NORMAL GENOMIC

• Sudhof TC
• alpha-Latrotoxin and its receptors: neurexins and CIRL/latrophilins.
• Annu Rev Neurosci. 2001; 24: 933-62
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• alpha-Latrotoxin, a potent neurotoxin from black widow spider venom, triggers synaptic vesicle exocytosis from presynaptic nerve terminals. alpha-Latrotoxin is a large protein toxin (120 kDa) that contains 22 ankyrin repeats. In stimulating exocytosis, alpha-latrotoxin binds to two distinct families of neuronal cell-surface receptors, neurexins and CLs (Cirl/latrophilins), which probably have a physiological function in synaptic cell adhesion. Binding of alpha-latrotoxin to these receptors does not in itself trigger exocytosis but serves to recruit the toxin to the synapse. Receptor-bound alpha-latrotoxin then inserts into the presynaptic plasma membrane to stimulate exocytosis by two distinct transmitter-specific mechanisms. Exocytosis of classical neurotransmitters (glutamate, GABA, acetylcholine) is induced in a calcium-independent manner by a direct intracellular action of alpha-latrotoxin, while exocytosis of catecholamines requires extracellular calcium. Elucidation of precisely how alpha-latrotoxin works is likely to provide major insight into how synaptic vesicle exocytosis is regulated, and how the release machineries of classical and catecholaminergic neurotransmitters differ.

• Volynski KE et al.
• Latrophilin, neurexin, and their signaling-deficient mutants facilitate alpha -latrotoxin insertion into membranes but are not involved in pore formation.
• J Biol Chem. 2000; 275: 41175-83
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• Pure alpha-latrotoxin is very inefficient at forming channels/pores in artificial lipid bilayers or in the plasma membrane of non-secretory cells. However, the toxin induces pores efficiently in COS-7 cells transfected with the heptahelical receptor latrophilin or the monotopic receptor neurexin. Signaling-deficient (truncated) mutants of latrophilin and latrophilin-neurexin hybrids also facilitate pore induction, which correlates with toxin binding irrespective of receptor structure. This rules out the involvement of signaling in pore formation. With any receptor, the alpha-latrotoxin pores are permeable to Ca(2+) and small molecules including fluorescein isothiocyanate and norepinephrine. Bound alpha-latrotoxin remains on the cell surface without penetrating completely into the cytosol. Higher temperatures facilitate insertion of the toxin into the plasma membrane, where it co-localizes with latrophilin (under all conditions) and with neurexin (in the presence of Ca(2+)). Interestingly, on subsequent removal of Ca(2+), alpha-latrotoxin dissociates from neurexin but remains in the membrane and continues to form pores. These receptor-independent pores are inhibited by anti-alpha-latrotoxin antibodies. Our results indicate that (i) alpha-latrotoxin is a pore-forming toxin, (ii) receptors that bind alpha-latrotoxin facilitate its insertion into the membrane, (iii) the receptors are not physically involved in the pore structure, (iv) alpha-latrotoxin pores may be independent of the receptors, and (v) pore formation does not require alpha-latrotoxin interaction with other neuronal proteins.

• Van Renterghem C, Iborra C, Martin-Moutot N, Lelianova V, Ushkaryov Y, Seagar M
• alpha-latrotoxin forms calcium-permeable membrane pores via interactions with latrophilin or neurexin.
• Eur J Neurosci. 2000; 12: 3953-62
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• In order to explore the mechanisms by which alpha-latrotoxin activates neurotransmitter release, we have characterized its effects by patch-clamp methods on cells heterologously expressing its receptors, latrophilin-1 or neurexin-Ialpha. Application of alpha-latrotoxin (1 nM) to cells expressing rat latrophilin or neurexin, but not mock-transfected cells, induced a cationic conductance. In cells expressing latrophilin, current development was slow in the absence of divalent cations, but was accelerated by Ca2+ or Mg2+. In cells expressing neurexin, alpha-latrotoxin did not elicit currents in the absence of Ca2+. The toxin-induced conductance was rectifying, persistent, permeable to monovalent and divalent cations, but blocked by La3+. Single-channel recording revealed a permanently open state, with the same unitary conductance irrespective of whether cells expressed latrophilin or neurexin. Therefore, while pore formation displayed differences consistent with the reported properties of alpha-latrotoxin binding to latrophilin and neurexin, the pores induced by alpha-latrotoxin had identical properties. These results suggest that after anchoring to either of its nerve terminal receptors, alpha-latrotoxin inserts into the membrane and constitutes a single type of transmembrane ion pore.

• Kreienkamp HJ, Zitzer H, Gundelfinger ED, Richter D, Bockers TM
• The calcium-independent receptor for alpha-latrotoxin from human and rodent brains interacts with members of the ProSAP/SSTRIP/Shank family of multidomain proteins.
• J Biol Chem. 2000; 275: 32387-90
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• Subtypes of the calcium-independent receptors for alpha-latrotoxin (CIRL1-3) define a distinct subgroup within the large family of the seven-transmembrane region cell surface receptors. The physiological function of CIRLs is unknown because neither extracellular ligands nor intracellular coupling proteins (G-proteins) have been identified. Using yeast two-hybrid screening, we identified a novel interaction between the C termini of CIRL1 and -2 and the PSD-95/discs large/ZO-1 (PDZ) domain of a recently discovered multidomain protein family (ProSAP/SSTRIP/Shank) present in human and rat brain. In vitro, CIRL1 and CIRL2 interacted strongly with the PDZ domain of ProSAP1. The specificity of this interaction has been verified by in vivo experiments using solubilized rat brain membrane fractions and ProSAP1 antibodies; only CIRL1, but not CIRL2, was co-immunoprecipitated with ProSAP1. In situ hybridization revealed that ProSAP1 and CIRL1 are co-expressed in the cortex, hippocampus, and cerebellum. Colocalization was also observed at the subcellular level, as both CIRL1 and ProSAP1 are enriched in the postsynaptic density fraction from rat brain. Expression of all three CIRL isoforms is highly regulated during postnatal brain development, with CIRL3 exhibiting its highest expression levels immediately after birth, followed by CIRL2 and finally CIRL1 in aged rats.

• Pescatori M, Grasso A
• Characterization of the epitope for 4C4.1 mAb on alpha-latrotoxin using phage display-peptide libraries: prevention of toxin-dependent 45Ca(2+) uptake in non-neuronal human embryonic cells transiently expressing latrophilin.
• Biochimie. 2000; 82: 909-14
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• alpha-Latrotoxin, a protein toxin present in the venom of black widow spider, interacts with membrane receptors of neurons and other secretory cells to stimulate exocytosis. Two types of receptors have been identified and cloned. Our attention has been focused on the calcium independent receptor, a G-protein coupled receptor, named latrophilin to see whether alpha-latrotoxin interaction was capable to produce an ionotropic effect, in alternative to the metabotropic hypothesis. Expression of latrophilin receptor is sufficient for the alpha-latrotoxin effect to become manifest. By inducing the transient expression of latrophilin receptor in non-neuronal human embryonic cells, we made them susceptible to toxin action as demonstrated by the increase in 45Ca(2+) accumulation detected after toxin treatment. Since the presence of a monoclonal antibody against alpha-latrotoxin (4C4.1 mAb) was able to obliterate toxin-dependent effects, we further investigated the nature of toxin-antibody interaction by characterization of the binding epitope using phage display-peptide libraries. A conformational epitope was recognized and partially localized on a region of the peptide toxin whereby a tetrameric structure is formed and inserted into the membrane of target cells where it functions as a pore.

• Hlubek MD, Stuenkel EL, Krasnoperov VG, Petrenko AG, Holz RW
• Calcium-independent receptor for alpha-latrotoxin and neurexin 1alpha [corrected] facilitate toxin-induced channel formation: evidence that channel formation results from tethering of toxin to membrane.
• Mol Pharmacol. 2000; 57: 519-28
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• alpha-Latrotoxin binding to the calcium-independent receptor for alpha-latrotoxin (CIRL-1), a putative G-protein-coupled receptor, stimulates secretion from chromaffin and PC12 cells. Using patch clamp techniques and microspectrofluorimetry, we demonstrate that the interaction of alpha-latrotoxin with CIRL-1 produces a high conductance channel that permits increases in cytosolic Ca(2+). alpha-Latrotoxin interaction with CIRL-1 transiently expressed in bovine chromaffin cells produced a 400-pS channel, which rarely closed under Ca(2+)-free conditions. The major effect of overexpressing CIRL-1 was to greatly increase the sensitivity of chromaffin cells to channel formation by alpha-latrotoxin. alpha-Latrotoxin interaction with CIRL-1 transiently overexpressed in non-neuronal human embryonic kidney 293 (HEK293) cells produced channels that were nearly identical with those observed in chromaffin cells. Channel currents were reduced by millimolar Ca(2+). At alpha-latrotoxin concentrations below 500 pM, channel formation occurred many seconds after binding of toxin to CIRL-1 indicating distinct steps in channel formation. In all cases there was a rapid, sequential addition of channels once the first channel appeared. An analysis of CIRL-1 mutants indicated that channel formation in HEK293 cells is unlikely to be transduced by a G-protein-dependent mechanism. alpha-Latrotoxin interaction with a fusion construct composed of the extracellular domain of CIRL-1 anchored to the membrane by the transmembrane domain of vesicular stomatitis virus glycoprotein, and with neurexin 1alpha, an alpha-latrotoxin receptor structurally unrelated to CIRL-1, produced channels virtually identical with those observed with wild-type CIRL-1. We propose that alpha-latrotoxin receptors recruit toxin to facilitate its insertion across the membrane and that alpha-latrotoxin itself controls the conductance properties of the channels it produces.

• Bittner MA
• Alpha-latrotoxin and its receptors CIRL (latrophilin) and neurexin 1 alpha mediate effects on secretion through multiple mechanisms.
• Biochimie. 2000; 82: 447-52
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• Alpha-Latrotoxin and its plasma membrane receptors cause a number of distinct effects in secretory cells. First, by tethering alpha-latrotoxin to the plasma membrane, CIRL/latrophilin and neurexin 1 alpha facilitate alpha-latrotoxin-induced channel formation. The stimulation of secretion by alpha-latrotoxin in neuroendocrine cells is a consequence of Ca(2+) influx through these alpha-latrotoxin-induced channels. In addition to channel formation, alpha-latrotoxin enhances secretion in permeabilized cells through interaction with the plasma membrane receptor CIRL/latrophilin. Finally, overexpression of CIRL/latrophilin inhibits Ca(2+)-dependent secretion in permeabilized chromaffin cells in the absence of alpha-latrotoxin. This effect represents a 'constitutive' action of the G-protein coupled receptor to specifically inhibit an ATP-dependent priming step in the secretory pathway. The effect suggests that the receptor may have an important modulatory role in synaptic transmission.

• Sugita S, Khvochtev M, Sudhof TC
• Neurexins are functional alpha-latrotoxin receptors.
• Neuron. 1999; 22: 489-96
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• Alpha-latrotoxin is a potent neurotoxin that triggers synaptic exocytosis. Surprisingly, two distinct neuronal receptors for alpha-latrotoxin have been described: CIRL/latrophilin 1 (CL1) and neurexin-1alpha. Alpha-latrotoxin is thought to trigger exocytosis by binding to CL1, while the role of neurexin 1alpha is uncertain. Using PC12 cells, we now demonstrate that neurexins indeed function as alpha-latrotoxin receptors that are at least as potent as CL1. Both alpha- and beta-neurexins represent autonomous alpha-latrotoxin receptors that are regulated by alternative splicing. Similar to CL1, truncated neurexins without intracellular sequences are fully active; therefore, neurexins and CL1 recruit alpha-latrotoxin but are not themselves involved in exocytosis. Thus, alpha-latrotoxin is unique among neurotoxins, because it utilizes two unrelated receptors, probably to amplify recruitment of alpha-latrotoxin to active sites.

• Matsushita H, Lelianova VG, Ushkaryov YA
• The latrophilin family: multiply spliced G protein-coupled receptors with differential tissue distribution.
• FEBS Lett. 1999; 443: 348-52
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• Latrophilin is a brain-specific Ca2+-independent receptor of alpha-latrotoxin, a potent presynaptic neurotoxin. We now report the finding of two novel latrophilin homologues. All three latrophilins are unusual G protein-coupled receptors. They exhibit strong similarities within their lectin, olfactomedin and transmembrane domains but possess variable C-termini. Latrophilins have up to seven sites of alternative splicing; some splice variants contain an altered third cytoplasmic loop or a truncated cytoplasmic tail. Only latrophilin-1 binds alpha-latrotoxin; it is abundant in brain and is present in endocrine cells. Latrophilin-3 is also brain-specific, whereas latrophilin-2 is ubiquitous. Together, latrophilins form a novel family of heterogeneous G protein-coupled receptors with distinct tissue distribution and functions.

• Martin NP, Leavitt LM, Sommers CM, Dumont ME
• Assembly of G protein-coupled receptors from fragments: identification of functional receptors with discontinuities in each of the loops connecting transmembrane segments.
• Biochemistry. 1999; 38: 682-95
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• The alpha-factor receptor of the yeast Saccharomyces cerevisiae is a member of the superfamily of G protein-coupled receptors that mediate signal transduction in response to sensory and chemical stimuli. All members of this superfamily contain seven predicted transmembrane segments. We have created a series of genes encoding alpha-factor receptors with amino- or carboxyl-terminal truncations at each of the loop regions connecting transmembrane segments. Split receptors containing a discontinuity in the peptide backbone were synthesized by coexpressing pairs of truncated receptor fragments in yeast. Complementary pairs of fragments split at sites within each of the cytoplasmic and extracellular loops were capable of assembling and transducing a signal in response to alpha-factor binding. One pair of noncomplementary fragments containing a deletion in the second intracellular loop of the receptor also yielded a functional receptor. Coexpression of certain combinations of overlapping fragments containing supernumerary transmembrane segments also led to formation of functional receptors, apparently because of proteolytic trimming of overlapping regions. Coexpression of truncated receptor fragments with full-length receptors had no effect on signaling by the full-length receptors. These results demonstrate the following: (1) Correct folding of the alpha-factor receptor does not require a covalent connection between any pair of transmembrane segments that are adjacent in the sequence. (2) Most of the second intracellular loop of the receptor is not required for function. (3) The structure of the receptor cannot, in most cases, tolerate the presence of extra transmembrane segments. (4) None of the truncated fragments of the alpha-factor receptor can efficiently oligomerize with normal receptors in such a way as to inhibit receptor function.

• Volynski KE, Nosyreva ED, Ushkaryov YA, Grishin EV
• Functional expression of alpha-latrotoxin in baculovirus system.
• FEBS Lett. 1999; 442: 25-8
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• To facilitate the study of the mechanism of alpha-latrotoxin action, it is necessary to create a biologically active recombinant toxin. Mature alpha-latrotoxin is naturally produced by post-translational cleavage, probably at two furin sites located at the N- and C-termini of the precursor. A recombinant baculovirus has now been constructed, which encodes the melittin signal peptide fused to the 130-kDa mature toxin between the furin sites. Insect cells, infected with this baculovirus, secreted recombinant alpha-latrotoxin. This was partially purified and proved indistinguishable from the natural toxin with respect to its molecular mass, immunostaining, toxicity to mice, binding to alpha-latrotoxin receptors (latrophilin or neurexin Ialpha) and electrophysiological recording in the mouse diaphragm. The successful expression of recombinant alpha-latrotoxin permits mutational analysis of the toxin.

• Henkel AW, Sankaranarayanan S
• Mechanisms of alpha-latrotoxin action.
• Cell Tissue Res. 1999; 296: 229-33
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• The major component of black widow spider venom, alpha-latrotoxin, triggers massive exocytosis in a variety of neurosecretory cells. An important trigger for exocytosis is the calcium influx via alpha-latrotoxin-induced channels in biological membranes. However, this mechanism fails to explain exocytosis which occurred in the complete absence of extracellular calcium. Recently, sophisticated biochemical and molecular techniques have led to the discovery of novel alpha latrotoxin-binding membrane receptors: neurexins and latrophilin/CIRL (calcium-independent receptor for alpha-latrotoxin). Neurexins are single transmembrane proteins which bind to alpha-latrotoxin in a calcium-dependent manner and also interact with the synaptic vesicle protein, synaptotagmin. On the other hand, latrophilin is a seven-transmembrane protein and belongs to the family of G-protein-coupled receptors. The multitude of effects of alpha-latrotoxin on exocytosis in different cell systems and the nature of its membrane targets are discussed in this article. The molecular details of how alpha-latrotoxin binding is transduced eventually to exocytosis remain to be elucidated.

• Ichtchenko K et al.
• A novel ubiquitously expressed alpha-latrotoxin receptor is a member of the CIRL family of G-protein-coupled receptors.
• J Biol Chem. 1999; 274: 5491-8
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• Poisoning with alpha-latrotoxin, a neurotoxic protein from black widow spider venom, results in a robust increase of spontaneous synaptic transmission and subsequent degeneration of affected nerve terminals. The neurotoxic action of alpha-latrotoxin involves extracellular binding to its high affinity receptors as a first step. One of these proteins, CIRL, is a neuronal G-protein-coupled receptor implicated in the regulation of secretion. We now demonstrate that CIRL has two close homologs with a similar domain structure and high degree of overall identity. These novel receptors, which we propose to name CIRL-2 and CIRL-3, together with CIRL (CIRL-1) belong to a recently identified subfamily of large orphan receptors with structural features typical of both G-protein-coupled receptors and cell adhesion proteins. Northern blotting experiments indicate that CIRL-2 is expressed ubiquitously with highest concentrations found in placenta, kidney, spleen, ovary, heart, and lung, whereas CIRL-3 is expressed predominantly in brain similarly to CIRL-1. It appears that CIRL-2 can also bind alpha-latrotoxin, although its affinity to the toxin is about 14 times less than that of CIRL-1. When overexpressed in chromaffin cells, CIRL-2 increases their sensitivity to alpha-latrotoxin stimulation but also inhibits Ca2+-regulated secretion. Thus, CIRL-2 is a functionally competent receptor of alpha-latrotoxin. Our findings suggest that although the nervous system is the primary target of low doses of alpha-latrotoxin, cells of other tissues are also susceptible to the toxic effects of alpha-latrotoxin because of the presence of CIRL-2, a low affinity receptor of the toxin.

• Rahman MA, Ashton AC, Meunier FA, Davletov BA, Dolly JO, Ushkaryov YA
• Norepinephrine exocytosis stimulated by alpha-latrotoxin requires both external and stored Ca2+ and is mediated by latrophilin, G proteins and phospholipase C.
• Philos Trans R Soc Lond B Biol Sci. 1999; 354: 379-86
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• alpha-latrotoxin (LTX) stimulates massive release of neurotransmitters by binding to a heptahelical transmembrane protein, latrophilin. Our experiments demonstrate that latrophilin is a G-protein-coupled receptor that specifically associates with heterotrimeric G proteins. The latrophilin-G protein complex is very stable in the presence of GDP but dissociates when incubated with GTP, suggesting a functional interaction. As revealed by immunostaining, latrophilin interacts with G alpha q/11 and G alpha o but not with G alpha s, G alpha i or G alpha z, indicating that this receptor may couple to several G proteins but it is not promiscuous. The mechanisms underlying LTX-evoked norepinephrine secretion from rat brain nerve terminals were also studied. In the presence of extracellular Ca2+, LTX triggers vesicular exocytosis because botulinum neurotoxins E, Cl or tetanus toxin inhibit the Ca(2+)-dependent component of the toxin-evoked release. Based on (i) the known involvement of G alpha q in the regulation of inositol-1,4,5-triphosphate generation and (ii) the requirement for Ca2+ in LTX action, we tested the effect of inhibitors of Ca2+ mobilization on the toxin-evoked norepinephrine release. It was found that aminosteroid U73122, which inhibits the coupling of G proteins to phospholipase C, blocks the Ca(2+)-dependent toxin's action. Thapsigargin, which depletes intracellular Ca2+ stores, also potently decreases the effect of LTX in the presence of extracellular Ca2+. On the other hand, clostridial neurotoxins or drugs interfering with Ca2+ metabolism do not inhibit the Ca2(+)-independent component of LTX-stimulated release. In the absence of Ca2+, the toxin induces in the presynaptic membrane non-selective pores permeable to small fluorescent dyes; these pores may allow efflux of neurotransmitters from the cytoplasm. Our results suggest that LTX stimulates norepinephrine exocytosis only in the presence of external Ca2+ provided intracellular Ca2+ stores are unperturbed and that latrophilin, G proteins and phospholipase C may mediate the mobilization of stored Ca2+, which then triggers secretion.

• Bittner MA, Krasnoperov VG, Stuenkel EL, Petrenko AG, Holz RW
• A Ca2+-independent receptor for alpha-latrotoxin, CIRL, mediates effects on secretion via multiple mechanisms.
• J Neurosci. 1998; 18: 2914-22
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• alpha-Latrotoxin (alpha-Ltx), a component of black widow spider venom, stimulates secretion from nerve terminals and from PC12 cells. In this study we examine the effects of expression of a newly cloned Ca2+-independent receptor for alpha-Ltx (CIRL) on secretion from bovine chromaffin cells. We first characterized the effect of alpha-Ltx on secretion from untransfected cells. alpha-Ltx, by binding in a Ca2+-independent manner to an endogenous receptor, causes subsequent Ca2+-dependent secretion from intact cells. The stimulation of secretion is correlated with Ca2+ influx caused by the toxin. In permeabilized cells in which the Ca2+ concentration is regulated by buffer, alpha-Ltx also enhances Ca2+-dependent secretion, indicating a direct role of the endogenous receptor in the secretory pathway. Expression of CIRL increased the sensitivity of intact and permeabilized cells to the effects of alpha-Ltx, demonstrating that this protein is functional in coupling to secretion. Importantly, in the absence of alpha-Ltx, the expression of CIRL specifically inhibited the ATP-dependent component of secretion in permeabilized cells without affecting the ATP-independent secretion. This suggests that this receptor modulates the normal function of the regulated secretory pathway and that alpha-Ltx may act by reversing the inhibitory effects of the receptor.

• Davletov BA et al.
• Vesicle exocytosis stimulated by alpha-latrotoxin is mediated by latrophilin and requires both external and stored Ca2+.
• EMBO J. 1998; 17: 3909-20
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• alpha-Latrotoxin (LTX) stimulates massive neurotransmitter release by two mechanisms: Ca2+-dependent and -independent. Our studies on norepinephrine secretion from nerve terminals now reveal the different molecular basis of these two actions. The Ca2+-dependent LTX-evoked vesicle exocytosis (abolished by botulinum neurotoxins) is 10-fold more sensitive to external Ca2+ than secretion triggered by depolarization or A23187; it does not, however, depend on the cation entry into terminals but requires intracellular Ca2+ and is blocked by drugs depleting Ca2+ stores and by inhibitors of phospholipase C (PLC). These data, together with binding studies, prove that latrophilin, which is linked to G proteins and inositol polyphosphate production, is the major functional LTX receptor. The Ca2+-independent LTX-stimulated release is not inhibited by botulinum neurotoxins or drugs interfering with Ca2+ metabolism and occurs via pores in the presynaptic membrane, large enough to allow efflux of neurotransmitters and other small molecules from the cytoplasm. Our results unite previously contradictory data about the toxin's effects and suggest that LTX-stimulated exocytosis depends upon the co-operative action of external and intracellular Ca2+ involving G proteins and PLC, whereas the Ca2+-independent release is largely non-vesicular.

• Dosil M, Giot L, Davis C, Konopka JB
• Dominant-negative mutations in the G-protein-coupled alpha-factor receptor map to the extracellular ends of the transmembrane segments.
• Mol Cell Biol. 1998; 18: 5981-91
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• G-protein-coupled receptors (GPCRs) transduce the signals for a wide range of hormonal and sensory stimuli by activating a heterotrimeric guanine nucleotide-binding protein (G protein). The analysis of loss-of-function and constitutively active receptor mutants has helped to reveal the functional properties of GPCRs and their role in human diseases. Here we describe the identification of a new class of mutants, dominant-negative mutants, for the yeast G-protein-coupled alpha-factor receptor (Ste2p). Sixteen dominant-negative receptor mutants were isolated based on their ability to inhibit the response to mating pheromone in cells that also express wild-type receptors. Detailed analysis of two of the strongest mutant receptors showed that, unlike other GPCR interfering mutants, they were properly localized at the plasma membrane and did not alter the stability or localization of wild-type receptors. Furthermore, their dominant-negative effect was inversely proportional to the relative amount of wild-type receptors and was reversed by overexpressing the G-protein subunits, suggesting that these mutants compete with the wild-type receptors for the G protein. Interestingly, the dominant-negative mutations are all located at the extracellular ends of the transmembrane segments, defining a novel region of the receptor that is important for receptor signaling. Altogether, our results identify residues of the alpha-factor receptor specifically involved in ligand binding and receptor activation and define a new mechanism by which GPCRs can be inactivated that has important implications for the evaluation of receptor mutations in other G-protein-coupled receptors.

• Geppert M et al.
• Neurexin I alpha is a major alpha-latrotoxin receptor that cooperates in alpha-latrotoxin action.
• J Biol Chem. 1998; 273: 1705-10
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• alpha-Latrotoxin is a potent neurotoxin from black widow spider venom that binds to presynaptic receptors and causes massive neurotransmitter release. A surprising finding was the biochemical description of two distinct cell surface proteins that bind alpha-latrotoxin with nanomolar affinities; Neurexin I alpha binds alpha-latrotoxin in a Ca(2+)-dependent manner, and CIRL/latrophilin binds in a Ca(2+)-independent manner. We have now generated and analyzed mice that lack neurexin I alpha to test its importance in alpha-latrotoxin action. alpha-Latrotoxin binding to brain membranes from mutant mice was decreased by almost 50% compared with wild type membranes; the decrease was almost entirely due to a loss of Ca(2+)-dependent alpha-latrotoxin binding sites. In cultured hippocampal neurons, alpha-latrotoxin was still capable of activating neurotransmission in the absence of neurexin I alpha. Direct measurements of [3H]glutamate release from synaptosomes, however, showed a major decrease in the amount of release triggered by alpha-latrotoxin in the presence of Ca2+. Thus neurexin I alpha is not essential for alpha-latrotoxin action but contributes to alpha-latrotoxin action when Ca2+ is present. Viewed as a whole, our results show that mice contain two distinct types of alpha-latrotoxin receptors with similar affinities and abundance but different properties and functions. The action of alpha-latrotoxin may therefore be mediated by independent parallel pathways, of which the CIRL/latrophilin pathway is sufficient for neurotransmitter release, whereas the neurexin I alpha pathway contributes to the Ca(2+)-dependent action of alpha-latrotoxin.

• Grishin EV
• Black widow spider toxins: the present and the future.
• Toxicon. 1998; 36: 1693-701
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• The venom of the black widow spider Latrodectus mactans tredisimguttatus was found to contain a family of high molecular weight toxic proteins inducing a sharp increase in transmitter secretion from the affected nerve endings, which are highly specific for vertebrates, or for insects, or for crustaceans. Along with the known alpha-latrotoxin, five latroinsectotoxins affecting the neurotransmitter release from presynaptic endings of insects and one latrocrustatoxin active only for crustaceans were isolated and studied in detail. Alpha-latrotoxin provokes a massive transmitter release from different nerve endings of vertebrates, whereas other toxins increase the secretion process either in insects or crustaceans. The cDNAs encoding the putative alpha-latrotoxin and two latroinsectotoxins (alpha-latroinsectotoxin and delta-latroinsectotoxin) precursors were cloned and sequenced. These toxins are polypeptides of about 1000 amino acids and share a high level of amino acid identity. Analysis of amino acid sequences of the three toxins reveals the central regions being almost entirely composed of series of ankyrin-like repeats. Taking into account the size and multifunctional properties of latrotoxin its molecule can be divided into several functional domains. Immunochemical experiments indicated the presence in the alpha-latrotoxin molecule of distinguishable functional domains responsible for ionophoric and secretogenic actions. The highly purified preparation of alpha-latrotoxin was shown to contain an additional component, a low molecular weight protein structurally related to crustacean hyperglycemic hormones. Several attempts were made to characterize and isolate alpha-latrotoxin receptor components. The existence of Ca-dependent and Ca-independent binding proteins was found in the presynaptic membrane preparations.

• Ichtchenko K, Khvotchev M, Kiyatkin N, Simpson L, Sugita S, Sudhof TC
• alpha-latrotoxin action probed with recombinant toxin: receptors recruit alpha-latrotoxin but do not transduce an exocytotic signal.
• EMBO J. 1998; 17: 6188-99
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• alpha-Latrotoxin stimulates neurotransmitter release probably by binding to two receptors, CIRL/latrophilin 1 (CL1) and neurexin Ialpha. We have now produced recombinant alpha-latrotoxin (LtxWT) that is as active as native alpha-latrotoxin in triggering synaptic release of glutamate, GABA and norepinephrine. We have also generated three alpha-latrotoxin mutants with substitutions in conserved cysteine residues, and a fourth mutant with a four-residue insertion. All four alpha-latrotoxin mutants were found to be unable to trigger release. Interestingly, the insertion mutant LtxN4C exhibited receptor-binding affinities identical to wild-type LtxWT, bound to CL1 and neurexin Ialpha as well as LtxWT, and similarly stimulated synaptic hydrolysis of phosphatidylinositolphosphates. Therefore, receptor binding by alpha-latrotoxin and stimulation of phospholipase C are insufficient to trigger exocytosis. This conclusion was confirmed in experiments with La3+ and Cd2+. La3+ blocked release triggered by LtxWT, whereas Cd2+ enhanced it. Both cations, however, had no effect on the stimulation by LtxWT of phosphatidylinositolphosphate hydrolysis. Our data show that receptor binding by alpha-latrotoxin and activation of phospholipase C do not by themselves trigger exocytosis. Thus receptors recruit alpha-latrotoxin to its point of action without activating exocytosis. Exocytosis probably requires an additional receptor-independent activity of alpha-latrotoxin that is selectively inhibited by the LtxN4C mutation and by La3+.

• Lang J, Ushkaryov Y, Grasso A, Wollheim CB
• Ca2+-independent insulin exocytosis induced by alpha-latrotoxin requires latrophilin, a G protein-coupled receptor.
• EMBO J. 1998; 17: 648-57
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• alpha-Latrotoxin (alpha-LTX) induces exocytosis of small synaptic vesicles (SSVs) in neuronal cells both by a calcium-independent mechanism and by opening cation-permeable pores. Since the basic molecular events regulating exocytosis in neurons and endocrine cells may be similar, we have used the exocytosis of insulin-containing large dense core vesicles (LDCVs) as a model system. In primary pancreatic beta-cells and in the derived cell lines INS-1 and MIN6, alpha-LTX increased insulin release in the absence of extracellular calcium, but the insulin-secreting cell lines HIT-T15 and RINm5F were unresponsive. alpha-LTX did not alter membrane potential or cytosolic calcium, and its stimulatory effect on exocytosis was still observed in pre-permeabilized INS-1 cells kept at 0.1 microM Ca2+. Consequently, pore formation or ion fluxes induced by alpha-LTX could be excluded. The Ca2+-independent alpha-LTX-binding protein, latrophilin, is a novel member of the secretin family of G protein-coupled receptors (GPCR). Sensitivity to alpha-LTX correlated with expression of latrophilin, but not with synaptotagmin I or neurexin Ialpha expression. Moreover, transient expression of latrophilin in HIT-T15 cells conferred alpha-LTX-induced exocytosis. Our results indicate that direct stimulation of exocytosis by a GPCR mediates the Ca2+-independent effects of alpha-LTX in the absence of altered ion fluxes. Therefore, direct regulation by receptor-activated heterotrimeric G proteins constitutes an important feature of the endocrine exocytosis of insulin-containing LDCVs and may also apply to SSV exocytosis in neurons.

• Krasnoperov VG et al.
• alpha-Latrotoxin stimulates exocytosis by the interaction with a neuronal G-protein-coupled receptor.
• Neuron. 1997; 18: 925-37
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• alpha-Latrotoxin is a potent stimulator of neurosecretion. Its action requires extracellular binding to high affinity presynaptic receptors. Neurexin I alpha was previously described as a high affinity alpha-latrotoxin receptor that binds the toxin only in the presence of calcium ions. Therefore, the interaction of alpha-latrotoxin with neurexin I alpha cannot explain how alpha-latrotoxin stimulates neurotransmitter release in the absence of calcium. We describe molecular cloning and functional expression of the calcium-independent receptor of alpha-latrotoxin (CIRL), which is a second high affinity alpha-latrotoxin receptor that may be the major mediator of alpha-latrotoxin's effects. CIRL appears to be a novel orphan G-protein-coupled receptor, a member of the secretin receptor family. In contrast with other known serpentine receptors, CIRL has two subunits of the 120 and 85 kDa that are the result of endogenous proteolytic cleavage of a precursor polypeptide. CIRL is found in brain where it is enriched in the striatum and cortex. Expression of CIRL in chromaffin cells increases the sensitivity of the cells to the effects of alpha-latrotoxin, demonstrating that this protein is functional in coupling to secretion. Syntaxin, a component of the fusion complex, copurifies with CIRL on an alpha-latrotoxin affinity column and forms stable complexes with this receptor in vitro. Interaction of CIRL with a specific presynaptic neurotoxin and with a component of the docking-fusion machinery suggests its role in regulation of neurosecretion.

• Feng Y, Gregor P
• Cloning of a novel member of the G protein-coupled receptor family related to peptide receptors.
• Biochem Biophys Res Commun. 1997; 231: 651-4
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• We have used PCR with degenerate oligonucleotide primers to clone novel members of the G protein-coupled receptor (GPCR) superfamily. We report here a novel gene, CEPR, which encodes a candidate receptor that is most similar to the peptide receptor family. The coding region of the human CEPR gene predicts a seven transmembrane domain (TM) receptor of 375 amino acids. CEPR has 28-30 percent amino acid identity to angiotensin II and interleukin 8 receptors, and slightly lower percent identity to many other GPCRs. Northern blot analysis reveals a 3.3 kb CEPR transcript in different regions of human brain and in various peripheral tissues. The ubiquitous tissue distribution of CEPR, its expression in early development, and its conservation in evolution indicate a potentially important biological function for this receptor and its putative peptide ligand.

• Lelianova VG et al.
• Alpha-latrotoxin receptor, latrophilin, is a novel member of the secretin family of G protein-coupled receptors.
• J Biol Chem. 1997; 272: 21504-8
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• alpha-Latrotoxin (LTX) stimulates massive exocytosis of synaptic vesicles and may help to elucidate the mechanism of regulation of neurosecretion. We have recently isolated latrophilin, the synaptic Ca2+-independent LTX receptor. Now we demonstrate that latrophilin is a novel member of the secretin family of G protein-coupled receptors that are involved in secretion. Northern blot analysis shows that latrophilin message is present only in neuronal tissue. Upon expression in COS cells, the cloned protein is indistinguishable from brain latrophilin and binds LTX with high affinity. Latrophilin physically interacts with a Galphao subunit of heterotrimeric G proteins, because the two proteins co-purify in a two-step affinity chromatography. Interestingly, extracellular domain of latrophilin is homologous to olfactomedin, a soluble neuronal protein thought to participate in odorant binding. Our findings suggest that latrophilin may bind unidentified endogenous ligands and transduce signals into nerve terminals, thus implicating G proteins in the control of synaptic vesicle exocytosis.

• David NE, Gee M, Andersen B, Naider F, Thorner J, Stevens RC
• Expression and purification of the Saccharomyces cerevisiae alpha-factor receptor (Ste2p), a 7-transmembrane-segment G protein-coupled receptor.
• J Biol Chem. 1997; 272: 15553-61
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• A plasmid vector was developed that permitted high-level expression of a functional form of the Saccharomyces cerevisiae alpha-factor receptor (the STE2 gene product) tagged at its C-terminal end with an epitope (FLAG) and a His6 tract. When expressed in yeast from this plasmid, Ste2p was produced at a level at least 3-fold higher than that reported previously for any other 7-transmembrane-segment receptor expressed in the same cells. For purification, isolated cell membranes containing the overexpressed receptor were solubilized with detergent under specific conditions and subjected to immobilized metal affinity chromatography. Yields as high as 1 mg of nearly homogeneous (95%) receptor were routinely obtained even from relatively small scale preparations (60 g of frozen cell paste). The purified receptor was reconstituted into artificial phospholipid vesicles. Radioligand binding studies demonstrated that the purified receptor, in the reconstituted vesicles, bound its tridecapeptide ligand (alpha-factor) with a KD (155 nM) consistent with the affinity expected for this receptor in the absence of its associated G protein. Efficient restoration of ligand binding activity upon reconstitution required the addition of solubilized membranes prepared from a yeast strain lacking the receptor. Sufficient amounts of active material can be obtained by this procedure to allow physical studies of this receptor and other 7-transmembrane-segment receptors expressed in this system.

• Konopka JB, Margarit SM, Dube P
• Mutation of Pro-258 in transmembrane domain 6 constitutively activates the G protein-coupled alpha-factor receptor.
• Proc Natl Acad Sci U S A. 1996; 93: 6764-9
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• The alpha-factor pheromone receptor stimulates MATa yeast cells to undergo conjugation. The receptor contains seven transmembrane domains that function in ligand binding and in transducing a signal to the cytoplasmic receptor sequences to mediate G protein activation. A genetic screen was used to isolate receptor mutations that constitutively signal in the absence of alpha-factor. The Pro-258-->Leu (P258L) mutation caused constitutive receptor signaling that was equivalent to about 45% of the maximum level observed in wild-type cells stimulated with alpha-factor. Mutations of both Pro-258 and the adjacent Ser-259 to Leu increased constitutive signaling to > or = 90% of the maximum level. Since Pro-258 occurs in the central portion of transmembrane domain 6, and since proline residues are expected to cause a kink in alpha-helical domains, the P258L mutation is predicted to alter the structure of transmembrane domain 6. The P258L mutation did not result in a global distortion of receptor structure because alpha-factor bound to the mutant receptors with high affinity and induced even higher levels of signaling. These results suggest that sequences surrounding Pro-258 may be involved in ligand activation of the receptor. Conformational changes in transmembrane domain 6 may effect a change in the adjacent sequences in the third intracellular loop that are thought to function in G protein activation. Greater than 90% of all G protein-coupled receptors contain a proline residue at a similar position in transmembrane domain 6, suggesting that this aspect of receptor activation may be conserved in other receptors.

• Davletov BA, Shamotienko OG, Lelianova VG, Grishin EV, Ushkaryov YA
• Isolation and biochemical characterization of a Ca2+-independent alpha-latrotoxin-binding protein.
• J Biol Chem. 1996; 271: 23239-45
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• alpha-Latrotoxin, a black widow spider neurotoxin, can bind to high affinity receptors on the presynaptic plasma membrane and stimulate massive neurotransmitter release in the absence of Ca2+. Neurexins, previously isolated as alpha-latrotoxin receptors, require Ca2+ for their interaction with the toxin and, thus, may not participate in the Ca2+-independent alpha-latrotoxin activity. We now report the isolation of a novel protein that binds alpha-latrotoxin with high affinity in the presence of various divalent cations (Ca2+, Mg2+, Ba2+, and Sr2+) as well as in EDTA. This protein, termed here latrophilin, has been purified from detergent-solubilized bovine brain membranes by affinity chromatography on immobilized alpha-latrotoxin and concentrated on a wheat germ agglutinin affinity column. The single polypeptide chain of latrophilin is N-glycosylated and has an apparent molecular weight of 120,000. Sucrose gradient centrifugations demonstrated that latrophilin and alpha-latrotoxin form a stable equimolar complex. In the presence of the toxin, anti-alpha-latrotoxin antibodies precipitated iodinated latrophilin, whose binding to immobilized toxin was characterized by a dissociation constant of 0.5-0.7 nM. This presumably membrane-bound protein is localized to and differentially distributed among neuronal tissues, with about four times more latrophilin expressed in the cerebral cortex than in the cerebellum; subcellular fractionation showed that the protein is highly enriched in synaptosomal plasma membranes. Our data suggest that latrophilin may represent the Ca2+-independent receptor and/or molecular target for alpha-latrotoxin.

• Roglic A, Prossnitz ER, Cavanagh SL, Pan Z, Zou A, Ye RD
• cDNA cloning of a novel G protein-coupled receptor with a large extracellular loop structure.
• Biochim Biophys Acta. 1996; 1305: 39-43
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• A cDNA designated as AZ3B has been isolated from a differentiated HL-6 0 cell cDNA library with a probe derived from the N-formyl peptide receptor gene. The 1.97-kb cDNA encodes a novel G protein-coupled receptor (GPCR) with 482 amino acids. In addition to the predicted 7 transmembrane domains common to all GPCRs, the protein encoded by AZ3B contains a large extracellular loop of approximately 172 amino acids between the fourth and the fifth transmembrane domains, a feature unique among the hundreds of GPCRs identified to date. High sequence homology exists between the AZ3B protein and a number of chemoattractant receptors in the amino-terminal 170 residues and the carboxyl-terminal 150 residues. Northern and flow cytometric analyses suggested that the AZ3B message and protein are widely expressed in several differentiated hematopoietic cell lines, in the lung, placenta, heart, and endothelial cells. We postulate that the AZ3B protein defines a distinct group of receptors within the GPCR superfamily.

• Krasnoperov VG, Beavis R, Chepurny OG, Little AR, Plotnikov AN, Petrenko AG
• The calcium-independent receptor of alpha-latrotoxin is not a neurexin.
• Biochem Biophys Res Commun. 1996; 227: 868-75
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• alpha-Latrotoxin (alpha-LTx), a vertebrate neurotoxin isolated from Black Widow Spider venom, causes massive spontaneous neurotransmitter release. The molecular mechanism(s) by which the toxin exerts its effect is largely unknown. Here we report identification and purification of a novel membrane receptor with high affinity for alpha-LTx. Unlike neurexin Ia, a previously described high affinity alpha-LTx receptor, this novel protein binds alpha-LTx independently of Ca2+ presence and therefore may be a mediator of the calcium-independent stimulation of neurotransmitter release by alpha-latrotoxin. The major protein component of calcium-independent alpha-LTx receptors is a novel M(r) 120,000 protein which does not belong to the neurexin family. Among several tissues tested, the M(r) 120,000 protein was found only in brain.

• Davletov BA, Krasnoperov V, Hata Y, Petrenko AG, Sudhof TC
• High affinity binding of alpha-latrotoxin to recombinant neurexin I alpha.
• J Biol Chem. 1995; 270: 23903-5
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• alpha-Latrotoxin is a potent neurotoxin from black widow spider venom that stimulates neurotransmitter release. alpha-Latrotoxin is thought to act by binding to a high affinity receptor on presynaptic nerve terminals. In previous studies, high affinity alpha-latrotoxin binding proteins were isolated and demonstrated to contain neurexin I alpha as a major component. Neurexin I alpha is a cell surface protein that exists in multiple differentially spliced isoforms and belongs to a large family of neuron-specific proteins. Using a series of neurexin I-IgG fusion proteins, we now show that recombinant neurexin I alpha binds alpha-latrotoxin directly with high affinity (Kd approximately 4 nM). Binding of alpha-latrotoxin to recombinant neurexin I alpha is dependent on Ca2+ (EC50 approximately 30 microM). Our data suggest that neurexin I alpha is a Ca(2+)-dependent high affinity receptor for alpha-latrotoxin.

• Schwartz TW
• Locating ligand-binding sites in 7TM receptors by protein engineering.
• Curr Opin Biotechnol. 1994; 5: 434-44
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• Over the past year, mutational analysis of peptide receptors has started to change our understanding of the interaction between G protein coupled receptors and their ligands, an area previously almost totally dominated by results from studies of monoamine receptors. A picture is currently emerging, in which small ligands appear to bind in three (more or less) overlapping ligand-binding pockets in between the transmembrane segments. In contrast, contact residues for peptide and protein ligands have mainly been found in exterior regions of peptide and protein receptors. It is also becoming increasingly clear that agonists and antagonists may interact in vastly different manners, even though they are competitive ligands for a common receptor.

• Pin JP, Joly C, Heinemann SF, Bockaert J
• Domains involved in the specificity of G protein activation in phospholipase C-coupled metabotropic glutamate receptors.
• EMBO J. 1994; 13: 342-8
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• G protein-coupled glutamate receptors (mGluR) have recently been characterized. These receptors have seven putative transmembrane domains, but display no sequence homology with the large family of G protein-coupled receptors. They constitute therefore a new family of receptors. Whereas mGluR1 and mGluR5 activate phospholipase C (PLC), mGluR2, mGluR3, mGluR4 and mGluR6 inhibit adenylyl cyclase (AC) activity. The third putative intracellular loop, which determines the G protein specificity in many G protein-coupled receptors, is highly conserved among mGluRs, and may therefore not be involved in the specific recognition of G proteins in this receptor family. By constructing chimeric receptors between the AC-coupled mGluR3 and the PLC-coupled mGluR1c, we report here that both the C-terminal end of the second intracellular loop and the segment located downstream of the seventh transmembrane domain are necessary for the specific activation of PLC by mGluR1c. These two segments are rich in basic residues and are likely to be amphipathic alpha-helices, two characteristics of the G protein interacting domains of all G protein-coupled receptors. This indicates that whereas no amino acid sequence homology between mGluRs and the other G protein-coupled receptors can be found, their G protein interacting domains have similar structural features.

• Petrenko AG et al.
• Polypeptide composition of the alpha-latrotoxin receptor. High affinity binding protein consists of a family of related high molecular weight polypeptides complexed to a low molecular weight protein.
• J Biol Chem. 1993; 268: 1860-7
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• alpha-Latrotoxin is a vertebrate neurotoxin from black widow spider venom that causes massive neurotransmitter release. In order to gain insight into the mechanism of action of alpha-latrotoxin, we have studied alpha-latrotoxin-binding proteins from bovine and rat brain. Proteins purified by affinity chromatography on immobilized alpha-latrotoxin were investigated. Two sets of proteins were isolated: 1) three polypeptides of M(r) 79,000, 65,000, and 43,000 that were eluted from immobilized alpha-latrotoxin by increasing KCl concentrations in the presence of Ca2+, and 2) a family of related proteins ranging in molecular weight from 160,000 to 220,000 and a low molecular weight component of M(r) 29,000 that were eluted from immobilized alpha-latrotoxin only after removal of Ca2+. Amino acid sequences of these proteins demonstrated that all of these proteins represent novel proteins except for the M(r) 65,000 polypeptide, which is identical with synaptotagmin (Petrenko, A. G., Perin, M. S., Davletov, B. A., Ushkaryov, Y. A., Geppert, M., and Sudhof, T. C. (1991) Nature 353, 65-68). Surprisingly, the M(r) 79,000 and 43,000 proteins were also found in tissues insensitive to alpha-latrotoxin action. Since these proteins do not bind 125I-alpha-latrotoxin with high affinity, their purification probably is not physiologically significant. On the other hand, the fractions containing the M(r) 160,000-220,000 and 29,000 polypeptides bound alpha-latrotoxin with high affinity. Sucrose gradient centrifugations and anion exchange chromatography suggested that most of the M(r) 160,000-220,000 proteins were complexed with the M(r) 29,000 protein. alpha-Latrotoxin binding correlated with the presence of the M(r) 160,000-220,000 proteins and M(r) 29,000 polypeptide, and alpha-latrotoxin formed stable complexes with the M(r) 160,000-220,000 proteins. Accordingly, the alpha-latrotoxin receptor consists of a high molecular weight protein (M(r) 160,000-220,000) that is complexed with one or several copies of an M(r) 29,000 polypeptide. In addition, the receptor is found in a less tight association with synaptotagmin but not with other polypeptides.

• Kiyatkin N, Dulubova I, Chekhovskaya I, Lipkin A, Grishin E
• Structure of the low molecular weight protein copurified with alpha-latrotoxin.
• Toxicon. 1992; 30: 771-4
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• Some samples of latrotoxin purified from the black widow spider venom contain two components: alpha-latrotoxin (M(r) approximately 130,000) and a low mol. wt protein with M(r) about 8000. Clones carrying the cDNA sequence for the low mol. wt protein copurified with alpha-latrotoxin were isolated from spider venom glands. Nucleotide sequence analysis of the cloned cDNA revealed the primary structure of the polypeptide to be 18 amino acids signal peptide and 70 amino acids protein chain with mol. wt of 7947 and pI of approximately 4.0. The protein exhibits certain structural homology with erabutoxin-a from the sea snake.

• Malgaroli A, DeCamilli P, Meldolesi J
• Distribution of alpha latrotoxin receptor in the rat brain by quantitative autoradiography: comparison with the nerve terminal protein, synapsin I.
• Neuroscience. 1989; 32: 393-404
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• Tissue slice autoradiography was employed to reveal the brain distribution of the receptor for alpha latrotoxin, the presynaptic neurotoxin of the black widow spider venom. The receptor distribution pattern was compared with that of a marker protein for nerve endings, synapsin I, a phosphoprotein known to be present within nerve terminals. The alpha latrotoxin receptor and synapsin I were detected in gray matter-containing regions but their relative amounts were not constant. In the cerebral cortex and in the caudatum their distribution was similar, while in the hippocampus they were both abundant, but their distribution varied: synapsin I labeling was heavier in CA4 and CA3, alpha latrotoxin receptor labeling in CA1 and dentate gyrus. A dissociation was also observed in the globus pallidus and in the lateral thalamic nuclear complex, where alpha latrotoxin receptor labeling was very weak. The most striking dissociation occurred in the cerebellum, where the molecular layer was strongly labeled for synapsin I, but almost unlabeled for the alpha latrotoxin receptor, which was more concentrated in the granular layer. Taken as a whole, the data appear compatible with a widespread localization of the alpha latrotoxin receptor at synapses. However, they also suggest that either some nerve terminals are insensitive to alpha latrotoxin, or the receptor for the toxin is not present at a similar concentration in all presynaptic plasma membranes.