RA
J Biol Chem. 1996; 271: 2380-2

2',5'-Dideoxyadenosine 3'-di- and triphosphates were tested as inhibitors of brain adenylyl cyclases. With an IC50 approximately 40 nM, 2',5'-dideoxy-3'-ATP is the most potent nonprotein synthetic regulator of adenylyl cyclases thus far described. Neither 2',5'-dideoxy-3'-ADP nor 2',5'-dideoxy-3'-ATP inhibited activity by competition with substrate, and the linear noncompetitive inhibition observed was consistent with interaction via a distinct domain. The availability of this ligand will permit the development of a variety of probes that will be extremely useful in investigating adenylyl cyclase structure and the role(s) that this class of compound may play in physiologically regulating cell function.

Molecular cloning of the human type VIII adenylyl cyclase.

Defer N, Marinx O, Stengel D, Danisova A, Iourgenko V, Matsuoka I, Caput D, Hanoune J
FEBS Lett. 1994; 351: 109-13

A cDNA coding for a human type VIII adenylyl cyclase has been isolated from human newborn brain-stem tissue. This cDNA is 6,005 bp long and encodes for a protein of 1251 amino acids, exhibiting the two sets of six transmembrane spanning regions and the hydrophobicity profile typical of other mammalian adenylyl cyclases. Comparison with the rat form shows that they share 97% identity in amino acids. Type VIII adenylyl cyclase is unique in that it has both a long carboxy terminal and a long amino terminal tail. This is the first report on a complete cDNA clone coding for a human adenylyl cyclase. The distribution and regulation of this particular adenylyl cyclase suggest that it may be involved in learning, in memory and in drug dependence.

The olfactory adenylyl cyclase type 3 is expressed in male germ cells.

Defer N, Marinx O, Poyard M, Lienard MO, Jegou B, Hanoune J
FEBS Lett. 1998; 424: 216-20

Elements of the olfactory pathway, such as receptors, receptor-desensitization machinery, and cyclic nucleotide-gated channels, are expressed in male germ cells. Here we report the expression, in rat testis, of both adenylyl cyclase type 3 (AC3) and the olfactory G protein subunit, G(alpha)olf. Both are expressed in the same sub-population of germ cells, pachytene spermatocytes to spermatids, and in residual bodies. Neither AC3 nor G(alpha)olf was found in Sertoli or in peritubular cells, as shown by Western blotting and immunocytochemical analyses. It thus appears that male germ cells contain all the elements of the signaling cascade present in olfactory cells.

[Molecular organization and the realization of what is possible.]

Debru C
Arch Physiol Biochem. 1996; 104: 775-81

Characterization of soluble guanylyl cyclase in transformed human non-pigmented epithelial cells.

Danziger RS, Star RA, Matsumoto S, Coca-Prados M, DeSantis L, Pang IH
Biochem Biophys Res Commun. 1993; 195: 958-62

Topical application of nitro vasodilators, such as sodium nitroprusside, reduces intraocular pressure. In brain and blood vessels, nitro vasodilators activate soluble guanylyl cyclases, producing cGMP. The location and molecular identity of ocular guanylyl cyclases are unknown. We studied transformed human non-pigmented ciliary epithelial cells, whose parental cells are responsible for the production of aqueous humor. Sodium nitroprusside increased cGMP 40 to 60 fold in a time and concentration dependent manner (EC50 40 to 100 microM). Methylene blue inhibited this effect (IC50 0.6 microM in the presence of 100 microM sodium nitroprusside). We also detected mRNA for the alpha 1 and beta 1, but not the beta 2, subunit isoforms of soluble guanylyl cyclase in these cells by Northern blotting. We conclude that transformed non-pigmented epithelial cells contain an active heterodimeric soluble guanylyl cyclase composed of at least alpha 1 and beta 1 subunits.

The adenylate cyclase catalytic domain of Streptomyces coelicolor is carboxy-terminal.

Danchin A, Pidoux J, Krin E, Thompson CJ, Ullmann A
FEMS Microbiol Lett. 1993; 114: 145-51

A DNA fragment of Streptomyces coelicolor encoding the carboxy-terminal catalytic domain of adenylate cyclase was cloned, sequenced and expressed in an Escherichia coli cya-defective strain where it produced nanomole levels of cAMP. The amino acid sequence of the enzyme displays similarities with the Brevibacterium liquefaciens pyruvate regulated adenylate cyclase.

Protein carboxylmethylase and germ cell adenylyl cyclase at specific stages of the spermatogenic cycle of the rat.

Cusan L, Gordeladze JO, Parvinen M, Clausen OP, Hansson V
Biol Reprod. 1981; 25: 915-9

Bifunctional structure of two adenylyl cyclases from the myxobacterium Stigmatella aurantiaca.

Coudart-Cavalli MP, Sismeiro O, Danchin A
Biochimie. 1997; 79: 757-67

Two adenylyl cyclase genes (cyaA and cyaB) from the myxobacterium Stigmatella aurantiaca were cloned by complementation of Escherichia coli mutants defective in the cya gene. cyaA codes for a protein of 424 amino acid residues (AC1), while cyaB encodes a protein of 352 residues (AC2). Both cyclases are sensitive to adenosine: cAMP production was strongly inhibited in E coli cells and cell extracts expressing these genes. AC1 comprises a hydrophobic domain of six transmembrane helices coupled to a cytoplasmic catalytic domain endowed with adenylyl cyclase activity. A 17 amino acid residue sequence, which is a signature of G-protein coupled receptors, as well as of slime mold Dictyostelium discoideum cyclic AMP receptors, was found in the membrane domain. AC2 displays features also indicating that it is a bifunctional enzyme. The domain located upstream from the catalytic adenylyl cyclase domain shows strong similarity to receiver modules of response regulators of two-component bacterial signaling systems. In vitro mutagenesis of conserved aspartate residues in this domain was shown to interfere with cAMP synthesis.

Adenylyl cyclases and the interaction between calcium and cAMP signalling.

Cooper DM, Mons N, Karpen JW
Nature. 1995; 374: 421-4

Adenylyl cyclase is the prototypical second messenger generator. Nearly all of the eight cloned adenylyl cyclases are regulated by one or other arm of the phospholipase C pathway. Functional and ultrastructural investigations have shown that adenylyl cyclases are intimately associated with sites of calcium ion entry into the cell. Oscillations in cellular cyclic AMP levels are predicted to arise because of feedback inhibition of adenylyl cyclase by Ca2+. Such findings inextricably intertwine cellular signalling by cAMP and internal Ca2+ and extend the known regulatory modes available to cAMP.

Ca(2+)-sensitive adenylyl cyclases.

Cooper DM, Karpen JW, Fagan KA, Mons NE
Adv Second Messenger Phosphoprotein Res. 1998; 32: 23-51

Ca(2+)-inhibited adenylyl cyclase in cardiac tissue.

Cooper DM, Brooker G
Trends Pharmacol Sci. 1993; 14: 34-6

Studies of the thermal inactivation of cardiac adenylyl cyclase: evidence for a conformational change in the reaction mechanism.

Colvin RA, Allen RA
Arch Biochem Biophys. 1991; 289: 337-42

Membrane bound cardiac adenylyl cyclase was shown to undergo a spontaneous and irreversible thermal inactivation with a t1/2 of approximately 10 min. The loss of activity could not be explained by the action of endogenous proteases. Repeated freeze-thaw of membrane preparations resulted in a much increased rate of thermal inactivation (t1/2 = approx. 2 min). ATP, adenylimidodiphosphate, ADP, and PPi protected the enzyme from thermal inactivation with dissociation constants (Kd) of 193, 5.04, 84.4, and 6.3 microM, respectively. 5'-AMP and cyclic AMP were ineffective as protectors at concentrations as high as 3 mM. Activators of adenylyl cyclase such as Mn2+, forskolin, 5-guanylylimidodiphosphate, and NaF and 9 mM Mg2+ protected against thermal inactivation with Kd of 16.8 microM, 8.81 microM, 0.23 microM and 1.04 mM, respectively. Mg2+ alone was without effect. Thermal inactivation was first order under all conditions tested. Arrhenius plots of the rate constants for inactivation vs temperature were linear. The increased stability of ligand bound adenylyl cyclase was shown to be associated with an increased free energy of activation (delta G 0). These data provide evidence for the existence of two distinct conformations of cardiac adenylyl cyclase based on different susceptibilities to thermal inactivation. These enzyme conformations, termed E1 and E2, may be important reaction intermediates. The thermal stability of E1 was highly influenced by the enzyme's membrane lipid environment. The formation of E2 from E1 was enhanced by interaction with substrate, PPi, activators of adenylyl cyclase, and by interaction with dissociated stimulatory guanine nucleotide binding protein-alpha beta gamma heterotrimers.

The regulatory diversity of the mammalian adenylyl cyclases.

Choi EJ, Xia Z, Villacres EC, Storm DR
Curr Opin Cell Biol. 1993; 5: 269-73

Clones for six mammalian adenylyl cyclases have recently been isolated. One of these enzymes, the type I calmodulin-sensitive adenylyl cyclase, is neurospecific and is implicated in neuroplasticity. We propose that the type I adenylyl cyclase may be important for learning and memory because it allows Ca(2+)-amplified cAMP signals, synergism between Ca2+ and cAMP-activated kinases, and positive feedback regulation of Ca2+ channels by cAMP-dependent protein kinase.

Ca(2+)-inhibitable adenylyl cyclase and pulmonary microvascular permeability.

Chetham PM, Guldemeester HA, Mons N, Brough GH, Bridges JP, Thompson WJ, Stevens T
Am J Physiol. 1997; 273: 2230-2230

Intracellular mechanisms responsible for endothelial cell disruption are unknown, although either elevated cytosolic Ca2+ ([Ca2+]i) or decreased adenosine 3',5'-cyclic monophosphate (cAMP) promotes permeability. Recent identification that Ca(2+)-inhibitable adenylyl cyclase establishes an inverse relationship between [Ca2+]i and cAMP in macrovascular endothelial cells provided a possible mechanism of development of permeability. However, these data utilized an in vitro model; lacking was evidence supporting 1) expression of Ca(2+)-inhibitable adenylyl cyclase in pulmonary microvascular endothelium and 2) Ca2+ inhibition of adenylyl cyclase and cAMP content as a paradigm for inflammatory mediator-induced permeability in the intact circulation. We therefore addressed these issues in microvascular endothelial cells derived from rat lung and in an isolated perfused rat lung preparation. Results demonstrate expression of a Ca(2+)-inhibitable adenylyl cyclase in microvascular endothelial cells. Furthermore, data suggest that Ca2+ inhibition of adenylyl cyclase is necessary for development of microvascular permeability in the intact circulation. We conclude Ca2+ inhibition of cAMP represents a critical step in genesis of microvascular permeability in the intact pulmonary circulation.

Regulation of adenylyl cyclase in the central nervous system.

Chern Y
Cell Signal. 2000; 12: 195-204

Adenylyl cyclases (ACs) are a family of enzymes that synthesize one of the major second messengers, cAMP, upon stimulation. Since the report of the first adenylyl cyclase (AC) gene in 1989, tremendous efforts have been devoted to identifying and characterizing more AC isozymes. In the past decade, significant knowledge regarding the basic structure, tissue distribution, and regulation of AC isozymes has been accumulated. Because members of the AC superfamily are tightly controlled by various signals, one of the most important impacts of these AC isozymes is their contribution to the complexity and fine-tuning of cellular signalling, especially in the central nervous system (CNS) where multiple signals constantly occur. This review focuses on recent progress toward understanding the physiological roles of ACs in the CNS.

A surface on the G protein beta-subunit involved in interactions with adenylyl cyclases.

Chen Y, Weng G, Li J, Harry A, Pieroni J, Dingus J, Hildebrandt JD, Guarnieri F, Weinstein H, Iyengar R
Proc Natl Acad Sci U S A. 1997; 94: 2711-4

Receptor activation of heterotrimeric G proteins dissociates G alpha from the G betagamma complex, allowing both to regulate effectors. Little is known about the effector-interaction regions of G betagamma. We had used molecular modeling to dock a peptide encoding the region of residues 956-982 of adenylyl cyclase (AC) 2 onto Gbeta to identify residues on Gbeta that may interact with effectors. Based on predictions from the model, we synthesized peptides encoding sequences of residues 86-105 (Gbeta 86-105) and 115-135 (Gbeta 115-135) from Gbeta. The Gbeta 86-105 peptide inhibited G betagamma stimulation of AC2 and blocked G betagamma inhibition of AC1 and by itself inhibited calmodulin-stimulated AC1, thus displaying partial agonist activity. Substitution of Met-101 with Asn in this peptide resulted in the loss of both the inhibitory and partial agonist activities. Most activities of the Gbeta 115-135 peptide were similar to those of Gbeta 86-105 but Gbeta 115-135 was less efficacious in blocking G betagamma inhibition of AC1. Substitution of Tyr-124 with Val in the Gbeta 115-135 peptide diminished all of its activities. These results identify the region encoded by amino acids 84-143 of Gbeta as a surface that is involved in transmitting signals to effectors.

Adenylyl cyclase 6 is selectively regulated by protein kinase A phosphorylation in a region involved in Galphas stimulation.

Chen Y, Harry A, Li J, Smit MJ, Bai X, Magnusson R, Pieroni JP, Weng G, Iyengar R
Proc Natl Acad Sci U S A. 1997; 94: 14100-4

Receptors activate adenylyl cyclases through the Galphas subunit. Previous studies from our laboratory have shown in certain cell types that express adenylyl cyclase 6 (AC6), heterologous desensitization included reduction of the capability of adenylyl cyclases to be stimulated by Galphas. Here we further analyze protein kinase A (PKA) effects on adenylyl cyclases. PKA treatment of recombinant AC6 in insect cell membranes results in a selective loss of stimulation by high (>10 nM) concentrations of Galphas. Similar treatment of AC1 or AC2 did not affect Galphas stimulation. Conversion of Ser-674 in AC6 to an Ala blocks PKA phosphorylation and PKA-mediated loss of Galphas stimulation. A peptide encoding the region 660-682 of AC6 blocks stimulation of AC6 and AC2 by high concentrations of Galphas. Substitution of Ser-674 to Asp in the peptide renders the peptide ineffective, indicating that the region 660-682 of AC6 is involved in regulation of signal transfer from Galphas. This region contains a conserved motif present in most adenylyl cyclases; however, the PKA phosphorylation site is unique to members of the AC6 family. These observations suggest a mechanism of how isoform selective regulatory diversity can be obtained within conserved regions involved in signal communication.

Soluble adenylyl cyclase as an evolutionarily conserved bicarbonate sensor.

Chen Y, Cann MJ, Litvin TN, Iourgenko V, Sinclair ML, Levin LR, Buck J
Science. 2000; 289: 625-8

Spermatozoa undergo a poorly understood activation process induced by bicarbonate and mediated by cyclic adenosine 3',5'-monophosphate (cAMP). It has been assumed that bicarbonate mediates its effects through changes in intracellular pH or membrane potential; however, we demonstrate here that bicarbonate directly stimulates mammalian soluble adenylyl cyclase (sAC) activity in vivo and in vitro in a pH-independent manner. sAC is most similar to adenylyl cyclases from cyanobacteria, and bicarbonate regulation of cyclase activity is conserved in these early forms of life. sAC is also expressed in other bicarbonate-responsive tissues, which suggests that bicarbonate regulation of cAMP signaling plays a fundamental role in many biological systems.

Molecular mechanism of cGMP-mediated smooth muscle relaxation.

Carvajal JA, Germain AM, Huidobro-Toro JP, Weiner CP
J Cell Physiol. 2000; 184: 409-20

Contraction and relaxation of smooth muscle is a tightly regulated process involving numerous endogenous substances and their intracellular second messengers. We examine the key role of cyclic guanosine monophosphate (cGMP) in mediating smooth muscle relaxation. We briefly review the current art regarding cGMP generation and degradation, while focusing on the recent identification of the molecular mechanisms underlying cGMP-mediated smooth muscle relaxation. cGMP-induced SM relaxation is mediated mainly by cGMP-dependent protein kinase activation. It involves several molecular events culminating in a reduction in intracellular Ca(2+) concentration and a decrease in the sensitivity of the contractile system to Ca(2+). We propose that the cGMP-induced decrease in Ca(2+) sensitivity is a strategic way to achieve "active relaxation" of the smooth muscle. In summary, we present compelling evidence supporting a key role for cGMP as a mediator of smooth muscle relaxation in physiological and pharmacological settings.

Guanylyl cyclase activity associated with putative bifunctional integral membrane proteins in Plasmodium falciparum.

Carucci DJ, Witney AA, Muhia DK, Warhurst DC, Schaap P, Meima M, Li JL, Taylor MC, Kelly JM, Baker DA
J Biol Chem. 2000; 275: 22147-56

We report here that guanylyl cyclase activity is associated with two large integral membrane proteins (PfGCalpha and PfGCbeta) in the human malaria parasite Plasmodium falciparum. Unusually, the proteins appear to be bifunctional; their amino-terminal regions have strong similarity with P-type ATPases, and the sequence and structure of the carboxyl-terminal regions conform to that of G protein-dependent adenylyl cyclases, with two sets of six transmembrane sequences, each followed by a catalytic domain (C1 and C2). However, amino acids that are enzymatically important and present in the C2 domain of mammalian adenylyl cyclases are located in the C1 domain of the P. falciparum proteins and vice versa. In addition, certain key residues in these domains are more characteristic of guanylyl cyclases. Consistent with this, guanylyl cyclase activity was obtained following expression of the catalytic domains of PfGCbeta in Escherichia coli. In P. falciparum, expression of both genes was detectable in the sexual but not the asexual blood stages of the life cycle, and PfGCalpha was localized to the parasite/parasitophorous vacuole membrane region of gametocytes. The profound structural differences identified between mammalian and parasite guanylyl cyclases suggest that aspects of this signaling pathway may be mechanistically distinct.

Terpenoid cyclases: design and function of electrophilic catalysts.

Cane DE
Ciba Found Symp. 1992; 171: 163-76

Terpenoid cyclases catalyse the cyclization of the universal acyclic precursors geranyl and farnesyl diphosphate to monoterpenes and sesquiterpenes, respectively. All such cyclases investigated to date are operationally soluble, moderately lipophilic proteins of relative molecular weight 40,000-100,000, requiring no cofactors other than a divalent metal, usually Mg2+ and occasionally Mn2+. The focus of most work has been on the mechanisms of the cyclization reactions themselves. It is currently proposed that the cyclase binds the acyclic substrate in a suitable conformation and initiates the cyclization by ionization of the labile allylic diphosphate moiety. The use of stereospecifically labelled substrates and analysis of the sites of labelling in the derived cyclization products has allowed the proposal of detailed cyclization mechanisms. Further insight into the architecture and function of the cyclase active site has come from the study of substrate and intermediate analogues designed to act as potential inhibitors or anomalous substrates of the normal cyclization reaction. Progress has also been made on the cloning of the relevant structural genes for sesquiterpene cyclases. This has led to new insights into the basic requirements for cyclase catalysis and specificity.

Effect of phosphatidylcholine structure on the adenylate cyclase activity of a murine fibroblast cell line.

Calorini L, Mugnai G, Mannini A, Ruggieri S
Lipids. 1993; 28: 727-30

To determine which structural characteristics of membrane phospholipids influence adenylate cyclase activity, we measured basal and sodium fluoride-or forskolin-stimulated activity in a murine fibroblast cell line, i.e., Balb/c3T3 cells grown in media supplemented with fetal calf serum (FCS), lipid-depleted FCS (LD-FCS) or LD-FCS complexed with different phosphatidylcholine (PC) molecular species. Cells grown in the presence of LD-FCS showed a substantial decrease in their basal and NaF-stimulated adenylate cyclase activities; however, their forskolin-stimulated activity was not altered, suggesting that the enzyme's catalytic site is not affected by changes in membrane lipids. Media supplemented with different LD-FCS/PC complexes were shown to prevent the LD-FCS-mediated reduction of basal and NaF-stimulated adenylate cyclase activity to different extents. Addition of cis-9-16:1, cis-9-18:1/cis-9-18:1 or cis-9-18:1/cis-9,12-18:2 sn-glycerophosphocholine (GPC) completely restored adenylate cyclase activity, while cis-11-18:1/cis-11-18:1 GPC was not effective and only a partial recovery was observed with 16:0/16:0, 16:0/cis-9-18:1 and trans-9-18:1 GPC. Considering the structural features of these seven PC molecular species, the findings suggest that an optimal lipid environment is conferred to the enzyme by the presence of two cis double bonds, each located in delta 9 position of the PC acyl chains. The limited effect of cis-9-16:1/cis-9-18:1 GPC and cis-9-18:1/cis-9-16:1 GPC suggests that an equal length of the terminal hydrocarbon chains extending beyond the delta 9 double bonds is also important.(ABSTRACT TRUNCATED AT 250 WORDS)

Splice variants of type VIII adenylyl cyclase. Differences in glycosylation and regulation by Ca2+/calmodulin.

Cali JJ, Parekh RS, Krupinski J
J Biol Chem. 1996; 271: 1089-95

Three alternatively spliced type VIII adenylyl cyclase messages have been identified by cDNA cloning and amplification from rat brain cDNA. Type VIII-A was previously referred to simply as type VIII (Cali, J. J., Zwaagstra, J. C., Mons, N., Cooper, D. M. F., and Krupinski, J. (1994) J. Biol. Chem. 269, 12190-12195). The types VIII-B and -C cDNAs differ from that of type VIII-A by deletion of 90 and 198 base pair exons, respectively, which encode a 30-amino acid extracellular domain with two consensus sites for N-linked glycosylation and a 66-amino acid cytoplasmic domain. Stable expression of types VIII-A, -B, and -C cDNAs in human embryonal kidney 293 (HEK-293) cells leads to the appearance of novel proteins, which are recognized by type VIII-specific antibodies and which co-migrate with immunoreactive species detected on immunoblots of rat brain membranes. Types VIII-A and -C are modified by N-linked glycosylation, while type VIII-B is insensitive to treatment with N-glycosidase F. An influx of extracellular Ca2+ stimulates cAMP accumulation in HEK-293 cells stably expressing type VIII-A, -B, or -C, but not in control cells. Adenylyl cyclase activity of each of the variants is stimulated by Ca2+/calmodulin and the EC50 for activation of type VIII-C is one fourth of that for either type VIII-A or -B. Type VIII-C also has a distinct Km for substrate, which is approximately 4-12-fold higher than that for types VIII-A or -B depending on whether Mn2+ or Mg2+ is the counterion for ATP. The differences in the structural and enzymatic properties of these three variants are discussed.

Regulation of adenylate cyclase by adenosine: characterization of the P-site.

Bushfield M, Johnson RA
Biochem Soc Trans. 1990; 18: 150-1

Cytosolic adenylyl cyclase defines a unique signaling molecule in mammals.

Buck J, Sinclair ML, Schapal L, Cann MJ, Levin LR
Proc Natl Acad Sci U S A. 1999; 96: 79-84

Mammals have nine differentially regulated isoforms of G protein-responsive transmembrane-spanning adenylyl cyclases. We now describe the existence of a distinct class of mammalian adenylyl cyclase that is soluble and insensitive to G protein or Forskolin regulation. Northern analysis indicates the gene encoding soluble adenylyl cyclase (sAC) is preferentially expressed in testis. As purified from rat testis cytosol, the active form of sAC appears to be a fragment derived from the full-length protein, suggesting a proteolytic mechanism for sAC activation. The two presumptive catalytic domains of sAC are closely related to cyanobacterial adenylyl cyclases, providing an evolutionary link between bacterial and mammalian signaling molecules.

G alpha 3 regulates the cAMP signaling system in Dictyostelium.

Brandon MA, Podgorski GJ
Mol Biol Cell. 1997; 8: 1677-85

The Dictyostelium discoideum developmental program is initiated by starvation and its progress depends on G-protein-regulated transmembrane signaling. Disruption of the Dictyostelium G-protein alpha-subunit G alpha 3 (g alpha 3-) blocks development unless the mutant is starved in the presence of artificial cAMP pulses. The function of G alpha 3 was investigated by examining the expression of several components of the cAMP transmembrane signaling system in the g alpha 3- mutant. cAMP receptor 1 protein, cyclic nucleotide phosphodiesterase, phosphodiesterase inhibitor, and aggregation-stage adenylyl cyclase mRNA expression were absent or greatly reduced when cells were starved without exogenously applied pulses of cAMP. However, cAMP receptor 1 protein and aggregation-stage adenylyl cyclase mRNA expression were restored by starving the g alpha 3- cells in the presence of exogenous cAMP pulses. Adenylyl cyclase activity was also reduced in g alpha 3- cells starved without exogenous cAMP pulses compared with similarly treated wild-type cells but was elevated to a level twofold greater than wild-type cells in g alpha 3- cells starved in the presence of exogenous cAMP pulses. These results suggest that G alpha 3 is essential in early development because it controls the expression of components of the transmembrane signaling system.

Pieces of the true grail: a G protein finds its target.

Bourne HR
Science. 1997; 278: 1898-9

Conformational transitions within the calmodulin-binding site of Bordetella pertussis adenylate cyclase studied by time-resolved fluorescence of Trp242 and circular dichroism.

Bouhss A, Vincent M, Munier H, Gilles AM, Takahashi M, Barzu O, Danchin A, Gallay J
Eur J Biochem. 1996; 237: 619-28

The sequence situated around Trp242 in Bordetella pertussis adenylate cyclase, a bifunctional protein of 1706 amino acid residues, forms the core of the calmodulin-binding site. Peptides varying in size and in affinity for calmodulin, and preserving the same sequence around Trp242 were analyzed by time-resolved fluorescence spectroscopy. Their dynamic properties were compared to those of the catalytic domain of B. pertussis adenylate cyclase corresponding to the first 400 amino acid residues of the protein and in which the Trp69 residue was replaced by Phe. The heterogeneity of the fluorescence intensity decays of Trp242 is likely due to the existence of conformers in equilibrium as is suggested by the effect of trifluoroethanol both on the secondary structure content and the lifetime distributions. Binding to calmodulin leads to striking effects on the lifetime distribution profiles by selecting a major excited state population and therefore one major conformer. Trp242 still presents some degree of rotational freedom in the complexes. The reduction of rotational freedom is more important for the shorter peptides than for the longest one. A similar selection of one major conformer with the same lifetime was also observed for the Trp242 in the mutant protein when bound to calmodulin, as in the complexes with the peptides. We conclude that the site of interaction of B. pertussis adenylate cyclase with calmodulin has similar conformational flexibility as that evidenced in the isolated peptides. This property of the molecule allows a better adjustment of the enzyme upon interaction with calmodulin.

Cooperative phenomena in binding and activation of Bordetella pertussis adenylate cyclase by calmodulin.

Bouhss A, Krin E, Munier H, Gilles AM, Danchin A, Glaser P, Barzu O
J Biol Chem. 1993; 268: 1690-4

The catalytic domain of Bordetella pertussis adenylate cyclase located within the first 400 amino acids of the protein can be cleaved by trypsin in two subdomains (T25 and T18) corresponding to ATP-(T25) and calmodulin (CaM)-(T18) binding sites. Reassociation of subdomains by CaM is a cooperative process, which is a unique case among CaM-activated enzymes. To understand better the molecular basis of this phenomenon, we used several approaches such as partial deletions of the adenylate cyclase gene, isolation of peptides of various size, and site-directed mutagenesis experiments. We found that a stretch of 72 amino acid residues overlapping the carboxyl terminus of T25 and the amino terminus of T18 accounts for 90% of the binding energy of adenylate cyclase-CaM complex. The hydrophobic "side" of the helical region situated around Trp242 plays a major role in the interaction of adenylate cyclase with CaM, whereas basic residues that alternate with acidic residues in bacterial enzyme play a much less important role. The amino-terminal half of the catalytic domain of adenylate cyclase contributes only 10% to the binding energy of CaM, whereas the last 130 amino acid residues are not at all involved in binding. However, these segments of adenylate cyclase might affect protein/protein interaction and catalysis by propagating conformational changes to the CaM-binding sequence which is located in the middle of the catalytic domain of bacterial enzyme.

Differential effects of ceramides upon adenylyl cyclase subtypes.

Bosel A, Pfeuffer T
FEBS Lett. 1998; 422: 209-12

Ceramides are reported to stimulate different effector systems, among them atypical protein kinases C (PKCs). When HEK 293 cells, stably expressing adenylyl cyclase type II (AC II), were treated with various ceramide derivatives, adenylyl cyclase activity was enhanced 8-15-fold. The stimulation by the most potent analog, C18/C24 ceramide, was comparable to that by the phorbolester TPA. The stimulatory effect of ceramide was not restricted to AC II, although the type I and type V enzymes were affected less dramatically. Unexpectedly, the dihydro derivatives of ceramides, generally serving as non-activating controls, exhibited only slightly lower stimulation than ceramides, whereas short-chain ceramides (e.g. C2) were without effect. The action of ceramides was at least partially inhibited by okadaic acid, suggesting involvement of a phosphatase. Furthermore, ceramides and TPA operated synergistically. While the PKC inhibitor staurosporine counteracted the action of phorbol-esters, it significantly (2.5x) enhanced the effect of ceramides.

Phorbol ester-induced sensitisation of adenylyl cyclase type II is related to phosphorylation of threonine 1057.

Bol GF, Gros C, Hulster A, Bosel A, Pfeuffer T
Biochem Biophys Res Commun. 1997; 237: 251-6

Following up the results from previous studies on chemical fragmentation of TPA-treated, [32P]phosphate labeled adenylyl cyclase type II (AC II) (Bol, G. F., Hulster, A., and Pfeuffer, T. in press) we have replaced serine 871 or threonine 1057 by alanine using site directed mutagenesis. Both mutants had unimpaired catalytic activity, however enhancement by phorbolester TPA was reduced by 60-80 % in the T1057A mutant, but not in the S871A mutant. The stimulation of adenylyl cyclase type II by betagamma subunits of heterotrimeric G-pro teins and that by PKC have been previously shown to be mutually exclusive (Zimmermann and Taussig (1996), J. Biol. Chem. 271, 27161-27166). This is in line with the present findings that AC II expressed in COS-1 cells was only barely stimulated (10%) by coexpressed betagamma-subunits in presence of TPA. Mutation of threonine 1057 to alanine however caused partial regain of betagamma-stimulation in the presence of TPA by 40%, as compared to that of WT adenylyl cyclase type II which was 70% in the absence of TPA. These data strongly implicate the importance of threonine 1057 as phosphate acceptor following PKC-mediated sensitisation of adenylyl cyclase type II.

Mutational analysis of the active site and antibody epitopes of the complement-inhibitory glycoprotein, CD59.

Bodian DL, Davis SJ, Morgan BP, Rushmere NK
J Exp Med. 1997; 185: 507-16

The Ly-6 superfamily of cell surface molecules includes CD59, a potent regulator of the complement system that protects host cells from the cytolytic action of the membrane attack complex (MAC). Although its mechanism of action is not well understood, CD59 is thought to prevent assembly of the MAC by binding to the C8 and/or C9 proteins of the nascent complex. Here a systematic, structure-based mutational approach has been used to determine the region(s) of CD59 required for its protective activity. Analysis of 16 CD59 mutants with single, highly nonconservative substitutions suggests that CD59 has a single active site that includes Trp-40, Arg-53, and Glu-56 of the glycosylated, membrane-distal face of the disk-like extra-cellular domain and, possibly, Asp-24 positioned at the edge of the domain. The putative active site includes residues conserved across species, consistent with the lack of strict homologous restriction previously observed in studies of CD59 function. Competition and mutational analyses of the epitopes of eight CD59-blocking and non-blocking monoclonal antibodies confirmed the location of the active site. Additional experiments showed that the expression and function of CD59 are both glycosylation independent.

Guanylyl cyclase C receptor: regulation of catalytic activity by ATP.

Bhandari R, Suguna K, Visweswariah SS
Biosci Rep. 1999; 19: 179-88

Guanylyl cyclase C (GCC), a member of the family of membrane bound guanylyl cyclases is the receptor for the heat-stable enterotoxin (ST) peptides and the guanylin family of endogenous peptides. GCC is activated upon ligand binding to increase intracellular cGMP levels, which in turn activates other downstream signalling events in the cell. GCC is also activated in vitro by nonionic detergents. We have used the T84 cell line as a model system to investigate the regulation of GCC activity by ATP. Ligand-stimulated GCC activity is potentiated in the presence of ATP, whereas detergent-stimulated activity is inhibited. The potentiation of GCC activity by ATP is dependent on the presence of Mg2+ ions, and is probably brought about by a direct binding of Mg-ATP to GCC. The protein kinase-like domain of GCC, which has earlier been shown to play a critical role in the regulation of GCC activity, may be a possible site for the binding of Mg-ATP to GCC.

Rhizobium meliloti adenylate cyclase: probing of a NTP-binding site common to cyclases and cation transporters.

Beuve A, Krin E, Danchin A
C R Acad Sci III. 1993; 316: 553-9

Alignments between amino acid sequences of eukaryotic adenylate (ACase) and guanylate (GCase) cyclases and the prokaryotic Rhizobium meliloti ACase revealed four conserved regions. Two were the target of site-directed mutagenesis. A positive charge at position 44 converted the enzyme to GCase, a negative charge at this position had no effect. A second modification indicated that residues 107 and 124 contribute to the nucleoside triphosphate binding pocket's conformation. This latter region was used to scan protein sequences data banks. A similar region was detected in the family of E1-E2 type ATPases. Topographical resemblance between these ATPases, eukaryotic ACases and several transporters suggest that they evolved from a common ancestor.

From adenylate cyclase to guanylate cyclase. Mutational analysis of a change in substrate specificity.

Beuve A, Danchin A
J Mol Biol. 1992; 225: 933-8

Adenylate and guanylate cyclases, having different but related substrates, are a paradigm for the study of substrate discrimination. A prokaryotic adenylate cyclase gene, phylogenetically related to eukaryotic counterparts, was screened for mutants remodelling the enzyme's specificity. In a first step, a mutant was selected displaying a significant level of guanylate cyclase activity. This was due to a point mutation destroying most of the adenylate cyclase activity. A second selection step restored most of the original activity. This resulted from an additional mutation in the same region, thus permitting the first identification of a functional domain in adenylate and guanylate cyclases.

Conversion of a guanylyl cyclase to an adenylyl cyclase.

Beuve A
Methods. 1999; 19: 545-50

Guanylyl cyclases catalyze the formation of cGMP from GTP, but display extensive identity at the catalytic domain primary amino acid level with the adenylyl cyclases. The recent solving of the crystal structures of soluble forms of adenylyl cyclase has resulted in predictions of those amino acids important for substrate specificity. Modeling of a membrane-bound homodimeric guanylyl cyclase predicted the comparable amino acids that would interact with the guanine ring. Based on these structural data, the replacement of three key residues in the heterodimeric form of soluble guanylyl cyclase has led to a complete conversion in substrate specificity. Furthermore, the mutant enzyme remained fully sensitive to sodium nitroprusside, a nitric oxide donor.

Determinants of potency and temperature-dependent function in the Aplysia bag cell peptides.

Berry RW, Hanu R, Redman RS, Kim JJ
Peptides. 1994; 15: 855-60

Structure-activity relationships were determined for the natural bag cell peptides (BCPs) and for a series of synthetic analogues in terms of their ability to stimulate (at 30 degrees C) and to inhibit (at 15 degrees C) bag cell adenyl cyclase. We found that the core RLRF motif shared by all these peptides is active in this assay, and is stimulatory. The histidine residue C-terminal to this motif in beta-BCP is superfluous in this respect. An electronegative residue C-terminal to RLRF is sufficient to induce temperature-dependent function. The Ala-Pro pair that is N-terminal to this motif in alpha-BCP increases potency, but does not alter function.

Modulation of adenylyl cyclase by Aplysia bag cell peptides: evidence for a common receptor.

Berry RW
Brain Res Mol Brain Res. 1996; 38: 176-8

A variant of the alpha 2 subunit of soluble guanylyl cyclase contains an insert homologous to a region within adenylyl cyclases and functions as a dominant negative protein.

Behrends S, Harteneck C, Schultz G, Koesling D
J Biol Chem. 1995; 270: 21109-13

A variant of the alpha 2 subunit of soluble guanylyl cyclase (alpha 2i) containing 31 additional amino acids was identified in a number of cell lines and tissues. The in-frame sequence of the insert was within the proposed catalytic domain of guanylyl cyclases and was homologous to a region within the putative catalytic domain of adenylyl cyclases. Messenger RNA for the new variant was detected in some but not all cell lines and tissues expressing the alpha 2 subunit. The novel form, as well as the alpha 2 subunit lacking the insert, were coexpressed with the beta 1 subunit in Sf9 and COS-7 cells; alpha 2/beta 1 coexpression yielded a NO-sensitive recombinant protein, whereas the coexpressed alpha 2i/beta 1 subunits exhibited no guanylyl or adenylyl cyclase activities. Because both subunits (alpha 2i/beta 1) copurified, the novel variant retains its ability to heterodimerize. In coexpression experiments, the alpha 2i subunit competed with the alpha 2 subunit for dimerization with the beta 1 subunit, thereby reducing alpha 2/beta 1-catalyzed guanylyl cyclase activity. These data show that the novel variant functions as a dominant negative protein and that post-transcriptional mRNA processing represents a potential mechanism for regulation of NO-sensitive guanylyl cyclase activity.

Differential modulation of adenylyl cyclases I and II by various G beta subunits.

Bayewitch ML, Avidor-Reiss T, Levy R, Pfeuffer T, Nevo I, Simonds WF, Vogel Z
J Biol Chem. 1998; 273: 2273-6

The accepted dogma concerning the regulation of adenylyl cyclase (AC) activity by G beta gamma dimers states that the various isoforms of AC respond differently to the presence of free G beta gamma. It has been demonstrated that AC I activity is inhibited and AC II activity is stimulated by G beta gamma subunits. This result does not address the possible differences in modulation that may exist among the different G beta gamma heterodimers. Six isoforms of G beta and 12 isoforms of G gamma have been cloned to date. We have established a cell transfection system in which G beta and G gamma cDNAs were cotransfected with either AC isoform I or II and the activity of these isoforms was determined. We found that while AC I activity was inhibited by both G beta 1/gamma 2 and G beta 5/gamma 2 combinations, AC II responded differentially and was stimulated by G beta 1/gamma 2 and inhibited by G beta 5/gamma 2. This finding demonstrates differential modulatory activity by different combinations of G beta gamma on the same AC isoform and demonstrates another level of complexity within the AC signaling system.

Inhibition of adenylyl cyclase isoforms V and VI by various Gbetagamma subunits.

Bayewitch ML, Avidor-Reiss T, Levy R, Pfeuffer T, Nevo I, Simonds WF, Vogel Z
FASEB J. 1998; 12: 1019-25

An intriguing development in the G-protein signaling field has been the finding that not only the Galpha subunit, but also Gbetagamma subunits, affect a number of downstream target molecules. One of the downstream targets of Gbetagamma is adenylyl cyclase, and it has been demonstrated that a number of isoforms of adenylyl cyclase can be either inhibited or stimulated by Gbetagamma subunits. Until now, adenylyl cyclase type I has been the only isoform reported to be inhibited by free Gbetagamma. Here we show by transient cotransfection into COS-7 cells of either adenylyl cyclase V or VI, together with Ggamma2 and various Gbeta subunits, that these two adenylyl cyclase isozymes are markedly inhibited by Gbetagamma. In addition, we show that Gbeta1 and Gbeta5 subunits differ in their activity. Gbeta1 transfected alone markedly inhibited adenylyl cylcase V and VI (probably by recruiting endogenous Ggamma subunits). On the other hand, Gbeta5 produced less inhibition of these isozymes, and its activity was enhanced by the addition of Ggamma2. These results demonstrate that adenylyl cyclase types V and VI are inhibited by Gbetagamma dimers and that Gbeta1 and Gbeta5 subunits differ in their capacity to regulate these adenylyl cyclase isozymes.

Adenylyl cyclases: a heterogeneous class of ATP-utilizing enzymes.

Barzu O, Danchin A
Prog Nucleic Acid Res Mol Biol. 1994; 49: 241-83

Interaction of sulfur-containing analogs of ATP with adenylate cyclase.

Bar HP, Simonson LP, Eckstein F
FEBS Lett. 1974; 41: 199-202

Identification of a specialized adenylyl cyclase that may mediate odorant detection.

Bakalyar HA, Reed RR
Science. 1990; 250: 1403-6

The mammalian olfactory system may transduce odorant information via a G protein-mediated adenosine 3',5'-monophosphate (cAMP) cascade. A newly discovered adenylyl cyclase, termed type III, has been cloned, and its expression was localized to olfactory neurons. The type III protein resides in the sensory neuronal cilia, which project into the nasal lumen and are accessible to airborne odorants. The enzymatic activity of the type III adenylyl cyclase appears to differ from nonsensory cyclases. The large difference seen between basal and stimulated activity for the type III enzyme could allow considerable modulation of the intracellular cAMP concentration. This property may represent one mechanism of achieving sensitivity in odorant perception.

Opiate-induced adenylyl cyclase superactivation is isozyme-specific.

Avidor-Reiss T, Nevo I, Saya D, Bayewitch M, Vogel Z
J Biol Chem. 1997; 272: 5040-7

While acute activation of inhibitory Gi/o-coupled receptors leads to inhibition of adenylyl cyclase, chronic activation of such receptors leads to an increase in cAMP accumulation. This phenomenon, observed in many cell types, has been referred to as adenylyl cyclase superactivation. At this stage, the mechanism leading to adenylyl cyclase superactivation and the nature of the isozyme(s) responsible for this phenomenon are largely unknown. Here we show that transfection of adenylyl cyclase isozymes into COS-7 cells results in an isozyme-specific increase in AC activity upon stimulation (e.g. with forskolin, ionomycin, or stimulatory receptor ligands). However, independently of the method used to activate specific adenylyl cyclase isozymes, acute activation of the mu-opioid receptor inhibited the activity of adenylyl cyclases I, V, VI, and VIII, while types II, IV, and VII were stimulated and type III was not affected. Chronic mu-opioid receptor activation followed by removal of the agonist was previously shown, in transfected COS-7 cells, to induce superactivation of adenylyl cyclase type V. Here we show that it also leads to superactivation of adenylyl cyclase types I, VI, and VIII, but not of type II, III, IV, or VII, demonstrating that the superactivation is isozyme-specific. Not only were isozymes II, IV, and VII not superactivated, but a reduction in the activities of these isozymes was actually observed upon chronic opiate exposure. These results suggest that the phenomena of tolerance and withdrawal involve specific adenylyl cyclase isozymes.

The bovine photoreceptor outer segment guanylate cyclase: purification, kinetic properties, and molecular size.

Aparicio JG, Applebury ML
Protein Expr Purif. 1995; 6: 501-11

A simple protocol was developed to isolate the integral membrane guanylate cyclase from bleached bovine photoreceptor outer segments. Hypotonic and hypertonic washes strip the photoreceptor outer segment membranes of peripheral proteins. The guanylate cyclase activity is solubilized by dodecyl-b-D-maltoside in a low salt concentration buffer. Phosphatidylcholine, glycerol, and dithiothreitol are used to stabilize the activity during chromatography. GTP-affinity chromatography achieves a 250-fold increase in specific activity over that of membranes stripped of peripheral proteins. From 100 retinas, the protocol yields 100-140 mg of purified guanylate cyclase composed of a 115-kDa subunit. The molar ratio of the guanylate cyclase to rhodopsin is estimated to be 1:440. A significant portion of the freshly solubilized enzyme behaves as a monomer with a Stokes radius of 48.7 A, whereas the purified protein forms homooligomers ranging from dimers to tetramers. These properties are similar to those of ANP and guanylin receptors, indicating that the photoreceptor protein shares characteristics of the membrane receptor guanylate cyclase family. For the physiological substrate MgGTP, the Km and Vmax are 1.07 +/- 0.20 mM and 3262 +/- 514 nmol cGMP min-1 mg-1, respectively, generating a turnover rate of approximately 3.9 nmol cGMP s-1 at physiological substrate concentrations. The relatively high Km suggests that in vivo changes in GTP concentration might modulate the rate of cGMP synthesis. These properties indicate that the photoreceptor membrane guanylate cyclase can sustain a rate of cGMP synthesis comparable to the dark-adapted (basal) rate of cGMP degradation by the cGMP phosphodiesterase.

Calcium control of adenylyl cyclase: the calcineurin connection.

Antoni FA, Smith SM, Simpson J, Rosie R, Fink G, Paterson JM
Adv Second Messenger Phosphoprotein Res. 1998; 32: 153-72

Molecular aspects of soluble guanylyl cyclase regulation.

Andreopoulos S, Papapetropoulos A
Gen Pharmacol. 2000; 34: 147-57

Soluble guanylyl cyclase (sGC) is a heterodimeric enzyme (comprised of alpha and beta subunits) that generates the intracellular second messenger cyclic guanosine monophosphate (cGMP) from guanosine triphosphate (GTP). cGMP is subsequently important for the regulation of protein kinases, ion channels, and phosphodiesterases. Since recent evidence has demonstrated that heterodimerization of the alpha/beta subunits is essential for basal and stimulated enzymatic activity, the existence of several types of isoforms for each of the two subunits, along with their varying degrees of expression in different tissues, implies that multiple regulatory mechanisms exist for sGC. Yet, progress in studying and clarifying the regulatory processes that can alter sGC expression and activity has only slowly started being elucidated. In the following paper, we elaborate on sGC structure, function, and distribution along with recently described signaling pathways that modulate sGC gene expression.

Importance of the region around lysine 196 for catalytic activity of adenylyl cyclase from Escherichia coli.

Amin N, Peterkofsky A
J Biol Chem. 1994; 269: 31074-9

Escherichia coli adenylyl cyclase contains no sequence that corresponds to the previously defined ATP/GTP binding consensus (A,G)XXXXGK(S,T). Using a search for lysine residues located adjacent to glycine residues, three regions that were possible candidates for part of the ATP binding site were identified. These were the residues located at positions 59, 90, and 196. A plasmid vector capable of overexpressing the cya gene under the control of the lambda PL promoter was mutated at these three loci to convert those lysine residues to methionine. Assays for catalytic activity of the mutated hyperexpressed proteins revealed that only the mutation at position 196 led to loss of activity. Photoaffinity labeling experiments using 8-azido-ATP provided evidence that the loss of activity was associated with a loss of the capability of the enzyme to bind ATP. A further series of replacement mutations in the hyperexpression vector was created at position 196. Assays of the adenylyl cyclase activity of the mutated proteins showed that replacement of lysine 196 by arginine led to minimal change in the activity. Replacements by histidine, glutamine, or glutamic acid resulted in approximately 10-20-fold reductions in the activity; replacements by methionine, isoleucine, or aspartic acid resulted in total loss of activity. When the mutated forms of the cya gene were expressed under the control of the cya promoter, the activity of the wild-type protein was higher than that of all the mutants, including the arginine replacement mutant. All of the mutants that retained activity also retained the capability of adenylyl cyclase to be stimulated by either inorganic orthophosphate or GTP. A helical wheel analysis of the region of adenylyl cyclase around lysine 196 revealed a structure compatible with an amphipathic helix with one face enriched with basic amino acid residues. Assays for adenylyl cyclase activity of a series of replacement mutations of residues on the hydrophilic face of the helix (R188I, R192I, G195I) as well as on the hydrophobic face (R197I) indicated that the R188I, G195I, and K196I replacement mutants were inactive, and R192I was approximately 30% as active as the wild-type, while the R197I mutant was equivalent to the wild-type control. A model is suggested for a unique binding motif in E. coli adenylyl cyclase in which there is a repetition of 3 basic residues on one face of a helix where there is an interaction with the three phosphate groups of ATP.

Requirement for GLY-60 of Escherichia coli adenylyl cyclase for ATP binding and catalytic activity.

Amin N, Peterkofsky A
Biochem Biophys Res Commun. 1992; 182: 1218-25

The region of Escherichia coli adenylyl cyclase spanned by glycine-55 to threonine-65 was tested for its importance for enzyme activity. Site-directed mutagenesis was used to replace glycine-55 and glycine-60 as well as lysine-59, leucine-63 and threonine-65 with other amino acids. While substitution of glycine-55 with aspartic acid produced no significant change in kinetic parameters, the change of glycine-60 to aspartic acid or asparagine eliminated binding to 8-azido-ATP and decreased the Vmax (two orders of magnitude) and Km (factor of four-five). Smaller effects on kinetic parameters were observed with substitutions of lysine-59, leucine-63 or threonine-65.

Three-dimensional structure of guanylyl cyclase activating protein-2, a calcium-sensitive modulator of photoreceptor guanylyl cyclases.

Ames JB, Dizhoor AM, Ikura M, Palczewski K, Stryer L
J Biol Chem. 1999; 274: 19329-37

Guanylyl cyclase activating protein-2 (GCAP-2) is a Ca2+-sensitive regulator of phototransduction in retinal photoreceptor cells. GCAP-2 activates retinal guanylyl cyclases at low Ca2+ concentration (<100 nM) and inhibits them at high Ca2+ (>500 nM). The light-induced lowering of the Ca2+ level from approximately 500 nM in the dark to approximately 50 nM following illumination is known to play a key role in visual recovery and adaptation. We report here the three-dimensional structure of unmyristoylated GCAP-2 with three bound Ca2+ ions as determined by nuclear magnetic resonance spectroscopy of recombinant, isotopically labeled protein. GCAP-2 contains four EF-hand motifs arranged in a compact tandem array like that seen previously in recoverin. The root mean square deviation of the main chain atoms in the EF-hand regions is 2.2 A in comparing the Ca2+-bound structures of GCAP-2 and recoverin. EF-1, as in recoverin, does not bind calcium because it contains a disabling Cys-Pro sequence. GCAP-2 differs from recoverin in that the calcium ion binds to EF-4 in addition to EF-2 and EF-3. A prominent exposed patch of hydrophobic residues formed by EF-1 and EF-2 (Leu24, Trp27, Phe31, Phe45, Phe48, Phe49, Tyr81, Val82, Leu85, and Leu89) may serve as a target-binding site for the transmission of calcium signals to guanylyl cyclase.

Synthesis of leucine enkephalin derivatives: structure-function studies.

Agarwal NS, Hruby VJ, Katz R, Klee W, Nirenberg M
Biochem Biophys Res Commun. 1977; 76: 129-35

Purification, characterization, and N-terminal amino acid sequence of the adenylyl cyclase-activating protease from bovine sperm.

Adeniran AJ, Shoshani I, Minuth M, Awad JA, Elce JS, Johnson RA
Biol Reprod. 1995; 52: 490-9

We previously reported the extraction of a factor from bovine sperm that activated adenylyl cyclases of rat brain and human platelets, and identified it as a trypsin-like protease that was referred to as "ninhibin." This proteolytic activity was purified to near homogeneity from an alkaline extract of washed sperm particles by sequential chromatography on p-aminobenzamidine agarose and CM-Sephadex. Purification was greater than 100-fold with nearly 30% recovery of protease activity exhibiting a major band of approximately 40 kDa. An approximately 45-kDa form of the protease was also evident in crude extracts and was preferentially isolated when the enzyme was prepared in the presence of a mixture of protease inhibitors. The larger form of the protease was substantially less effective in stimulating adenylyl cyclase than was the smaller form; it is likely to be a zymogen form from which the smaller, more active form is derived. Purified forms of acrosin and ninhibin exhibited similar mobilities on PAGE, similar capacities for activating adenylyl cyclase, similar patterns of proteolytic fragmentation, and similar immunoblot patterns obtained with an antibody against purified bovine acrosin. More importantly, the N-terminal amino acid sequence of bovine ninhibin was found to be identical with that of bovine acrosin and caprine acrosin and more than 75% identical with porcine acrosin. The data support the conclusion that the adenylyl cyclase-activating protease previously referred to as ninhibin is, in fact, acrosin.