SMART MODE:

NORMAL GENOMIC

• Piddini E, Schmid JA, de Martin R, Dotti CG
• The Ras-like GTPase Gem is involved in cell shape remodelling and interacts with the novel kinesin-like protein KIF9.
• EMBO J. 2001; 20: 4076-87
• Display abstract

• Gem belongs to the Rad/Gem/Kir (RGK) subfamily of Ras-related GTPases, which also comprises Rem, Rem2 and Ges. The RGK family members Ges and Rem have been shown to produce endothelial cell sprouting and reorganization of the actin cytoskeleton upon overexpression. Here we show that high intracellular Gem levels promote profound changes in cell morphology and we investigate how this phenotype arises dynamically. We also show that this effect requires intact microtubules and microfilaments, and that Gem is associated with both cytoskeletal components. In order to investigate the mechanisms of Gem recruitment to the cytoskeleton, we performed a yeast two-hybrid screen and identified a novel kinesin-like protein, termed KIF9, as a new Gem interacting partner. We further show that Gem and KIF9 interact by co-immunoprecipitation. Furthermore, Gem and KIF9 display identical patterns of gene expression in different tissues and developmental stages. The Gem- KIF9 interaction reported here is the first molecular link between RGK family members and the microtubule cytoskeleton.

• Chatah NE, Abrams CS
• G-protein-coupled receptor activation induces the membrane translocation and activation of phosphatidylinositol-4-phosphate 5-kinase ialpha by a rac- and rho-dependent pathway.
• J Biol Chem. 2001; 276: 34059-65
• Display abstract

• Phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) mediates cell motility and changes in cell shape in response to extracellular stimuli. In platelets, it is synthesized from PI4P by PIP5K in response to stimulation of a G-protein-coupled receptor by an agonist, such as the thrombin. In the present study, we have addressed the pathway that induces PIP5K Ialpha activation following the addition of thrombin. Under resting condition expressed PIP5K Ialpha was predominantly localized in a perinuclear distribution. After stimulation of the thrombin receptor, PAR1, or overexpression of a constitutively active variant of Galpha(q), PIP5K Ialpha translocated to the plasma membrane. Movement of PIP5K Ialpha to the cell membrane was dependent on both GTP-bound Rac and Rho, but not Arf, because: 1) inactive GDP-bound variants of either Rac or Rho blocked the translocation induced by constitutively active Galpha(q), 2) constitutively GTP-bound active variants of Rac or Rho induced PIP5K Ialpha translocation in the absence of other stimuli, and 3) constitutively active variants of Arf1 or Arf6 failed to induce membrane translocation of PIP5K Ialpha. In addition, a dominant negative variant of Rho blocked the PIP5K Ialpha membrane translocation induced by constitutively active Rac, whereas dominant negative variants of either Rac or Arf6 failed to block PIP5K Ialpha membrane translocation induced by constitutively active Rho. This implies that the effect on PIP5K Ialpha by Rac is indirect, and requires the activation of Rho. In contrast to the findings with PIP5K Ialpha, the related lipid kinase PIP4K failed to undergo translocation after stimulation by small GTP-binding proteins Rac or Rho. We also tested whether membrane localization of PIP5K Ialpha correlated with an increase in its lipid kinase activity and found that co-expressing of PIP5K Ialpha with either constitutively active Galpha(q), Rac, or Rho led to a 5- to 7-fold increase in PIP5K Ialpha activity. Thus, these findings suggest that stimulation of a G-protein-coupled receptor (PAR1) leads to the sequential activation of Galpha(q), Rac, Rho, and PIP5K Ialpha. Once activated and translocated to the cell membrane, PIP5K Ialpha becomes available to phosphorylate PI4P to generate PI4,5P(2) on the plasma membrane.

• Allin C, Ahmadian MR, Wittinghofer A, Gerwert K
• Monitoring the GAP catalyzed H-Ras GTPase reaction at atomic resolution in real time.
• Proc Natl Acad Sci U S A. 2001; 98: 7754-9
• Display abstract

• The molecular reaction mechanism of the GTPase-activating protein (GAP)-catalyzed GTP hydrolysis by Ras was investigated by time resolved Fourier transform infrared (FTIR) difference spectroscopy using caged GTP (P(3)-1-(2-nitro)phenylethyl guanosine 5'-O-triphosphate) as photolabile trigger. This approach provides the complete GTPase reaction pathway with time resolution of milliseconds at the atomic level. Up to now, one structural model of the GAP x Ras x GDP x AlF(x) transition state analog is known, which represents a "snap shot" along the reaction-pathway. As now revealed, binding of GAP to Ras x GTP shifts negative charge from the gamma to beta phosphate. Such a shift was already identified by FTIR in GTP because of Ras binding and is now shown to be enhanced by GAP binding. Because the charge distribution of the GAP x Ras x GTP complex thus resembles a more dissociative-like transition state and is more like that in GDP, the activation free energy is reduced. An intermediate is observed on the reaction pathway that appears when the bond between beta and gamma phosphate is cleaved. In the intermediate, the released P(i) is strongly bound to the protein and surprisingly shows bands typical of those seen for phosphorylated enzyme intermediates. All these results provide a mechanistic picture that is different from the intrinsic GTPase reaction of Ras. FTIR analysis reveals the release of P(i) from the protein complex as the rate-limiting step for the GAP-catalyzed reaction. The approach presented allows the study not only of single proteins but of protein-protein interactions without intrinsic chromophores, in the non-crystalline state, in real time at the atomic level.

• van Nieuw Amerongen GP, van Hinsbergh VW
• Cytoskeletal effects of rho-like small guanine nucleotide-binding proteins in the vascular system.
• Arterioscler Thromb Vasc Biol. 2001; 21: 300-11
• Display abstract

• Rho-like small GTPases, with their main representatives (Rho, Rac, and Cdc42), have been recognized in the past decade as key regulators of the F-actin cytoskeleton. Rho-like small GTPases are now known to play a major role in vascular processes caused by changes in the actin cytoskeleton, such as smooth muscle cell contraction, endothelial permeability, platelet activation, and leukocyte migration. Data are now accumulating regarding the involvement of Rho GTPases in vascular disorders associated with vascular remodeling, altered cell contractility, and cell migration. The unraveling of signal transduction pathways used by the Rho-like GTPases revealed many upstream regulators and downstream effector molecules, and their number is still growing. An important action of Rho, Rac, and Cdc42 is their ability to regulate the phosphorylation status of the myosin light chain, a major regulator of actin-myosin interaction. Present knowledge of the Rho-like small GTPases has resulted in the development of promising new strategies for the treatment of many vascular disorders, including hypertension, vasospasms, and vascular leakage.

• Takai Y, Sasaki T, Matozaki T
• Small GTP-binding proteins.
• Physiol Rev. 2001; 81: 153-208
• Display abstract

• Small GTP-binding proteins (G proteins) exist in eukaryotes from yeast to human and constitute a superfamily consisting of more than 100 members. This superfamily is structurally classified into at least five families: the Ras, Rho, Rab, Sar1/Arf, and Ran families. They regulate a wide variety of cell functions as biological timers (biotimers) that initiate and terminate specific cell functions and determine the periods of time for the continuation of the specific cell functions. They furthermore play key roles in not only temporal but also spatial determination of specific cell functions. The Ras family regulates gene expression, the Rho family regulates cytoskeletal reorganization and gene expression, the Rab and Sar1/Arf families regulate vesicle trafficking, and the Ran family regulates nucleocytoplasmic transport and microtubule organization. Many upstream regulators and downstream effectors of small G proteins have been isolated, and their modes of activation and action have gradually been elucidated. Cascades and cross-talks of small G proteins have also been clarified. In this review, functions of small G proteins and their modes of activation and action are described.

• Furukawa Y et al.
• Isolation of a novel human gene, ARHGAP9, encoding a rho-GTPase activating protein.
• Biochem Biophys Res Commun. 2001; 284: 643-9
• Display abstract

• Members of the Rho family of small guanosine triphosphatases (Rho-GTPases) have emerged as key coordinators of signaling pathways leading to remodeling of the actin cytoskeleton, a process that plays a critical role in cell adhesion and migration. However, the precise regulatory mechanisms remain to be elucidated. Here we report isolation of a novel human gene, ARHGAP9, which encodes a protein containing a Rho-GTPase activating protein (Rho-GAP) domain, a src-homology 3 (SH3) domain, a pleckstrin homology (PH) region, and a WW domain. In vitro, the recombinant protein revealed substantial GAP activity toward Cdc42Hs and Rac1, and less toward RhoA. The transcript was predominantly expressed in peripheral blood leukocytes, spleen, and thymus. Exogenous expression of the entire coding region of ARHGAP9 into human leukemia KG-1 cells repressed adhesion of the cells to fibronectin and collagen IV. Our results indicate that ARHGAP9 is involved in regulating adhesion of hematopoietic cells to extracellular matrix. Copyright 2001 Academic Press.

• Merithew E, Hatherly S, Dumas JJ, Lawe DC, Heller-Harrison R, Lambright DG
• Structural plasticity of an invariant hydrophobic triad in the switch regions of Rab GTPases is a determinant of effector recognition.
• J Biol Chem. 2001; 276: 13982-8
• Display abstract

• Rab GTPases function as regulatory components of an evolutionarily conserved machinery that mediates docking, priming, and fusion of vesicles with intracellular membranes. We have previously shown that the active conformation of Rab3A is stabilized by a substantial hydrophobic interface between the putative conformational switch regions (Dumas, J. J., Zhu, Z., Connolly, J. L., and Lambright, D. G. (1999) Structure 7, 413-423). A triad of invariant hydrophobic residues at this switch interface (Phe-59, Trp-76, and Tyr-91) represents a major interaction determinant between the switch regions of Rab3A and the Rab3A-specific effector Rabphilin3A (Ostermeier, C., and Brunger, A. T. (1999) Cell 96, 363-374). Here, we report the crystal structure of the active form of Rab5C, a prototypical endocytic Rab GTPase. As is true for Rab3A, the active conformation of Rab5C is stabilized by a hydrophobic interface between the switch regions. However, the conformation of the invariant hydrophobic triad (residues Phe-58, Trp-75, and Tyr-90 in Rab5C) is dramatically altered such that the resulting surface is noncomplementary to the switch interaction epitope of Rabphilin3A. This structural rearrangement reflects a set of nonconservative substitutions in the hydrophobic core between the central beta sheet and the alpha2 helix. These observations demonstrate that structural plasticity involving an invariant hydrophobic triad at the switch interface contributes to the mechanism by which effectors recognize distinct Rab subfamilies. Thus, the active conformation of the switch regions conveys information about the identity of a particular Rab GTPase as well as the state of the bound nucleotide.

• Park JH, Jensen BC, Kifer CT, Parsons M
• A novel nucleolar G-protein conserved in eukaryotes.
• J Cell Sci. 2001; 114: 173-185
• Display abstract

• We describe here a novel, evolutionarily conserved set of predicted G-proteins. The founding member of this family, TbNOG1, was identified in a two-hybrid screen as a protein that interacts with NOPP44/46, a nucleolar phosphoprotein of Trypanosoma brucei. The biological relevance of the interaction was verified by co-localization and co-immunoprecipitation. TbNOG1 localized to the trypanosome nucleolus and interacted with domains of NOPP44/46 that are found in several other nucleolar proteins. Genes encoding proteins highly related to TbNOG1 are present in yeast and metazoa, and related G domains are found in bacteria. We show that NOG1 proteins in humans and Saccharomyces cerevisae are also nucleolar. The S. cerevisae NOG1 gene is essential for cell viability, and mutations in the predicted G motifs abrogate function. Together these data suggest that NOG1 may play an important role in nucleolar functions. The GTP-binding region of TbNOG1 is similar to those of Obg and DRG proteins, which, together with NOG, form a newly recognized family of G-proteins, herein named ODN. The ODN family differs significantly from other G-protein families, and shows several diagnostic sequence characteristics. All organisms appear to possess an ODN gene, pointing to the biological significance of this family of G-proteins.

• Padmanabhan S, Freymann DM
• The conformation of bound gmppnp suggests a mechanism for gating the active site of the srp gtpase.
• Structure (Camb). 2001; 9: 859-67
• Display abstract

• Background: The signal recognition particle (SRP) is a phylogenetically conserved ribonucleoprotein that mediates cotranslational targeting of secreted and membrane proteins to the membrane. Targeting is regulated by GTP binding and hydrolysis events that require direct interaction between structurally homologous "NG" GTPase domains of the SRP signal recognition subunit and its membrane-associated receptor, SRalpha. Structures of both the apo and GDP bound NG domains of the prokaryotic SRP54 homolog, Ffh, and the prokaryotic receptor homolog, FtsY, have been determined. The structural basis for the GTP-dependent interaction between the two proteins, however, remains unknown.Results: We report here two structures of the NG GTPase of Ffh from Thermus aquaticus bound to the nonhydrolyzable GTP analog GMPPNP. Both structures reveal an unexpected binding mode in which the beta-phosphate is kinked away from the binding site and magnesium is not bound. Binding of the GTP analog in the canonical conformation found in other GTPase structures is precluded by constriction of the phosphate binding P loop. The structural difference between the Ffh complex and other GTPases suggests a specific conformational change that must accompany movement of the nucleotide from an "inactive" to an "active" binding mode.Conclusions: Conserved side chains of the GTPase sequence motifs unique to the SRP subfamily may function to gate formation of the active GTP bound conformation. Exposed hydrophobic residues provide an interaction surface that may allow regulation of the GTP binding conformation, and thus activation of the GTPase, during the association of SRP with its receptor.

• Abbracchio MP et al.
• The A3 adenosine receptor induces cytoskeleton rearrangement in human astrocytoma cells via a specific action on Rho proteins.
• Ann N Y Acad Sci. 2001; 939: 63-73
• Display abstract

• In previous studies, we have demonstrated that exposure of astroglial cells to A3 adenosine receptor agonists results in dual actions on cell survival, with "trophic" and antiapoptotic effects at nanomolar concentrations and induction of cell death at micromolar agonist concentrations. The protective actions of A3 agonists have been associated with a reinforcement of the actin cytoskeleton, which likely results in increased resistance of cells to cytotoxic stimuli. The molecular mechanisms at the basis of this effect and the signalling pathway(s) linking the A3 receptor to the actin cytoskeleton have never been elucidated. Based on previous literature data suggesting that the actin cytoskeleton is controlled by small GTP-binding proteins of the Rho family, in the study reported here we investigated the involvement of these proteins in the effects induced by A3 agonists on human astrocytoma ADF cells. The presence of the A3 adenosine receptor in these cells has been confirmed by immunoblotting analysis. As expected, exposure of human astrocytoma ADF cells to nanomolar concentrations of the selective A3 agonist 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (CI-IB-MECA) resulted in formation of thick actin positive stress fibers. Preexposure of cells to the C3B toxin that inactivates Rho-proteins completely prevented the actin changes induced by CI-IB-MECA. Exposure to the A3 agonist also resulted in significant reduction of Rho-GDI, an inhibitory protein known to maintain Rho proteins in their inactive state, suggesting a potentiation of Rho-mediated effects. This effect was fully counteracted by the concomitant exposure to the selective A3 receptor antagonist MRS1191. These results suggest that the reinforcement of the actin cytoskeleton induced by A3 receptor agonists is mediated by an interference with the activation/inactivation cycle of Rho proteins, which may, therefore, represent a biological target for the identification of novel neuroprotective strategies.

• Prakash B, Renault L, Praefcke GJ, Herrmann C, Wittinghofer A
• Triphosphate structure of guanylate-binding protein 1 and implications for nucleotide binding and GTPase mechanism.
• EMBO J. 2000; 19: 4555-64
• Display abstract

• The interferon-gamma-induced guanylate-binding protein 1 (GBP1) belongs to a special class of large GTP- binding proteins of 60-100 kDa with unique characteristics. Here we present the structure of human GBP1 in complex with the non-hydrolysable GTP analogue GppNHp. Basic features of guanine nucleotide binding, such as the P-loop orientation and the Mg(2+) co-ordination, are analogous to those of Ras-related and heterotrimeric GTP-binding proteins. However, the glycosidic bond and thus the orientation of the guanine base and its interaction with the protein are very different. Furthermore, two unique regions around the base and the phosphate-binding areas, the guanine and the phosphate caps, respectively, give the nucleotide-binding site a unique appearance not found in the canonical GTP-binding proteins. The phosphate cap, which constitutes the region analogous to switch I, completely shields the phosphate-binding site from solvent such that a potential GTPase-activating protein cannot approach. This has consequences for the GTPase mechanism of hGBP1 and possibly of other large GTP-binding proteins.

• Christopher RA, Guan JL
• To move or not: how a cell responds (Review).
• Int J Mol Med. 2000; 5: 575-81
• Display abstract

• The ability of a cell to migrate is dependent on the cooperative effects of many intracellular signaling events triggered through cell surface receptors (integrins), internal cellular mechanics, and the extracellular matrix. Many proteins have been identified as being involved in cell migration but the regulatory mechanisms involved in cell migration still remain unclear. This review summarizes studies involving various signal transduction pathways and their involvement in the regulation of cell migration. The focus is on cell adhesion sites (focal adhesions) and the actin cytoskeleton.

• Rak A et al.
• Crystal structure of the GAP domain of Gyp1p: first insights into interaction with Ypt/Rab proteins.
• EMBO J. 2000; 19: 5105-13
• Display abstract

• We present the 1.9 A resolution crystal structure of the catalytic domain of Gyp1p, a specific GTPase activating protein (GAP) for Ypt proteins, the yeast homologues of Rab proteins, which are involved in vesicular transport. Gyp1p is a member of a large family of eukaryotic proteins with shared sequence motifs. Previously, no structural information was available for any member of this class of proteins. The GAP domain of Gyp1p was found to be fully alpha-helical. However, the observed fold does not superimpose with other alpha-helical GAPs (e.g. Ras- and Cdc42/Rho-GAP). The conserved and catalytically crucial arginine residue, identified by mutational analysis, is in a comparable position to the arginine finger in the Ras- and Cdc42-GAPs, suggesting that Gyp1p utilizes an arginine finger in the GAP reaction, in analogy to Ras- and Cdc42-GAPs. A model for the interaction between Gyp1p and the Ypt protein satisfying biochemical data is given.

• Scita G et al.
• Signaling from Ras to Rac and beyond: not just a matter of GEFs.
• EMBO J. 2000; 19: 2393-8
• Display abstract

• Members of a family of intracellular molecular switches, the small GTPases, sense modifications of the extracellular environment and transduce them into a variety of homeostatic signals. Among small GTPases, Ras and the Rho family of proteins hierarchically and/or coordinately regulate signaling pathways leading to phenotypes as important as proliferation, differentiation and apoptosis. Ras and Rho-GTPases are organized in a complex network of functional interactions, whose molecular mechanisms are being elucidated. Starting from the simple concept of linear cascades of events (GTPase-->activator--> GTPase), the work of several laboratories is uncovering an increasingly complex scenario in which upstream regulators of GTPases also function as downstream effectors and influence the precise biological outcome. Furthermore, small GTPases assemble into macromolecular machineries that include upstream activators, downstream effectors, regulators and perhaps even final biochemical targets. We are starting to understand how these macromolecular complexes work and how they are regulated and targeted to their proper subcellular localization. Ultimately, the acquisition of a cogent picture of the various levels of integration and regulation in small GTPase-mediated signaling should define the physiology of early signal transduction events and the pathological implication of its subversion.

• Valster AH, Hepler PK, Chernoff J
• Plant GTPases: the Rhos in bloom.
• Trends Cell Biol. 2000; 10: 141-6
• Display abstract

• In animal cells and in fungi, small GTP-binding proteins of the Rho family have well-established roles in morphogenesis, cell-cycle progression, gene transcription and the generation of superoxide anions. The presence of these proteins in plant cells, however, has been established only recently, and the role of Rho GTPases in plants is now coming into view. Already, it is apparent that there are both striking similarities and fascinating differences in how Rho GTPases are regulated and used in plant versus animal and fungal cells. These new findings define certain core properties that might be common to members of this protein family in all eukaryotes.

• Hoffman GR, Nassar N, Cerione RA
• Structure of the Rho family GTP-binding protein Cdc42 in complex with the multifunctional regulator RhoGDI.
• Cell. 2000; 100: 345-56
• Display abstract

• The RhoGDI proteins serve as key multifunctional regulators of Rho family GTP-binding proteins. The 2.6 A X-ray crystallographic structure of the Cdc42/RhoGDI complex reveals two important sites of interaction between GDI and Cdc42. First, the amino-terminal regulatory arm of the GDI binds to the switch I and II domains of Cdc42 leading to the inhibition of both GDP dissociation and GTP hydrolysis. Second, the geranylgeranyl moiety of Cdc42 inserts into a hydrophobic pocket within the immunoglobulin-like domain of the GDI molecule leading to membrane release. The structural data demonstrate how GDIs serve as negative regulators of small GTP-binding proteins and how the isoprenoid moiety is utilized in this critical regulatory interaction.

• Szaszi K, Grinstein S, Orlowski J, Kapus A
• Regulation of the epithelial Na(+) /H(+) exchanger isoform by the cytoskeleton.
• Cell Physiol Biochem. 2000; 10: 265-72
• Display abstract

• Members of the Na(+)/H(+) exchanger (NHE) family mediate electroneutral countertransport of H(+) for Na(+) across cellular membranes. The six known isoforms mediate transepithelial Na(+) transport processes and housekeeping functions such as the regulation of cellular and organellar pH and volume. NHE3 is found primarily in the apical membrane of epithelial cells of the kidney and gastrointestinal tract, where it mediates Na(+) (re)absorption. Its fine regulation, whether by hormones that utilize cAMP as a signalling mechanism, or by physical parameters such as the cell volume, provides the adjustments necessary for the maintenance of systemic salt and fluid balance. Although the exact molecular mechanism of this control is unknown, two major modes of regulation have been invoked: 1) alteration of the number of cell surface transporters by changes in the rate of endocytosis and/or exocytosis and 2) regulation of the intrinsic activity of the individual exchangers. NHE3 requires an intact cytoskeleton for its optimal function. Pharmacological interference with actin polymerization or myosin phosphorylation markedly inhibits the exchanger, without altering the number of transporters exposed at the surface. This effect is isoform specific and is mediated by the cytoplasmic tail of the transporter. The small GTP-binding protein, RhoA and its downstream effector, Rho kinase regulate NHE3, possibly by controlling the level of myosin phosphorylation, that in turn determines the organization of actin. The cytoskeleton may not only be involved in the maintenance of the basal rate of transport, but is also likely to sense physical alterations and transmit signals to modulate NHE3 activity, thus providing fast and effective control of the exchanger. Copyright 2000 S. Karger AG, Basel.

• Wittinghofer A, Gierschik P
• Highlight: GTP binding proteins--central regulators in cell biology.
• Biol Chem. 2000; 381: 355-355

• Hopkins AM, Li D, Mrsny RJ, Walsh SV, Nusrat A
• Modulation of tight junction function by G protein-coupled events.
• Adv Drug Deliv Rev. 2000; 41: 329-40
• Display abstract

• Small G proteins or GTPases comprise a growing family of signal transduction molecules with inducible properties dependent upon reversible interactions with guanine nucleotides. Activation status of the proteins is characterized by preferential affinity for triphosphorylated guanine nucleotides, initiating signaling events that control fundamental processes involved in cell migration and contraction. Termination of small G protein signaling activity is in part achieved through intrinsic GTPase activity, which catalyzes the removal of GTP and its replacement with functionally inactive GDP. Recent investigations have implicated various small G proteins as messengers that control cell-cell contact between scaffold proteins and the actin cytoskeleton, suggesting an intrinsic mechanism for the regulation of paracellular permeability in polarized epithelial and endothelial cells. This review will examine current evidence for the control of tight junction permeability by small G proteins, and speculate upon future directions that may be of value in further exploring the biological importance of these key mediators.

• Steele-Mortimer O, Knodler LA, Finlay BB
• Poisons, ruffles and rockets: bacterial pathogens and the host cell cytoskeleton.
• Traffic. 2000; 1: 107-18
• Display abstract

• The cytoskeleton of eukaryotic cells is affected by a number of bacterial and viral pathogens. In this review we consider three recurring themes of cytoskeletal involvement in bacterial pathogenesis: 1) the effect of bacterial toxins on actin-regulating small GTP-binding proteins; 2) the invasion of non-phagocytic cells by the bacterial induction of ruffles at the plasma membrane; 3) the formation of actin tails and pedestals by intracellular and extracellular bacteria, respectively. Considerable progress has been made recently in the characterization of these processes. It is becoming clear that bacterial pathogens have developed a variety of sophisticated mechanisms for utilizing the complex cytoskeletal system of host cells. These bacterially-induced processes are now providing unique insights into the regulation of fundamental eukaryotic mechanisms.

• Ayscough KR
• Endocytosis and the development of cell polarity in yeast require a dynamic F-actin cytoskeleton.
• Curr Biol. 2000; 10: 1587-90
• Display abstract

• Studies using drugs that cause the disassembly of filamentous actin (F-actin) have demonstrated the importance of an intact actin cytoskeleton for polarised secretion by yeast cells [1,2]. To address the level of dynamic turnover needed for such processes, however, drugs or mutants that confer stabilising properties on F-actin are needed. Jasplakinolide is the only readily available drug that stabilises F-actin structures both in vivo and in vitro [3-6]. Yeast strains have been generated in which two of the ABC multidrug resistance transporter genes have been deleted, rendering normally jasplakinolide-resistant yeast cells sensitive to its effects. Treatment of these cells with jasplakinolide caused rapid and dramatic effects on the actin cytoskeleton, resulting in the accumulation of single large actin structures in cells. These structures, however, still contained components that are normally associated with cortical actin patches. A dynamic actin cytoskeleton was found to be critical for the generation of cell polarity and endocytosis.

• Galan JE, Fu Y
• Modulation of actin cytoskeleton by Salmonella GTPase activating protein SptP.
• Methods Enzymol. 2000; 325: 496-504

• Mullins RD
• How WASP-family proteins and the Arp2/3 complex convert intracellular signals into cytoskeletal structures.
• Curr Opin Cell Biol. 2000; 12: 91-6
• Display abstract

• In most cells, the structure of the actin cytoskeleton is regulated by Rho-family G proteins. Recent work has outlined a highly conserved signaling pathway from G protein activation to actin assembly. The key downstream components are WASP family proteins - adaptor molecules that bind multiple signaling and cytoskeletal proteins - and the Arp2/3 complex - a multi-functional protein complex that nucleates and crosslinks actin filaments.

• Nakayama AY, Luo L
• Intracellular signaling pathways that regulate dendritic spine morphogenesis.
• Hippocampus. 2000; 10: 582-6
• Display abstract

• Rac is a member of the Rho family of small GTPases and acts as a molecular switch. When GTP-bound, Rac binds specific effectors to induce downstream signaling events, including actin cytoskeletal rearrangements (Hall, Science 1998;279:509-514). Herein we review the recent evidence suggesting that Rac is involved in the morphogenesis of dendritic spines (Luo et al., Nature 1996;379:837-840; Nakayama et al., J Neurosci 2000; 20:5329-5338). In addition, we discuss how Rac activity is regulated by guanine nucleotide exchange factors, which may be further regulated by extracellular factors. Thus, the Rac signal transduction pathway may provide links between extracellular ligands or synaptic activity and the regulation of the actin cytoskeleton in spine morphogenesis.

• Hall A, Nobes CD
• Rho GTPases: molecular switches that control the organization and dynamics of the actin cytoskeleton.
• Philos Trans R Soc Lond B Biol Sci. 2000; 355: 965-70
• Display abstract

• The actin cytoskeleton plays a fundamental role in all eukaryotic cells it is a major determinant of cell morphology and polarity and the assembly and disassembly of filamentous actin structures provides a driving force for dynamic processes such as cell motility, phagocytosis, growth cone guidance and cytokinesis. The ability to reorganize actin filaments is a fundamental property of embryonic cells during development; the shape changes accompanying gastrulation and dorsal closure, for example, are dependent on the plasticity of the actin cytoskeleton, while the ability of cells or cell extensions, such as axons, to migrate within the developing embryo requires rapid and spatially organized changes to the actin cytoskeleton in response to the external environment. Work in mammalian cells over the last decade has demonstrated the central role played by the highly conserved Rho family of small GTPases in signal transduction pathways that link plasma membrane receptors to the organization of the actin cytoskeleton.

• Chimini G, Chavrier P
• Function of Rho family proteins in actin dynamics during phagocytosis and engulfment.
• Nat Cell Biol. 2000; 2: 1916-1916
• Display abstract

• Phagocytosis is the uptake of large particles by cells by a mechanism that is based on local rearrangement of the actin microfilament cytoskeleton. In higher organisms, phagocytic cells are essential for host defence against invading pathogens, and phagocytosis contributes to inflammation and the immune response. In addition, engulfment, defined as the phagocytic clearance of cell corpses generated by programmed cell death or apoptosis, has an essential role in tissue homeostasis. Although morphologically distinct phagocytic events can be observed depending on the type of surface receptor engaged, work over the past two years has revealed the essential underlying role of Rho family proteins and their downstream effectors in controlling actin dynamics during phagocytosis.

• Ellis S, Mellor H
• The novel Rho-family GTPase rif regulates coordinated actin-based membrane rearrangements.
• Curr Biol. 2000; 10: 1387-90
• Display abstract

• Small GTPases of the Rho family have a critical role in controlling cell morphology, motility and adhesion through dynamic regulation of the actin cytoskeleton [1,2]. Individual Rho GTPases have been shown to regulate distinct components of the cytoskeletal architecture; RhoA stimulates the bundling of actin filaments into stress fibres [3], Rac reorganises actin to produce membrane sheets or lamellipodia [4] and Cdc42 causes the formation of thin, actin-rich surface projections called filopodia [5]. We have isolated a new Rho-family GTPase, Rif (Rho in filopodia), and shown that it represents an alternative signalling route to the generation of filopodial structures. Coordinated regulation of Rho-family GTPases can be used to generate more complicated actin rearrangements, such as those underlying cell migration [6]. In addition to inducing filopodia, Rif functions cooperatively with Cdc42 and Rac to generate additional structures, increasing the diversity of actin-based morphology.

• Endo T
• [Rho family small G proteins antagonizing RhoA]
• Seikagaku. 2000; 72: 469-72

• Stebbins CE, Galan JE
• Modulation of host signaling by a bacterial mimic: structure of the Salmonella effector SptP bound to Rac1.
• Mol Cell. 2000; 6: 1449-60
• Display abstract

• Salmonella spp. utilize a specialized protein secretion system to deliver a battery of effector proteins into host cells. Several of these effectors stimulate Cdc42- and Rac1-dependent cytoskeletal changes that promote bacterial internalization. These potentially cytotoxic alterations are rapidly reversed by the effector SptP, a tyrosine phosphatase and GTPase activating protein (GAP) that targets Cdc42 and Rac1. The 2.3 A resolution crystal structure of an SptP-Rac1 transition state complex reveals an unusual GAP architecture that mimics host functional homologs. The phosphatase domain possesses a conserved active site but distinct surface properties. Binding to Rac1 induces a dramatic stabilization in SptP of a four-helix bundle that makes extensive contacts with the Switch I and Switch II regions of the GTPase.

• Boquet P, Sansonetti PJ, Tran Van Nhieu G
• Rho GTP-binding proteins as targets for microbial pathogens.
• Prog Mol Subcell Biol. 1999; 22: 183-99

• Ostermeier C, Brunger AT
• Structural basis of Rab effector specificity: crystal structure of the small G protein Rab3A complexed with the effector domain of rabphilin-3A.
• Cell. 1999; 96: 363-74
• Display abstract

• The small G protein Rab3A plays an important role in the regulation of neurotransmitter release. The crystal structure of activated Rab3A/GTP/Mg2+ bound to the effector domain of rabphilin-3A was solved to 2.6 A resolution. Rabphilin-3A contacts Rab3A in two distinct areas. The first interface involves the Rab3A switch I and switch II regions, which are sensitive to the nucleotide-binding state of Rab3A. The second interface consists of a deep pocket in Rab3A that interacts with a SGAWFF structural element of rabphilin-3A. Sequence and structure analysis, and biochemical data suggest that this pocket, or Rab complementarity-determining region (RabCDR), establishes a specific interaction between each Rab protein and its effectors. RabCDRs could be major determinants of effector specificity during vesicle trafficking and fusion.

• Coleman DE, Sprang SR
• Structure of Gialpha1.GppNHp, autoinhibition in a galpha protein-substrate complex.
• J Biol Chem. 1999; 274: 16669-72
• Display abstract

• The structure of the G protein Gialpha1 complexed with the nonhydrolyzable GTP analog guanosine-5'-(betagamma-imino)triphosphate (GppNHp) has been determined at a resolution of 1.5 A. In the active site of Gialpha1. GppNHp, a water molecule is hydrogen bonded to the side chain of Glu43 and to an oxygen atom of the gamma-phosphate group. The side chain of the essential catalytic residue Gln204 assumes a conformation which is distinctly different from that observed in complexes with either guanosine 5'-O-3-thiotriphosphate or the transition state analog GDP.AlF4-. Hydrogen bonding and steric interactions position Gln204 such that it interacts with a presumptive nucleophilic water molecule, but cannot interact with the pentacoordinate transition state. Gln204 must be released from this auto-inhibited state to participate in catalysis. RGS proteins may accelerate the rate of GTP hydrolysis by G protein alpha subunits, in part, by inserting an amino acid side chain into the site occupied by Gln204, thereby destabilizing the auto-inhibited state of Galpha.

• Nobes CD, Hall A
• Rho GTPases control polarity, protrusion, and adhesion during cell movement.
• J Cell Biol. 1999; 144: 1235-44
• Display abstract

• Cell movement is essential during embryogenesis to establish tissue patterns and to drive morphogenetic pathways and in the adult for tissue repair and to direct cells to sites of infection. Animal cells move by crawling and the driving force is derived primarily from the coordinated assembly and disassembly of actin filaments. The small GTPases, Rho, Rac, and Cdc42, regulate the organization of actin filaments and we have analyzed their contributions to the movement of primary embryo fibroblasts in an in vitro wound healing assay. Rac is essential for the protrusion of lamellipodia and for forward movement. Cdc42 is required to maintain cell polarity, which includes the localization of lamellipodial activity to the leading edge and the reorientation of the Golgi apparatus in the direction of movement. Rho is required to maintain cell adhesion during movement, but stress fibers and focal adhesions are not required. Finally, Ras regulates focal adhesion and stress fiber turnover and this is essential for cell movement. We conclude that the signal transduction pathways controlled by the four small GTPases, Rho, Rac, Cdc42, and Ras, cooperate to promote cell movement.

• Dumas JJ, Zhu Z, Connolly JL, Lambright DG
• Structural basis of activation and GTP hydrolysis in Rab proteins.
• Structure Fold Des. 1999; 7: 413-23
• Display abstract

• BACKGROUND: Rab proteins comprise a large family of GTPases that regulate vesicle trafficking. Despite conservation of critical residues involved in nucleotide binding and hydrolysis, Rab proteins exhibit low sequence identity with other GTPases, and the structural basis for Rab function remains poorly characterized. RESULTS: The 2. 0 A crystal structure of GppNHp-bound Rab3A reveals the structural determinants that stabilize the active conformation and regulate GTPase activity. The active conformation is stabilized by extensive hydrophobic contacts between the switch I and switch II regions. Serine residues in the phosphate-binding loop (P loop) and switch I region mediate unexpected interactions with the gamma phosphate of GTP that have not been observed in previous GTPase structures. Residues implicated in the interaction with effectors and regulatory factors map to a common face of the protein. The electrostatic potential at the surface of Rab3A indicates a non-uniform distribution of charged and nonpolar residues. CONCLUSIONS: The major structural determinants of the active conformation involve residues that are conserved throughout the Rab family, indicating a common mode of activation. Novel interactions with the gamma phosphate impose stereochemical constraints on the mechanism of GTP hydrolysis and provide a structural explanation for the large variation of GTPase activity within the Rab family. An asymmetric distribution of charged and nonpolar residues suggests a plausible orientation with respect to vesicle membranes, positioning predominantly hydrophobic surfaces for interaction with membrane-associated effectors and regulatory factors. Thus, the structure of Rab3A establishes a framework for understanding the molecular mechanisms underlying the function of Rab GTPases.

• Kaibuchi K, Kuroda S, Amano M
• Regulation of the cytoskeleton and cell adhesion by the Rho family GTPases in mammalian cells.
• Annu Rev Biochem. 1999; 68: 459-86
• Display abstract

• Members of the Rho family of small Ras-like GTPases--including RhoA, -B, and -C, Rac1 and -2, and Cdc42--exhibit guanine nucleotide-binding activity and function as molecular switches, cycling between an inactive GDP-bound state and an active GTP-bound state. The Rho family GTPases participate in regulation of the actin cytoskeleton and cell adhesion through specific targets. Identification and characterization of these targets have begun to clarify how the Rho family GTPases act to regulate cytoskeletal structure and cell-cell and cell-substratum contacts in mammalian cells. The Rho family GTPases are also involved in regulation of smooth muscle contraction, cell morphology, cell motility, neurite retraction, and cytokinesis. However, the molecular mechanisms by which the Rho family GTPases participate in the regulation of such processes are not well established.

• Kato M et al.
• Role of Rho small GTP binding protein in the regulation of actin cytoskeleton in hepatic stellate cells.
• J Hepatol. 1999; 31: 91-9
• Display abstract

• BACKGROUND/AIMS: In the fibrotic response to liver injury, hepatic stellate cells are activated, leading to the myofibroblastic cell shape, with actin cytoskeletal reorganization and increased extracellular matrix production. The reorganization of actin cytoskeleton suggests that the small GTP binding protein Rho might modulate the process of this myofibroblastic change. The aim of this study was to investigate the role of Rho in the phenotypic changes of hepatic stellate cells. METHODS: The phenotypic changes were investigated by the overexpression of Rho regulator, Rho GDI or dominant negative mutant of Rho in mouse hepatic stellate cell line, GRX cells. In activated rat hepatic stellate cells, the effects of microinjection of Botulinus toxin C3, which is the specific inhibitor for Rho, were analyzed. Furthermore, the effect of C3 on the type I collagen accumulation in hepatic stellate cells was investigated. RESULTS: Overexpression of Rho GDI or the dominant negative mutant of Rho caused the shrinkage cell shape and suppressed stress fiber formation. Microinjection of toxin C3 caused a markedly distorted cell shape and the disappearance of stress fibers in rat stellate cells. In addition, C3 strongly suppressed collagen accumulation in activated stellate cells. CONCLUSIONS: These results suggest that Rho regulates the actin cytoskeletal reorganization, and may be implicated in the collagen accumulation in activated stellate cells. These findings provide evidence for the role of Rho in the myofibroblastic phenotype in hepatic stellate cells.

• Rudolph MG, Wittinghofer A, Vetter IR
• Nucleotide binding to the G12V-mutant of Cdc42 investigated by X-ray diffraction and fluorescence spectroscopy: two different nucleotide states in one crystal.
• Protein Sci. 1999; 8: 778-87
• Display abstract

• The 2.5 A crystal structure of the full length human placental isoform of the Gly12 to Val mutant Cdc42 protein (Cdc42(G12V)) bound to both GDP/Mg2+ and GDPNH2 (guanosine-5'-diphospho-beta-amidate) is reported. The crystal contains two molecules in the asymmetric unit, of which one has bound GDP/Mg2+, while the other has bound GDPNH2 without a Mg2+ ion. Crystallization of the protein was induced via hydrolysis of the Cdc42 x GppNHp complex by the presence of contaminating alkaline phosphatase activity in combination with the crystallization conditions. This prompted us to compare the binding characteristics of GDPNH2 vs. GDP. The amino group of GDPNH2 drastically reduces the affinity to Cdc42 in comparison with that of GDP, causes the loss of the Mg2+ ion, and apparently also increases the conformational flexibility of the protein as seen in the crystal. Both the switch I and switch II regions are visible in the electron density of the GDP-bound molecule, but not in the molecule bound to GDPNH2. The C-terminus containing the CaaX-motif is partly ordered in both molecules due to an intramolecular disulfide bond formed between Cys105/Cys188 and Cys305/Cys388, respectively.

• Zeng J, Treutlein HR, Simonson T
• Molecular dynamics simulations of the Ras:Raf and Rap:Raf complexes.
• Proteins. 1999; 35: 89-100
• Display abstract

• The protein Raf is an immediate downstream target of Ras in the MAP kinase signalling pathway. The complex of Ras with the Ras-binding domain (RBD) of Raf has been modelled by homology to the (E30D,K31E)-Rap1A:RBD complex, and both have been subjected to multiple molecular dynamics simulations in solution. While both complexes are stable, several rearrangements occur in the Ras:RBD simulations: the RBD loop 100-109 moves closer to Ras, Arg73 in the RBD moves towards Ras to form a salt bridge with Ras-Asp33, and Loop 4 of the Ras switch II region shifts upwards toward the RBD. The Ras:RBD interactions (including the RBD-Arg73 interaction) are consistent with available NMR and mutagenesis data on the Ras: RBD complex in solution. The Ras switch II region does not interact directly with the RBD, although indirect interactions exist through the effector domain and bridging water molecules. No large-scale RBD motion is seen in the Ras:RBD complex, compared to the Rap:RBD complex, to suggest an allosteric activation of Raf by Ras. This may be because the Raf kinase domain (whose structure is unknown) is not included in the model.

• Schoenwaelder SM, Burridge K
• Bidirectional signaling between the cytoskeleton and integrins.
• Curr Opin Cell Biol. 1999; 11: 274-86
• Display abstract

• Clustering of integrins into focal adhesions and focal complexes is regulated by the actin cytoskeleton. In turn, actin dynamics are governed by Rho family GTPases. Integrin-mediated adhesion activates these GTPases, triggering assembly of filopodia, lamellipodia and stress fibers. In the past few years, signaling pathways have begun to be identified that promote focal adhesion disassembly and integrin dispersal. Many of these pathways result in decreased myosin-mediated cell contractility.

• Kjoller L, Hall A
• Signaling to Rho GTPases.
• Exp Cell Res. 1999; 253: 166-79
• Display abstract

• Rho GTPases regulate many important processes in all eukaryotic cells, including the organization of the actin cytoskeleton, gene transcription, cell cycle progression, and membrane trafficking. Their activity is regulated by signals originating from different classes of surface receptors including G-protein-coupled receptors, tyrosine kinase receptors, cytokine receptors, and adhesion receptors. Recent work has identified multiple mechanisms by which receptors can signal to Rho GTPases and this will be the major focus of this review. In addition, there is growing evidence for cross-talk within the Rho GTPase family as well as between the Rho and Ras GTPase families. These signaling networks are thought to provide the cooperative and coordinated interactions that are crucial for regulating complex biological processes such as cell migration.

• Goldberg J
• Structural and functional analysis of the ARF1-ARFGAP complex reveals a role for coatomer in GTP hydrolysis.
• Cell. 1999; 96: 893-902
• Display abstract

• The crystal structure of the complex of ARF1 GTPase bound to GDP and the catalytic domain of ARF GTPase-activating protein (ARFGAP) has been determined at 1.95 A resolution. The ARFGAP molecule binds to switch 2 and helix alpha3 to orient ARF1 residues for catalysis, but it supplies neither arginine nor other amino acid side chains to the GTPase active site. In the complex, the effector-binding region appears to be unobstructed, suggesting that ARFGAP could stimulate GTP hydrolysis while ARF1 maintains an interaction with its effector, the coatomer complex of COPI-coated vesicles. Biochemical experiments show that coatomer directly participates in the GTPase reaction, accelerating GTP hydrolysis a further 1000-fold in an ARFGAP-dependent manner. Thus, a tripartite complex controls the GTP hydrolysis reaction triggering disassembly of COPI vesicle coats.

• Zhang B, Zhang Y, Collins CC, Johnson DI, Zheng Y
• A built-in arginine finger triggers the self-stimulatory GTPase-activating activity of rho family GTPases.
• J Biol Chem. 1999; 274: 2609-12
• Display abstract

• Signal transduction through the Rho family GTPases requires regulated cycling of the GTPases between the active GTP-bound state and the inactive GDP-bound state. Rho family members containing an arginine residue at position 186 in the C-terminal polybasic region were found to possess a self-stimulatory GTPase-activating protein (GAP) activity through homophilic interaction, resulting in significantly enhanced intrinsic GTPase activities. This arginine residue functions effectively as an "arginine finger" in the GTPase activating reaction to confer the catalytic GAP activity but is not essential for the homophilic binding interactions of Rho family proteins. The arginine 186-mediated negative regulation seems to be absent from Cdc42, a Rho family member important for cell-division cycle regulation, of lower eukaryotes, yet appears to be a part of the turn-off machinery of Cdc42 from higher eukaryotes. Introduction of the arginine 186 mutation into S. cerevisiae CDC42 led to phenotypes consistent with down-regulated CDC42 function. Thus, specific Rho family GTPases may utilize a built-in arginine finger, in addition to RhoGAPs, for negative regulation.

• Robinson NG, Guo L, Imai J, Toh-E A, Matsui Y, Tamanoi F
• Rho3 of Saccharomyces cerevisiae, which regulates the actin cytoskeleton and exocytosis, is a GTPase which interacts with Myo2 and Exo70.
• Mol Cell Biol. 1999; 19: 3580-7
• Display abstract

• The Rho3 protein plays a critical role in the budding yeast Saccharomyces cerevisiae by directing proper cell growth. Rho3 appears to influence cell growth by regulating polarized secretion and the actin cytoskeleton, since rho3 mutants exhibit large rounded cells with an aberrant actin cytoskeleton. To gain insights into how Rho3 influences these events, we have carried out a yeast two-hybrid screen using an S. cerevisiae cDNA library to identify proteins interacting with Rho3. Two proteins, Exo70 and Myo2, were identified in this screen. Interactions with these two proteins are greatly reduced or abolished when mutations are introduced into the Rho3 effector domain. In addition, a type of mutation known to produce dominant negative mutants of Rho proteins abolished the interaction with both of these proteins. In contrast, Rho3 did not interact with protein kinase C (Pkc1), an effector of another Rho family protein, Rho1, nor did Rho1 interact with Exo70 or Myo2. Rho3 did interact with Bni1, another effector of Rho1, but less efficiently than with Rho1. The interaction between Rho3 and Exo70 and between Rho3 and Myo2 was also demonstrated with purified proteins. The interaction between Exo70 and Rho3 in vitro was dependent on the presence of GTP, since Rho3 complexed with guanosine 5'-O-(3-thiotriphosphate) interacted more efficiently with Exo70 than Rho3 complexed with guanosine 5'-O-(3-thiodiphosphate). Overlapping subcellular localization of the Rho3 and Exo70 proteins was demonstrated by indirect immunofluorescence. In addition, patterns of localization of both Exo70 and Rho3 were altered when a dominant active allele of RHO3, RHO3(E129,A131), which causes a morphological abnormality, was expressed. These results provide a direct molecular basis for the action of Rho3 on exocytosis and the actin cytoskeleton.

• Goldschmidt-Clermont PJ, Moldovan L
• Stress, superoxide, and signal transduction.
• Gene Expr. 1999; 7: 255-60
• Display abstract

• A variety of stressful events can trigger the production of free radicals by exposed cells. For years, the effect of such highly reactive radicals was expected to be damaging to cells, altering their biology irreversibly. However, many recent reports have shown that reactive oxygen species can have additional functions, and contribute to important signaling pathways to regulate key biological responses, including cell migration, mitosis, and apoptosis. With this review, we address the role of the small GTP binding protein, Rac, as a regulatory protein that controls superoxide production, and the effect of superoxide and derived oxidants in cell signaling.

• Fukata Y, Oshiro N, Kaibuchi K
• Activation of moesin and adducin by Rho-kinase downstream of Rho.
• Biophys Chem. 1999; 82: 139-47
• Display abstract

• The Rho GTPase (Rho) is a member of the Rho family, which belongs to the Ras superfamily of GTP-binding proteins. Like other GTP-binding proteins, Rho exists in two conformational states, an inactive GDP-bound form and an active GTP-bound form. Active Rho interacts with specific effectors to regulate the actin cytoskeleton and to mediate a variety of biological functions in cells. Rho-associated kinase (Rho-kinase) is the most studied Rho-effector, and studies of its biochemical and cell biological functions have provided us with useful information for understanding the molecular mechanisms of the actions of Rho. This review aims to summarize the roles of Rho and Rho-kinase in the regulation of the cytoskeletons.

• Olofsson B
• Rho guanine dissociation inhibitors: pivotal molecules in cellular signalling.
• Cell Signal. 1999; 11: 545-54
• Display abstract

• The small G proteins of the Ras family act as bimodal relays in the transfer of intracellular signals. This is a dynamic phenomenon involving a cascade of protein-protein interactions modulated by chemical modifications, structural rearrangements and intracellular relocalisations. Most of the small G proteins could be operationally defined as proteins having two conformational states, each of which interacts with different cellular partners. These two states are determined by the nature of the bound nucleotide, GDP or GTP. This capacity to cycle between a GDP-bound conformation and a GTP-bound conformation enables them to filter, to amplify or to temporise the upstream signals that they receive. Thus the control of this cycle is crucial. Membrane anchoring of the proteins in the Ras family is a prerequisite for their activity. Most of the proteins in the Rho/Rac and Rab subfamilies of Ras proteins cycle between cytosol and membranes. Then the control of membrane association/dissociation is an other important regulation level. This review will describe one family of crucial regulators acting on proteins in the Rho/Rac family-the Rho guanine nucleotide dissociation inhibitors, or RhoGDIs. As yet, only three RhoGDIs have been described: RhoGDI-1, RhoGDI-2 (or D4/Ly-GDI) and RhoGDI-3. RhoGDI 1 and 2 are cytosolic and participate in the regulation of both the GDP/GTP cycle and the membrane association/dissociation cycle of Rho/Rac proteins. The non-cytosolic RhoGDI-3 seems to act in a slightly different way.

• Kaibuchi K
• Regulation of cytoskeleton and cell adhesion by Rho targets.
• Prog Mol Subcell Biol. 1999; 22: 23-38

• Seasholtz TM, Majumdar M, Brown JH
• Rho as a mediator of G protein-coupled receptor signaling.
• Mol Pharmacol. 1999; 55: 949-56

• Ridley AJ
• Rho family proteins and regulation of the actin cytoskeleton.
• Prog Mol Subcell Biol. 1999; 22: 1-22

• Menetrey J, Cherfils J
• Structure of the small G protein Rap2 in a non-catalytic complex with GTP.
• Proteins. 1999; 37: 465-73
• Display abstract

• We report a novel crystal form of the small G protein Rap2A in complex with GTP which has no GTPase activity in the crystal. The asymmetric unit contains two complexes which show that a conserved switch I residue, Tyr 32, contributes an extra hydrogen bond to the gamma-phosphate of GTP as compared to related structures with GTP analogs. Since GTP is not hydrolyzed in the crystal, this interaction is unlikely to contribute to the intrinsic GTPase activity. The comparison of other G protein structures to the Rap2-GTP complex suggests that an equivalent interaction is likely to exist in their GTP form, whether unbound or bound to an effector. This interaction has to be released to allow the GAP-activated GTPase, and presumably the intrinsic GTPase activity as well. We also discuss the definition of the flexible regions and their hinges in the light of this structure and the expanding database of G protein structures. We propose that the switch I and switch II undergo either partial or complete disorder-to-order transitions according to their cellular status, thus defining a complex energy landscape comprising more than two conformational states. We observe in addition that the region connecting the switch I and switch II is flexible in Rap2 and other G proteins. This region may be important for protein-protein interactions and possibly behave as a conformational lever arm, as characterized for Arf. Taken together, these observations suggest that the structural mechanisms of small G proteins are significantly driven by entropy-based free energy changes.

• Aspenstrom P
• The Rho GTPases have multiple effects on the actin cytoskeleton.
• Exp Cell Res. 1999; 246: 20-5
• Display abstract

• The importance for the Rho GTPases Rho, Rac, and Cdc42 for the heterogeneity of the actin cytoskeleton is well documented. However, there is now an emerging view that they also have important roles in signaling pathways that control gene transcription, cell cycle regulation, apoptosis, and tumor progression. Furthermore, not only has the number of pathways which involve Rho GTPases increased, the number of Rho family members and their regulating proteins has increased considerably. This raises important questions regarding how specificity is achieved in cell signaling employing Rho GTPases.

• Settleman J
• Rho GTPases in development.
• Prog Mol Subcell Biol. 1999; 22: 201-29
• Display abstract

• It is becoming increasingly clear that the complex family of Rho-related GTPases and their associated regulators and targets are essential mediators of a variety of morphogenetic events required for normal development of multicellular organisms. It is worth noting that the results obtained thus far indicate that the Rho family proteins are largely associated with the regulation of morphogenesis, as opposed to other essential developmental processes such as cell proliferation and cell fate determination. Accumulating evidence also suggests that the role of these proteins and their associated signaling pathways in morphogenesis is in many, but not necessarily all, cases related to their ability to affect the organization of the actin cytoskeleton. Thus, these in vivo observations have served to corroborate similar findings in numerous cultured cell studies. As described, the power of genetics, particularly in Drosophila and C. elegans, has been critical to the recent identification and functional characterization of several Rho family signaling components. Moreover, evidence suggests that the highly evolutionarily conserved structures of many of these proteins translate into conservation of function as well. Thus, it will be possible, in many cases, to extrapolate the findings in the simple systems described herein to higher eukaryotes, including humans. Expanding use of these genetic model systems to dissect Rho-mediated signaling pathways in vivo will undoubtedly lead to a flood of new insights into the organization and function of these pathways in the coming years, especially in development. As the C. elegans genome sequencing effort nears completion and with the Drosophila genome project well underway, the identification of novel relevant genes will proceed with even greater speed. In addition, the rapidly expanding use of mouse knockout strategies, combined with recent developments in the associated knockout technology, will also contribute greatly to the investigation of mammalain Rho signaling pathways and their roles in development.

• Sasaki T, Takai Y
• The Rho small G protein family-Rho GDI system as a temporal and spatial determinant for cytoskeletal control.
• Biochem Biophys Res Commun. 1998; 245: 641-5
• Display abstract

• Recent extensive studies have clarified the functions of the small G protein superfamily, which consists of the Ras, Rho, Rab, Arf, Sar1, and Ran families (for reviews, Refs, 1 and 2). The Ras family regulates gene expression at least through the MAP kinase cascade; the Rho family mainly regulates reorganization of the actin cytoskeleton; the Rab, Arf, and Sar1 families regulate intracellular vesicle trafficking; and the Ran family regulates nuclear transport. Of these cellular functions, reorganization of the actin cytoskeleton, seen in the formation of filopodia, lamellipodia, and ruffles during cell motility, dynamically occurs at specific sites of cells. To regulate this type of dynamic cellular functions, temporal and spatial determination mechanisms of signal transduction would be important. Like other G proteins, small G proteins cycle between the GDP-bound inactive and GTP-bound active forms (1,2). They receive upstream signals through their regulators and transduce signals to downstream targets while they stay in the GTP-bound form. Thus, G proteins serve as timers. There are at least three types of regulators for small G proteins: GDP/GTP exchange protein (GEP) which stimulates conversion from the GDP-bound form to the GTP-bound form; GDP dissociation inhibitor (GDI) which inhibits this reaction; and GTPase activating protein (GAP) which stimulates conversion from the GTP-bound form to the GDP-bound form. Of these regulators, GDI has thus far been found for the Rho and Rab families. We have recently found that the Rho family-Rho GDI system plays an important role in spatial determination in the actin cytoskeletal control (3-6). We briefly describe here this function of the Rho family-Rho GDI system.

• Fields TA
• Identification of a GTPase activating protein specific for the heterotrimeric G protein, Gz.
• Cell Signal. 1998; 10: 43-8
• Display abstract

• Gz is a member of the Gi family of trimeric G proteins whose precise signalling function has not been defined. It can be distinguished from other members of the family by several interesting biochemical properties of its alpha subunit, Gzalpha. One particularly intriguing property is its extremely slow GTPase activity; its kcat for GTP hydrolysis is as much as 200-fold less than other Galpha's. Since there is evidence that cellular factors can accelerate the GTPase activities of Galpha subunits, we have suspected that cells expressing Gzalpha may contain a GTPase-activating protein, or GAP, that would enhance its hydrolytic ability. Using purified Gzalpha-GTP as a substrate, we have identified and characterized such a GAP that acts on Gzalpha, which we have termed Gz-GAP. The protein responsible for this activity is specific for Gzalpha and is found in the membrane fraction of bovine brain and in several other tissues that express Gz. Since G protein effectors are in many cases capable of stimulating the GTPases rate of Galpha subunits, we speculate that a novel effector for Gz is responsible for the activity.

• Cherfils J et al.
• Structure of the Sec7 domain of the Arf exchange factor ARNO.
• Nature. 1998; 392: 101-5
• Display abstract

• Small G proteins switch from a resting, GDP-bound state to an active, GTP-bound state. As spontaneous GDP release is slow, guanine-nucleotide-exchange factors (GEFs) are required to promote fast activation of small G proteins through replacement of GDP with GTP in vivo. Families of GEFs with no sequence similarity to other GEF families have now been assigned to most families of small G proteins. In the case of the small G protein Arf1, the exchange of bound GDP for GTP promotes the coating of secretory vesicles in Golgi traffic. An exchange factor for human Arf1, ARNO, and two closely related proteins, named cytohesin 1 and GPS1, have been identified. These three proteins are modular proteins with an amino-terminal coiled-coil, a central Sec7-like domain and a carboxy-terminal pleckstrin homology domain. The Sec7 domain contains the exchange-factor activity. It was first found in Sec7, a yeast protein involved in secretion, and is present in several other proteins, including the yeast exchange factors for Arf, Geal and Gea2. Here we report the crystal structure of the Sec7 domain of human ARNO at 2 A resolution and the identification of the site of interaction of ARNO with Arf.

• Treisman R, Alberts AS, Sahai E
• Regulation of SRF activity by Rho family GTPases.
• Cold Spring Harb Symp Quant Biol. 1998; 63: 643-51

• Clark EA, King WG, Brugge JS, Symons M, Hynes RO
• Integrin-mediated signals regulated by members of the rho family of GTPases.
• J Cell Biol. 1998; 142: 573-86
• Display abstract

• The organization of the actin cytoskeleton can be regulated by soluble factors that trigger signal transduction events involving the Rho family of GTPases. Since adhesive interactions are also capable of organizing the actin-based cytoskeleton, we examined the role of Cdc42-, Rac-, and Rho-dependent signaling pathways in regulating the cytoskeleton during integrin-mediated adhesion and cell spreading using dominant-inhibitory mutants of these GTPases. When Rat1 cells initially adhere to the extracellular matrix protein fibronectin, punctate focal complexes form at the cell periphery. Concomitant with focal complex formation, we observed some phosphorylation of the focal adhesion kinase (FAK) and Src, which occurred independently of Rho family GTPases. However, subsequent phosphorylation of FAK and paxillin occurs in a Rho-dependent manner. Moreover, we found Rho dependence of the assembly of large focal adhesions from which actin stress fibers radiate. Initial adhesion to fibronectin also stimulates membrane ruffling; we show that this ruffling is independent of Rho but is dependent on both Cdc42 and Rac. Furthermore, we observed that Cdc42 controls the integrin-dependent activation of extracellular signal-regulated kinase 2 and of Akt, a kinase whose activity has been demonstrated to be dependent on phosphatidylinositol (PI) 3-kinase. Since Rac-dependent membrane ruffling can be stimulated by PI 3-kinase, it appears that Cdc42, PI 3-kinase, and Rac lie on a distinct pathway that regulates adhesion-induced membrane ruffling. In contrast to the differential regulation of integrin-mediated signaling by Cdc42, Rac, and Rho, we observed that all three GTPases regulate cell spreading, an event that may indirectly control cellular architecture. Therefore, several separable signaling pathways regulated by different members of the Rho family of GTPases converge to control adhesion-dependent changes in the organization of the cytoskeleton, changes that regulate cell morphology and behavior.

• Grishin AV, Gerasimovskaia EV, Starikova MG, Panchenko MP, Nikashin AV, Tkachuk VA
• [Interaction of the beta1-subunit of G-proteins with the actin cytoskeleton]
• Vopr Med Khim. 1998; 44: 347-52
• Display abstract

• Using immunoblotting with specific antibodies, we have identified beta 1- and beta 2-subunits of Gi-proteins in membrane and cytosolic fractions of pig lung. It has been shown that beta 1-subunit is present both in membrane and cytosolic fractions, whereas beta 2-subunit is associated only with membranes. Activation of membrane-bound G proteins with non-hydrolysable GTP analogues have led to partial release from membrane of beta 1-, but not beta 2-subunits. When depolymerisation of F-actin during fractionation was prevented, both beta 1- and beta 2-subunits were found in fraction containing total membranes and F-actin. Dialysis of this fraction into low ionic strength buffer caused depolymerization of the bulk of actin and release of about 1/4 of beta 1-subunits into solution. The data presented here suggest that distribution of beta 1-subunits between membrane and cytosol could depend on the state of actin cytoskeleton.

• Apanovitch DM, Slep KC, Sigler PB, Dohlman HG
• Sst2 is a GTPase-activating protein for Gpa1: purification and characterization of a cognate RGS-Galpha protein pair in yeast.
• Biochemistry. 1998; 37: 4815-22
• Display abstract

• Genetic studies in the yeast Saccharomyces cerevisiae have shown that SST2 promotes pheromone desensitization in vivo. Sst2 is the founding member of the RGS (regulators of G protein signaling) family of proteins, which in mammals act as GAPs (GTPase activating proteins) for several subfamilies of Galpha proteins in vitro. A similar activity for Sst2 has not been demonstrated, and it is not self-evident from sequence homology arguments alone. Here we describe the purification of Sst2 and its cognate Galpha protein (Gpa1) in yeast, and demonstrate Sst2-stimulated Gpa1 GTPase activity. His-tagged versions of Sst2 and Gpa1 were expressed in E. coli, and purified using Ni2+-agarose and ion exchange chromatography. Time-course binding experiments reveal that Sst2 does not affect the binding or release of guanine nucleotides. Similarly, steady-state GTPase assays reveal that Sst2 does not alter the overall rate of hydrolysis, including the rate-limiting nucleotide exchange step. Single-turnover GTPase assays reveal, however, that Sst2 is a potent stimulator of GTP hydrolysis. Sst2 also exhibits GAP activity for mammalian Goalpha, and the mammalian RGS protein GAIP exhibits GAP activity for Gpa1. Finally, we show that Sst2 binds with highest affinity to the transition state of Gpa1 (GDP-AlF4--bound), and with much lower affinity to the inactive (GDP-bound) and active (GTPgammaS-bound) conformations. These experiments represent the first biochemical characterization of Gpa1 and Sst2, and provide a molecular basis for their well-established biological roles in signaling and desensitization.

• Cabib E, Drgonova J, Drgon T
• Role of small G proteins in yeast cell polarization and wall biosynthesis.
• Annu Rev Biochem. 1998; 67: 307-33
• Display abstract

• In the vegetative (mitotic) cycle and during sexual conjugation, yeast cells display polarized growth, giving rise to a bud or to a mating projection, respectively. In both cases one can distinguish three steps in these processes: choice of a growth site, organization of the growth site, and actual growth and morphogenesis. In all three steps, small GTP-binding proteins (G proteins) and their regulators play essential signaling functions. For the choice of a bud site, Bud1, a small G protein, Bud2, a negative regulator of Bud1, and Bud5, an activator, are all required. If any of them is defective, the cell loses its ability to select a proper bud position and buds randomly. In the organization of the bud site or of the site in which a mating projection appears, Cdc42, its activator Cdc24, and its negative regulators play a fundamental role. In the absence of Cdc42 or Cdc24, the actin cytoskeleton does not become organized and budding does not take place. Finally, another small G protein, Rho1, is required for activity of beta (1-->3)glucan synthase, the enzyme that catalyzes the synthesis of the major structural component of the yeast cell wall. In all of the above processes, G proteins can work as molecular switches because of their ability to shift between an active GTP-bound state and an inactive GDP-bound state.

• Valentijn JA, Jamieson JD
• On the role of rab GTPases: what can be learned from the developing pancreas.
• Biochem Biophys Res Commun. 1998; 243: 331-6

• Gamblin SJ, Smerdon SJ
• GTPase-activating proteins and their complexes.
• Curr Opin Struct Biol. 1998; 8: 195-201
• Display abstract

• In the past year, crystallographic structures for four complexes of GTPase-activating proteins (GAPs) with their target G proteins have been described and substantially enhance our understanding of how these proteins function. GAPs specific for the Rho and Ras families of small G proteins insert an arginine residue into the active site of the G protein, stabilise its switch regions and share an underlying topological relationship. The complex of a heterotrimeric G protein with its activating protein shows that the latter protein does not participate directly in the hydrolytic reaction and has a different structure of RhoGAP and RasGAP.

• Bae CD, Min DS, Fleming IN, Exton JH
• Determination of interaction sites on the small G protein RhoA for phospholipase D.
• J Biol Chem. 1998; 273: 11596-604
• Display abstract

• Phospholipase D (PLD) has been identified as a target of small G proteins of the Rho family. The present study was directed at defining the interaction sites of RhoA with rat brain PLD in vitro using chimeric proteins between RhoA and Ha-Ras or Cdc42Hs and point mutations. The switch I region of RhoA, which is the common effector domain of Ras-like G proteins, was a crucial interaction site for PLD. Mutations in conserved amino acids (Tyr34, Thr37, Phe39) totally abolished PLD activation, while mutations in Val38 or Tyr42 caused partial loss. Two additional sites were responsible for the differential PLD activation ability between RhoA and Cdc42Hs. Changing Asp76 in the switch II region of RhoA to the corresponding amino acid in Cdc42Hs led to partial loss of PLD activation. A chimeric protein with the N-terminal third of Cdc42Hs changed to RhoA showed enhanced PLD activation. Analysis of other Rho/Ha-Ras chimeric proteins and mutations indicated that Gln52 adjacent to the switch II region is responsible for this gain of function. In conclusion, the present study shows that conserved amino acids in the switch I region of RhoA are major PLD interaction sites and that residues in the switch II and internal regions are responsible for the differential activation of PLD by RhoA and Cdc42Hs.

• Shuster CB, Herman IM
• The mechanics of vascular cell motility.
• Microcirculation. 1998; 5: 239-57
• Display abstract

• Alterations in vascular cell shape and motility occur during developmental processes and in response to injury. Similarly, during tumor vascularization and atherogenesis, endothelial and smooth muscle cells undergo motile and proliferative responses to extracellular cues. Recent inroads into our understanding of signal transduction have identified several candidate pathways by which the extracellular matrix- and growth factor-mediated stimulation of vascular cell motility may be mediated. The multiple and divergent extracellular stimuli that stimulate vascular motile responses may converge on the cytoskeleton via a family of ras-related GTPases. Biochemical analyses as well as examination of cytoskeletal dynamics in vivo indicate that actin polymerization at the forward aspects of spreading cytoplasm is capable of driving forward protrusion formation in the absence of a conventional actin motor. Actin polymerization at the plasma membrane of leading lamellae may be mediated both by de novo nucleation of actin filaments and the generation of free filament ends by uncapping the barbed ends of existing actin filaments. This review summarizes the most recent findings in extracellular-cytoskeletal-signal transduction, therein, providing a framework to explain the remarkable remodeling seen in the vasculature during developmental and disease-related processes.

• Zohn IM, Campbell SL, Khosravi-Far R, Rossman KL, Der CJ
• Rho family proteins and Ras transformation: the RHOad less traveled gets congested.
• Oncogene. 1998; 17: 1415-38
• Display abstract

• The Rho family of small GTPases has attracted considerable research interest over the past 5 years. During this time, we have witnessed a remarkable increase in our knowledge of the biochemistry and biology of these Ras-related proteins. Thus, Rho family proteins have begun to rival, if not overshadow, interest in their more celebrated cousins, the Ras oncogene proteins. The fascination in Rho family proteins is fueled primarily by two major observations. First, like Ras, Rho family proteins serve as guanine nucleotide-regulated binary switches that control signaling pathways that in turn regulate diverse cellular processes. Rho family proteins are key components in cellular processes that control the organization of the actin cytoskeleton, activate kinase cascades, regulate gene expression, regulate membrane trafficking, promote growth transformation and induce apoptosis. Second, at least five Rho family proteins have been implicated as critical regulators of oncogenic Ras transformation. Thus, it is suspected that Rho family proteins contribute significantly to the aberrant growth properties of Ras-transformed cells. Rho family proteins are also critical mediators of the transforming actions of other transforming proteins and include Dbl family oncogene proteins, G protein-coupled receptors and G protein alpha subunits. Thus, Rho family proteins may be key components for the transforming actions of diverse oncogene proteins. Major aims of Rho family protein studies are to define the molecular mechanism by which Rho family proteins regulate such a diverse spectrum of cellular behavior. These efforts may reveal novel targets for the development of anti-Ras and anti-cancer drugs.

• Kigawa T, Endo M, Ito Y, Shirouzu M, Kikuchi A, Yokoyama S
• Solution structure of the Ras-binding domain of RGL.
• FEBS Lett. 1998; 441: 413-8
• Display abstract

• The RGL protein, a homolog of the Ral GDP dissociation stimulator (RalGDS), has been identified as a downstream effector of Ras. In the present study, the solution structure of the Ras-binding domain of RGL (RGL-RBD) was determined by NMR spectroscopy. The overall fold of RGL-RBD consists of a five-stranded beta-sheet and two alpha-helices, which is the same topology as that of RalGDS-RBD. The backbone chemical shift perturbation of RGL-RBD upon interaction with the GTP analog-bound Ras was also examined. The solution structure of RGL-RBD, especially around some of the Ras-interacting residues, is appreciably different from that of RalGDS-RBD.

• Helliwell SB, Schmidt A, Ohya Y, Hall MN
• The Rho1 effector Pkc1, but not Bni1, mediates signalling from Tor2 to the actin cytoskeleton.
• Curr Biol. 1998; 8: 1211-4
• Display abstract

• In Saccharomyces cerevisiae, the phosphatidylinositol kinase homologue Tor2 controls the cell-cycle-dependent organisation of the actin cytoskeleton by activating the small GTPase Rho1 via the exchange factor Rom2 [1,2]. Four Rho1 effectors are known, protein kinase C 1 (Pkc1), the formin-family protein Bni1, the glucan synthase Fks and the signalling protein Skn7 [2,3]. Rho1 has been suggested to signal to the actin cytoskeleton via Bni1 and Pkc1; rho1 mutants have never been shown to have defects in actin organisation, however [2,4]. We have further investigated the role of Rho1 in controlling actin organisation and have analysed which of the Rho1 effectors mediates Tor2 signalling to the actin cytoskeleton. We show that some, but not all, rho1 temperature-sensitive (rho1ts) mutants arrest growth with a disorganised actin cytoskeleton. Both the growth defect and the actin organisation defect of the rho1-2ts mutant were suppressed by upregulation of Pkc1 but not by upregulation of Bni1, Fks or Skn7. Overexpression of Pkc1, but not overexpression of Bni1, Fks or Skn7, also rescued a tor2ts mutant, and deletion of BNI1 or SKN7 did not prevent the suppression of the tor2ts mutation by overexpressed Rom2. Furthermore, overexpression of the Pkc1-controlled mitogen-activated protein (MAP) kinase Mpk1 suppressed the actin defect of tor2ts and rho1-2ts mutants. Thus, Tor2 signals to the actin cytoskeleton via Rho1, Pkc1 and the cell integrity MAP kinase cascade.

• Yee HF Jr
• Rho directs activation-associated changes in rat hepatic stellate cell morphology via regulation of the actin cytoskeleton.
• Hepatology. 1998; 28: 843-50
• Display abstract

• Hepatic stellate cell activation, thought to play a key role in fibrosis of the liver, is characterized by changes in cellular morphology. The intracellular signals regulating morphological alterations associated with stellate cell activation are uncertain. The ras-like guanosine triphosphate-binding protein, rho, has recently emerged as an important regulator of the actin cytoskeleton, and consequently cell morphology. The aim of this study was to test the hypothesis that rho signaling pathways direct activation-associated morphological changes in stellate cells by regulating the actin cytoskeleton. The morphology and actin cytoskeleton of primary rat hepatic stellate cells were studied with phase contrast, differential interference contrast, and epifluorescence microscopy. Immunohistochemistry and immunoblot analysis were used to examine rho expression and activity, respectively. Quiescent and activated stellate cells were investigated in the absence and presence of C3 transferase, a bacterial toxin that specifically inhibits rho. Stellate cell activation was characterized by the development of prominent intracellular fibers, and the loss of dendrite-like processes and perinuclear retinoid droplets. Moreover, activation was accompanied by the formation of prominent actin stress fibers and focal adhesions. Both rho expression and activity were demonstrated in stellate cells. C3 transferase blocked and reversed, both activation-associated morphological alterations and activation-associated changes in the actin cytoskeleton, in quiescent and activated stellate cells, respectively. These results indicate that rho directs activation-associated changes in rat hepatic stellate cell morphology via regulation of the actin cytoskeleton.

• Saito Y
• [Structures and functions of small GTPase and heterotrimeric G proteins]
• Nippon Rinsho. 1998; 56: 1750-5
• Display abstract

• Small GTPase ras and heterotrimeric G proteins composed of alpha, beta and gamma subunits are members of a superfamily of regulatory GTP hydrolases. They function as molecular switches which cycle between an inactive GDP-bound state and an active GTP-bound state, and are involved in regulatory biological processes from the outside of the cell to its interior. Binding of GTP triggers conformational changes in switch regions, which enable alpha subunit and ras to interact with effector molecules. Beta gamma dimers dissociated from alpha subunit are signaling molecules in their own rights. These G proteins activate various signal transduction pathways including activation of MAP kinases, phosphoinositide 3-kinases and small GTPases.

• Wall MA, Posner BA, Sprang SR
• Structural basis of activity and subunit recognition in G protein heterotrimers.
• Structure. 1998; 6: 1169-83
• Display abstract

• BACKGROUND: Inactive heterotrimeric G proteins are composed of a GDP-bound alpha subunit (Galpha) and a stable heterodimer of Gbeta and Ggamma subunits. Upon stimulation by a receptor, Galpha subunits exchange GDP for GTP and dissociate from Gbetagamma, both Galpha and Gbetagamma then interact with downstream effectors. Isoforms of Galpha, Gbeta and Ggamma potentially give rise to many heterotrimeric combinations, limited in part by amino acid sequence differences that lead to selective interactions. The mechanism by which GTP promotes Gbetagamma dissociation is incompletely understood. The Gly203-->Ala mutant of Gialpha1 binds and hydrolyzes GTP normally but does not dissociate from Gbetagamma, demonstrating that GTP binding and activation can be uncoupled. Structural data are therefore important for understanding activation and subunit recognition in G protein heterotrimers. RESULTS: The structures of the native (Gialpha1beta1gamma2) heterotrimer and that formed with Gly203-->AlaGialpha1 have been determined to resolutions of 2.3 A and 2.4 A, respectively, and reveal previously unobserved segments at the Ggamma2 C terminus. The Gly203-->Ala mutation alters the conformation of the N terminus of the switch II region (Val201-Ala203), but not the global structure of the heterotrimer. The N termini of Gbeta and Ggamma form a rigid coiled coil that packs at varying angles against the beta propeller of Gbeta. Conformational differences in the CD loop of beta blade 2 of Gbeta mediate isoform-specific contacts with Galpha. CONCLUSIONS: The Gly203-->Ala mutation in Gialpha1 blocks the conformational changes in switch II that are required to release Gbetagamma upon binding GTP. The interface between the ras-like domain of Galpha and the beta propeller of Gbeta appears to be conserved in all G protein heterotrimers. Sequence variation at the Gbeta-Galpha interface between the N-terminal helix of Galpha and the CD loop of beta blade 2 of Gbeta1 (residues 127-135) could mediate isoform-specific contacts. The specificity of Gbeta and Ggamma interactions is largely determined by sequence variation in the contact region between helix 2 of Ggamma and the surface of Gbeta.

• Ridley AJ
• Mammalian cell microinjection assay to study the function of Rac and Rho.
• Methods Mol Biol. 1998; 84: 153-60

• Pennisi E
• Structure of key cytoskeletal protein tubulin revealed.
• Science. 1998; 279: 176-7

• Ji TH, Grossmann M, Ji I
• G protein-coupled receptors. I. Diversity of receptor-ligand interactions.
• J Biol Chem. 1998; 273: 17299-302

• Zor T, Bar-Yaacov M, Elgavish S, Shaanan B, Selinger Z
• Rescue of a mutant G-protein by substrate-assisted catalysis.
• Eur J Biochem. 1997; 249: 330-6
• Display abstract

• Signaling by guanine-nucleotide-binding proteins (G-proteins) occurs when they are charged with GTP, while hydrolysis of the bound nucleotide turns the signaling off. Despite a wealth of biochemical and structural information, the mechanism of GTP hydrolysis by G-proteins remains controversial. We have employed substrate-assisted catalysis as a novel approach to study catalysis by G-proteins. In these studies, we have used diaminobenzophenone-phosphonoamidate-GTP, a unique GTP analog bearing the functional groups that are missing in the GTPase-deficient [Leu227]G(s alpha) mutant. This mutant, found in various human tumors, fails to hydrolyze GTP for an extended period. In contrast, the GTP analog is hydrolyzed by this mutant and by the wild-type enzyme at the same rate. On the other hand, modification of G(s alpha) by cholera toxin, which catalyses ADP-ribosylation of Arg201 of G(s alpha), decreased the rates of hydrolysis of both GTP and its analog by 95%. These results attest to the specificity of the GTP analog as a unique substrate for the [Leu227]G(s alpha) mutant and to the essential role of Gln227 in GTP hydrolysis. Furthermore, the finding that the GTP analog was hydrolyzed at the same rate as GTP by the wild-type enzyme, favors a model in which formation of a pentavalent transition state intermediate, presumably stabilized by the catalytic glutamine, is not the rate-limiting step of the GTPase reaction.

• Leeuwen FN, Kain HE, Kammen RA, Michiels F, Kranenburg OW, Collard JG
• The guanine nucleotide exchange factor Tiam1 affects neuronal morphology; opposing roles for the small GTPases Rac and Rho.
• J Cell Biol. 1997; 139: 797-807
• Display abstract

• The invasion-inducing T-lymphoma invasion and metastasis 1 (Tiam1) protein functions as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac1. Differentiation-dependent expression of Tiam1 in the developing brain suggests a role for this GEF and its effector Rac1 in the control of neuronal morphology. Here we show that overexpression of Tiam1 induces cell spreading and affects neurite outgrowth in N1E-115 neuroblastoma cells. These effects are Rac-dependent and strongly promoted by laminin. Overexpression of Tiam1 recruits the alpha 6 beta 1 integrin, a laminin receptor, to specific adhesive contacts at the cell periphery, which are different from focal contacts. Cells overexpressing Tiam1 no longer respond to lysophosphatidic acid- induced neurite retraction and cell rounding, processes mediated by Rho, suggesting that Tiam1-induced activation of Rac antagonizes Rho signaling. This inhibition can be overcome by coexpression of constitutively active RhoA, which may indicate that regulation occurs at the level of Rho or upstream. Conversely, neurite formation induced by Tiam1 or Rac1 is further promoted by inactivating Rho. These results demonstrate that Rac- and Rho-mediated pathways oppose each other during neurite formation and that a balance between these pathways determines neuronal morphology. Furthermore, our data underscore the potential role of Tiam1 as a specific regulator of Rac during neurite formation and illustrate the importance of reciprocal interactions between the cytoskeleton and the extracellular matrix during this process.

• Kubler E, Mosch HU, Rupp S, Lisanti MP
• Gpa2p, a G-protein alpha-subunit, regulates growth and pseudohyphal development in Saccharomyces cerevisiae via a cAMP-dependent mechanism.
• J Biol Chem. 1997; 272: 20321-3
• Display abstract

• The small GTP-binding protein Ras and heterotrimeric G-proteins are key regulators of growth and development in eukaryotic cells. In mammalian cells, Ras functions to regulate the mitogen-activated protein kinase pathway in response to growth factors, whereas many heterotrimeric GTP-binding protein alpha-subunits modulate cAMP levels through adenylyl cyclase as a consequence of hormonal action. In contrast, in the yeast Saccharomyces cerevisiae, it is the Ras1 and Ras2 proteins that regulate adenylyl cyclase. Of the two yeast G-protein alpha-subunits (GPA1 and GPA2), only GPA1 has been well studied and shown to negatively regulate the mitogen-activated protein kinase pathway upon pheromone stimulation. In this report, we show that deletion of the GPA2 gene encoding the other yeast G-protein alpha-subunit leads to a defect in pseudohyphal development. Also, the GPA2 gene is indispensable for normal growth in the absence of Ras2p. Both of these phenotypes can be rescued by deletion of the PDE2 gene product, which inactivates cAMP by cleavage, suggesting that these phenotypes can be attributed to low levels of intracellular cAMP. In support of this notion, addition of exogenous cAMP to the growth media was also sufficient to rescue the phenotype of a GPA2 deletion strain. Taken together, our results directly demonstrate that a G-protein alpha-subunit can regulate the growth and pseudohyphal development of S. cerevisiae via a cAMP-dependent mechanism. Heterologous expression of mammalian G-protein alpha-subunits in these yeast GPA2 deletion strains could provide a valuable tool for the mutational analysis of mammalian G-protein function in an in vivo null setting.

• Kaibuchi K
• [Regulation of cytoskeleton and cell adhesion by small GTPases]
• Seikagaku. 1997; 69: 16-29

• Schmidt A, Bickle M, Beck T, Hall MN
• The yeast phosphatidylinositol kinase homolog TOR2 activates RHO1 and RHO2 via the exchange factor ROM2.
• Cell. 1997; 88: 531-42
• Display abstract

• The Saccharomyces cerevisiae phosphatidylinositol kinase homolog TOR2 is required for organization of the actin cytoskeleton. Overexpression of RHO1 or RHO2, encoding Rho-like GTPases, or ROM2, encoding a GDP/GTP exchange factor for RHO1 and RHO2, suppresses a tor2 mutation. Deletion of SAC7, a gene originally identified as a suppressor of an actin mutation, also suppresses a tor2 mutation. SAC7 is a novel GTPase-activating protein for RHO1. ROM2 exchange activity is reduced in a tor2 mutant, and overexpression of ROM2 lacking its PH domain can no longer suppress a tor2 mutation. Thus, TOR2 signals to the actin cytoskeleton through a GTPase switch composed of RHO1, RHO2, ROM2, and SAC7. TOR2 activates this switch via ROM2, possibly via the ROM2 PH domain.

• Tanaka K
• [Regulation and modes of action of Ras and Rho small GTP-binding proteins in budding yeast]
• Seikagaku. 1997; 69: 979-90

• Narumiya S, Ishizaki T, Watanabe N
• Rho effectors and reorganization of actin cytoskeleton.
• FEBS Lett. 1997; 410: 68-72
• Display abstract

• The small GTPase Rho regulates several actomyosin-based cellular processes such as cell adhesion, cytokinesis and contraction. The biochemical mechanisms of these actions remain unknown. Recently, several GTP-Rho binding proteins were isolated. Among them, p140mDia and p160ROCK appear to work as Rho effectors mediating its action on the cytoskeleton. p140mDia induces actin polymerization by recruiting an actin binding protein, profilin, to the site of Rho action. p160ROCK induces focal adhesions and stress fibers by activating integrin and clustering them by the use of myosin-based contractility.

• Vincent S, Settleman J
• The PRK2 kinase is a potential effector target of both Rho and Rac GTPases and regulates actin cytoskeletal organization.
• Mol Cell Biol. 1997; 17: 2247-56
• Display abstract

• The Ras-related Rho family GTPases mediate signal transduction pathways that regulate a variety of cellular processes. Like Ras, the Rho proteins (which include Rho, Rac, and CDC42) interact directly with protein kinases, which are likely to serve as downstream effector targets of the activated GTPase. Activated RhoA has recently been reported to interact directly with several protein kinases, p120 PKN, p150 ROK alpha and -beta, p160 ROCK, and p164 Rho kinase. Here, we describe the purification of a novel Rho-associated kinase, p140, which appears to be the major Rho-associated kinase activity in most tissues. Peptide microsequencing revealed that p140 is probably identical to the previously reported PRK2 kinase, a close relative of PKN. However, unlike the previously described Rho-binding kinases, which are Rho specific, p140 associates with Rac as well as Rho. Moreover, the interaction of p140 with Rho in vitro is nucleotide independent, whereas the interaction with Rac is completely GTP dependent. The association of p140 with either GTPase promotes kinase activity substantially, and expression of a kinase-deficient form of p140 in microinjected fibroblasts disrupts actin stress fibers. These results indicate that p140 may be a shared kinase target of both Rho and Rac GTPases that mediates their effects on rearrangements of the actin cytoskeleton.

• Mulholland J, Wesp A, Riezman H, Botstein D
• Yeast actin cytoskeleton mutants accumulate a new class of Golgi-derived secretary vesicle.
• Mol Biol Cell. 1997; 8: 1481-99
• Display abstract

• Many yeast actin cytoskeleton mutants accumulate large secretory vesicles and exhibit phenotypes consistent with defects in polarized growth. This, together with actin's polarized organization, has suggested a role for the actin cytoskeleton in the vectorial transport of late secretory vesicles to the plasma membrane. By using ultrastructural and biochemical analysis, we have characterized defects manifested by mutations in the SLA2 gene (also known as the END4 gene), previously found to affect both the organization of the actin cytoskeleton and endocytosis in yeast. Defects in cell wall morphology, accumulated vesicles, and protein secretion kinetics were found in sla2 mutants similar to defects found in act1 mutants. Vesicles that accumulate in the sla2 and act1 mutants are immunoreactive with antibodies directed against the small GTPase Ypt1p but not with antibodies directed against the homologous Sec4p found on classical "late" secretory vesicles. In contrast, the late-acting secretory mutants sec1-1 and sec6-4 are shown to accumulate anti-Sec4p-positive secretory vesicles as well as vesicles that are immunoreactive with antibodies directed against Ypt1p. The late sec mutant sec4-8 is also shown to accumulate Ypt1p-containing vesicles and to exhibit defects in actin cytoskeleton organization. These results indicate the existence of at least two classes of morphologically similar, late secretory vesicles (associated with Ypt1p+ and Sec4p+, respectively), one of which appears to accumulate when the actin cytoskeleton is disorganized.

• Keep NH et al.
• A modulator of rho family G proteins, rhoGDI, binds these G proteins via an immunoglobulin-like domain and a flexible N-terminal arm.
• Structure. 1997; 5: 623-33
• Display abstract

• BACKGROUND: The rho family of small G proteins, including rho, rac and cdc42, are involved in many cellular processes, including cell transformation by ras and the organization of the actin cytoskeleton. Additionally, rac has a role in the regulation of phagocyte NADPH oxidase. Guanine nucleotide dissociation inhibitors (GDIs) of the rhoGDI family bind to these G proteins and regulate their activity by preventing nucleotide dissociation and by controlling their interaction with membranes. RESULTS: We report the structure of rhoGDI, determined by a combination of X-ray crystallography and NMR spectroscopy. NMR spectroscopy and selective proteolysis show that the N-terminal 50-60 residues of rhoGDI are flexible and unstructured in solution. The 2.5 A crystal structure of the folded core of rhoGDI, comprising residues 59-204, shows it to have an immunoglobulin-like fold, with an unprecedented insertion of two short beta strands and a 310 helix. There is an unusual pocket between the beta sheets of the immunoglobulin fold which may bind the C-terminal isoprenyl group of rac. NMR spectroscopy shows that the N-terminal arm is necessary for binding rac, although it remains largely flexible even in the complex. CONCLUSIONS: The rhoGDI structure is notable for the existence of both a structured and a highly flexible domain, both of which appear to be required for the interaction with rac. The immunoglobulin-like fold of the structured domain is unusual for a cytoplasmic protein. The presence of equivalent cleavage sites in rhoGDI and the closely related D4/Ly-GDI (rhoGDI-2) suggest that proteolytic cleavage between the flexible and structured regions of rhoGDI may have a role in the regulation of the activity of members of this family. There is no detectable similarity between the structure of rhoGDI and the recently reported structure of rabGDI, which performs the same function as rhoGDI for the rab family of small G proteins.

• Rebstein PJ, Cardelli J, Weeks G, Spiegelman GB
• Mutational analysis of the role of Rap1 in regulating cytoskeletal function in Dictyostelium.
• Exp Cell Res. 1997; 231: 276-83
• Display abstract

• It was shown previously that increased expression of the ras-related rap1 gene in Dictyostelium discoideum altered cell morphology (Rebstein et al., Dev. Genet., 1993, 14, 347-355). Vegetative Rap1 transformants were more flattened and spread than parental Ax2 cells and had increased F-actin near the cell periphery. In addition, Rap1 cells were inhibited in the rapid cell contraction that occurs upon refeeding with nutrient media. In this communication, we show that expression of Rap also markedly reduces the contraction response that occurs upon addition of azide to vegetative cells. The changes in cell morphology, the refeeding contraction response, and the azide contraction response have been used to analyze mutants of Rap1 generated by site-directed mutagenesis. The substitution G12V, predicted to increase the proportion of protein binding GTP, did not alter the effect of Rap on cell morphology or on its ability to inhibit the contraction response to azide, but modestly enhanced the ability of Rap1 to inhibit cell rounding in response to nutrient media. The substitution S17N, predicted to restrict the protein to the GDP-bound state, did not produce the flattened cell morphology and abolished the inhibitory effects of Rap in the two cell contraction assays. These results are consistent with a requirement of GTP binding for the Rap-induced effects. Transformants carrying the Rap-S17N protein had a more polar morphology than the parental Ax2 cells, suggesting the possibility that Rap-S17N interferes with the ability of endogenous Rap to regulate the cytoskeleton. Substitutions at amino acid 38, within the presumptive effector domain, reduced but did not abolish the effects of Rap1 on cell contraction, while the substitution T61Q had no effect on Rap1 activity. Taken together, the results suggest that Rap may have multiple regulatory effects on cytoskeletal function.

• Braga VM, Machesky LM, Hall A, Hotchin NA
• The small GTPases Rho and Rac are required for the establishment of cadherin-dependent cell-cell contacts.
• J Cell Biol. 1997; 137: 1421-31
• Display abstract

• Cadherins are calcium-dependent cell-cell adhesion molecules that require the interaction of the cytoplasmic tail with the actin cytoskeleton for adhesive activity. Because of the functional relationship between cadherin receptors and actin filament organization, we investigated whether members of the Rho family of small GTPases are necessary for cadherin adhesion. In fibroblasts, the Rho family members Rho and Rac regulate actin polymerization to produce stress fibers and lamellipodia, respectively. In epithelial cells, we demonstrate that Rho and Rac are required for the establishment of cadherin-mediated cell-cell adhesion and the actin reorganization necessary to stabilize the receptors at sites of intercellular junctions. Blocking endogenous Rho or Rac selectively removed cadherin complexes from junctions induced for up to 3 h, while desmosomes were not perturbed. In addition, withdrawal of cadherins from intercellular junctions temporally precedes the removal of CD44 and integrins, other microfilament-associated receptors. Our data showed that the concerted action of Rho and Rac modulate the establishment of cadherin adhesion: a constitutively active form of Rac was not sufficient to stabilize cadherindependent cell-cell contacts when endogenous Rho was inhibited. Upon induction of calcium-dependent intercellular adhesion, there was a rapid accumulation of actin at sites of cell-cell contacts, which was prevented by blocking cadherin function, Rho or Rac activity. However, if cadherin complexes are clustered by specific antibodies attached to beads, actin recruitment to the receptors was perturbed by inhibiting Rac but not Rho. Our results provide new insights into the role of the small GTPases in the cadherin-dependent cell- cell contact formation and the remodelling of actin filaments in epithelial cells.

• Sprang SR
• GAP into the breach.
• Science. 1997; 277: 329-30

• Ridley AJ
• The GTP-binding protein Rho.
• Int J Biochem Cell Biol. 1997; 29: 1225-9
• Display abstract

• RhoA, RhoB and RhoC are three closely related proteins, and are members of the Ras super-family of small GTP-binding proteins. They bind and hydrolyse GTP, and are active in the GTP-bound form. Their activity in cells is regulated by exchange factors, GTPase activating proteins and guanine nucleotide dissociation inhibitors. Several potential downstream target proteins for Rho proteins have been identified, including protein kinases and adaptor-type proteins. Rho proteins regulate actin cytoskeletal organization; for example in fibroblasts RhoA induces the formation of actin stress fibres. Rho proteins are also involved in regulating secretion, pinocytosis and clathrin coat-mediated endocytosis, transcriptional activation and stimulation of DNA synthesis. In addition, there is evidence that Rho proteins can play a role in cell transformation, and thus Rho proteins or components of their signalling pathways may be potential targets for the development of anti-cancer therapies.

• Geyer M, Herrmann C, Wohlgemuth S, Wittinghofer A, Kalbitzer HR
• Structure of the Ras-binding domain of RalGEF and implications for Ras binding and signalling.
• Nat Struct Biol. 1997; 4: 694-9
• Display abstract

• The solution structure of the Ras-binding domain (RBD) of Ral guanine-nucleotide exchange factor RalGEF was solved by NMR spectroscopy. The overall structure is similar to that of Raf-RBD, another effector of Ras, although the sequence identity is only 13%. 15N chemical shifts changes in the complex of RalGEF-RBD with Ras indicate an interaction similar to the intermolecular beta-sheet observed for the complex between Ras and Raf-RBD.

• Dharmawardhane S, Bokoch GM
• Rho GTPases and leukocyte cytoskeletal regulation.
• Curr Opin Hematol. 1997; 4: 12-8
• Display abstract

• The Rho GTPases (Rho, Rac, and Cdc42) regulate assembly of the actin cytoskeleton in many cells, including leukocytes. Recent work in identifying the protein targets of these GTPases is providing greater insight into the mechanisms used by cells to control cytoskeletal dynamics for a variety of purposes.

• Ahmadian MR, Mittal R, Hall A, Wittinghofer A
• Aluminum fluoride associates with the small guanine nucleotide binding proteins.
• FEBS Lett. 1997; 408: 315-8
• Display abstract

• AlF4- has long been known to associate with and activate the GDP-bound alpha subunits of heterotrimeric G-proteins. Recently the small guanine nucleotide binding protein Ras has also been shown to associate with AlF4- in the presence of stoichiometric amounts of its GTPase activating protein (GAP). Here we present the isolation of a stable Ras x GDP- x AlF4- x GAP ternary complex by gel filtration. In addition, we generalise the association of AlF4- with the small GTP-binding proteins by demonstrating ternary complex formation for the Cdc42, Rap and Ran proteins in the presence of their respective GAP proteins.

• Fiorentini C et al.
• Escherichia coli cytotoxic necrotizing factor 1 (CNF1), a toxin that activates the Rho GTPase.
• J Biol Chem. 1997; 272: 19532-7
• Display abstract

• Cytotoxic necrotizing factor 1 (CNF1), a 110-kDa protein toxin from pathogenic Escherichia coli induces actin reorganization into stress fibers and retraction fibers in human epithelial cultured cells allowing them to spread. CNF1 is acting in the cytosol since microinjection of the toxin into HEp-2 cells mimics the effects of the externally applied CNF1. Incubation in vitro of CNF1 with recombinant small GTPases induces a modification of Rho (but not of Rac, Cdc42, Ras, or Rab6) as demonstrated by a discrete increase in the apparent molecular weight of the molecule. Preincubation of cells with CNF1 impairs the cytotoxic effects of Clostridium difficile toxin B, which inactivates Rho but not those of Clostridium sordellii LT toxin, which inhibits Ras and Rac. As shown for Rho-GTP, CNF1 activates, in a time- and dose-dependent manner, a cytoskeleton-associated phosphatidylinositol 4-phosphate 5-kinase. However, neither the phosphatidylinositol 4,5-bisphosphate (PIP2) nor the phosphatidylinositol 3,4-bisphosphate (PI 3,4-P2) or 3,4,5-trisphosphate (PIP3) cellular content were found increased in CNF1 treated HEp-2 cells. Cellular effects of CNF1 were not blocked by LY294002, a stable inhibitor of the phosphoinositide 3-kinase. Incubation of HEp-2 cells with CNF1 induces relocalization of myosin 2 in stress fibers but not in retraction fibers. Altogether, our data indicate that CNF1 is a toxin that selectively activates the Rho GTP-binding protein, thus inducing contractility and cell spreading.

• Tisi D, Teahan C, Greasley S, Bax B, Neu M, Jhoti H
• Common themes and surprising differences in small G-proteins.
• Biochem Soc Trans. 1997; 25: 989-91

• Ridley AJ
• Signalling by Rho family proteins.
• Biochem Soc Trans. 1997; 25: 1005-10

• Shibasaki Y, Ishihara H, Kizuki N, Asano T, Oka Y, Yazaki Y
• Massive actin polymerization induced by phosphatidylinositol-4-phosphate 5-kinase in vivo.
• J Biol Chem. 1997; 272: 7578-81
• Display abstract

• The Rho family GTP-binding proteins have been known to mediate extracellular signals to the actin cytoskeleton. Although several Rho interacting proteins have been found, downstream signals have yet to be determined. Many actin-binding proteins are known to be regulated by phosphatidylinositol 4,5-bisphosphate in vitro. Rho has been shown to enhance the activity of phosphatidylinositol-4-phosphate 5-kinase (PI4P5K), the phosphatidylinositol 4,5-bisphosphate synthesizing enzyme. Recently we isolated several isoforms of type I PI4P5K. Here we report that PI4P5K Ialpha induces massive actin polymerization resembling "pine needles" in COS-7 cells in vivo. When truncated from the C terminus to amino acid 308 of PI4P5K Ialpha, both kinase activity and actin polymerizing activity were lost. Although the dominant negative form of Rho, RhoN19, alone decreased actin fibers, those induced by PI4P5K were not affected by the coexpression of RhoN19. These results suggest that PI4P5K is located downstream from Rho and mediates signals for actin polymerization through its phosphatidylinositol-4-phosphate 5-kinase activity.

• Aktories K
• Rho proteins: targets for bacterial toxins.
• Trends Microbiol. 1997; 5: 282-8
• Display abstract

• GTP-binding proteins of the Rho family are regulators of the actin cytoskeleton and molecular switches in various signal transduction pathways. The Rho proteins are targets for bacterial protein toxins that either inactivate GTPases by ADP-ribosylation or glucosylation, or activate them by deamidation. Rho proteins play essential roles in host cell invasion by bacteria.

• Koch G, Tanaka K, Masuda T, Yamochi W, Nonaka H, Takai Y
• Association of the Rho family small GTP-binding proteins with Rho GDP dissociation inhibitor (Rho GDI) in Saccharomyces cerevisiae.
• Oncogene. 1997; 15: 417-22
• Display abstract

• The small GTP-binding proteins of the Rho family, consisting of the Rho, Rac, and Cdc42 subfamilies, are implicated in various cell functions, such as cell shape change, cell motility and cytokinesis, through reorganization of actin cytoskeleton. Rho GDI is a general regulator which forms a complex with the GDP-bound inactive form of the Rho family members and inhibits their activation. We have purified Rho GDI from the yeast Saccharomyces cerevisiae, cloned its gene, and named it RDII (Rho GD). In this study, we have further characterized yeast Rho GDI. Rho GDI was found in the cytosol by immunoblot and immunofluorescence microscopic analyses. Rho1p and Cdc42p were co-immunoprecipitated with Rho GDI from the cytosol. This immunoprecipitated Rho1p was mainly bound to GDP. In the disruption mutant of Rho GDI, which did not show any apparent phenotype, both Rho1p and Cdc42p were also present in the cytosol. These results indicate that yeast Rho GDI possesses properties similar to those of mammalian Rho GDI, and that there is a cytosolic factor which functionally substitutes for Rho GDI in yeast.

• Valentijn JA, LaCivita DQ, Gumkowski FD, Jamieson JD
• Rab4 associates with the actin terminal web in developing rat pancreatic acinar cells.
• Eur J Cell Biol. 1997; 72: 1-8
• Display abstract

• Rab4 is a small GTP-binding protein that has been implicated in the regulation of membrane traffic and recycling of transferrin receptors and GLUT4 transporters along the endocytic pathway. Here we present data that suggest a novel and very different role for rab4 during development in the rat exocrine pancreas. On immunoblots of pancreatic homogenates, a dramatic increase in rab4 expression occurred over the first 40 h after birth, concomitant with the time of acquisition of stimulus-secretion coupling. Following high-speed centrifugation of postnuclear supernatants prepared from 1-day neonatal pancreatic homogenates, rab4 partitioned into a Triton X-100 insoluble particulate fraction and was partially solubilized upon extraction with 0.1 M Na2CO3, pH 11.5, or 1 M KCl, suggesting that rab4 was not an integral membrane protein. This was confirmed by Triton X-114 extractions of post-nuclear supernatants showing that rab4 partitioned into the aqueous phase of Triton X-114, which is indicative of a lack of isoprenylation. Confocal and electron microscopic immunocytochemistry revealed that rab4 colocalized with the actin terminal web and microvilli in the apical region of the exocrine acinar cells. In view of these findings, we propose that rab4 is involved in the maturation of the regulated secretory pathway in pancreatic acinar cells through an interaction with the apical actin cytoskeleton.

• Koch G, Benz C, Schmidt G, Olenik C, Aktories K
• Role of Rho protein in lovastatin-induced breakdown of actin cytoskeleton.
• J Pharmacol Exp Ther. 1997; 283: 901-9
• Display abstract

• The Rho GTPases are involved in actin cytoskeleton organization and signal transduction. They need polyisoprenylation for membrane association and activation. Lovastatin, a hydroxymethylglutaryl coenzyme A inhibitor, prevents isoprene synthesis and thereby lipid modification of the Rho protein carboxy terminus. Because lovastatin causes rounding up of cultured cells, we investigated whether the compound acts on the actin cytoskeleton through Rho proteins. Lovastatin treatment decreased F-actin content in a time- and concentration-dependent manner. G-actin content remained unchanged. In lovastatin-treated NIH 3T3 cells, the amount of Rho protein which was ADP-ribosylated by Clostridium botulinum exoenzyme C3 decreased in membranes and increased in the cytosol fraction. Cycloheximide prevented lovastatin-induced rounding up of cells. However, after microinjection or direct application of exoenzyme C3, cells treated with cycloheximide and lovastatin rounded up again. On the contrary, lovastatin-treated, round Swiss 3T3 cells reverted to a flat morphology when microinjected with dominant active RhoA (Val14RhoA). Escherichia coli cytotoxic necrotizing factor (CNF1) which activates Rho proteins caused flattening of round, lovastatin-treated NIH 3T3 cells. These results suggest that lovastatin affects the actin cytoskeleton through inactivation of Rho proteins.

• Cherfils J et al.
• Crystal structures of the small G protein Rap2A in complex with its substrate GTP, with GDP and with GTPgammaS.
• EMBO J. 1997; 16: 5582-91
• Display abstract

• The small G protein Rap2A has been crystallized in complex with GDP, GTP and GTPgammaS. The Rap2A-GTP complex is the first structure of a small G protein with its natural ligand GTP. It shows that the hydroxyl group of Tyr32 forms a hydrogen bond with the gamma-phosphate of GTP and with Gly13. This interaction does not exist in the Rap2A-GTPgammaS complex. Tyr32 is conserved in many small G proteins, which probably also form this hydrogen bond with GTP. In addition, Tyr32 is structurally equivalent to a conserved arginine that binds GTP in trimeric G proteins. The actual participation of Tyr32 in GTP hydrolysis is not yet clear, but several possible roles are discussed. The conformational changes between the GDP and GTP complexes are located essentially in the switch I and II regions as described for the related oncoprotein H-Ras. However, the mobile segments vary in length and in the amplitude of movement. This suggests that even though similar regions might be involved in the GDP-GTP cycle of small G proteins, the details of the changes will be different for each G protein and will ensure the specificity of its interaction with a given set of cellular proteins.

• Mackay DJ, Esch F, Furthmayr H, Hall A
• Rho- and rac-dependent assembly of focal adhesion complexes and actin filaments in permeabilized fibroblasts: an essential role for ezrin/radixin/moesin proteins.
• J Cell Biol. 1997; 138: 927-38
• Display abstract

• The small GTPases Rho and Rac regulate actin filament assembly and the formation of integrin adhesion complexes to produce stress fibers and lamellipodia, respectively, in mammalian cells. Although numerous candidate effectors that might mediate these responses have been identified using the yeast two-hybrid and affinity purification techniques, their cellular roles remain unclear. We now describe a biological assay that allows components of the Rho and Rac signaling pathways to be identified. Permeabilization of serum-starved Swiss 3T3 cells with digitonin in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) induces both actin filament and focal adhesion complex assembly through activation of endogenous Rho and Rac. These responses are lost when GTPgammaS is added 6 min after permeabilization, but can be reconstituted using concentrated cytosolic extracts. We have achieved a 10,000-fold purification of the activity present in pig brain cytosol and protein sequence analysis shows it to contain moesin. Using recombinant proteins, we show that moesin and its close relatives ezrin and radixin can reconstitute stress fiber assembly, cortical actin polymerization and focal complex formation in response to activation of Rho and Rac.

• Wei Y et al.
• Crystal structure of RhoA-GDP and its functional implications.
• Nat Struct Biol. 1997; 4: 699-703
• Display abstract

• RhoA, a ubiquitous intracellular GTPase, mediates cytoskeletal responses to extracellular signals. A 2.1 A resolution crystal structure of the human RhoA-GDP complex shows unique stereochemistry in the switch I region, which results in a novel mode of Mg2+ binding.

• Takahashi K et al.
• Direct interaction of the Rho GDP dissociation inhibitor with ezrin/radixin/moesin initiates the activation of the Rho small G protein.
• J Biol Chem. 1997; 272: 23371-5
• Display abstract

• The Rho GDP dissociation inhibitor (GDI) forms a complex with the GDP-bound form of the Rho family small G proteins and inhibits their activation. The GDP-bound form complexed with Rho GDI is not activated by the GDP/GTP exchange factor for the Rho family members, suggesting the presence of another factor necessary for this activation. We have reported that the Rho subfamily members regulate the ezrin/radixin/moesin (ERM)-CD44 system, implicated in reorganization of actin filaments. Here we report that Rho GDI directly interacts with ERM, initiating the activation of the Rho subfamily members by reducing the Rho GDI activity. These results suggest that ERM as well as Rho GDI and the Rho GDP/GTP exchange factor are involved in the activation of the Rho subfamily members, which then regulate reorganization of actin filaments through the ERM system.

• Machesky LM, Hall A
• Role of actin polymerization and adhesion to extracellular matrix in Rac- and Rho-induced cytoskeletal reorganization.
• J Cell Biol. 1997; 138: 913-26
• Display abstract

• Most animal cells use a combination of actin-myosin-based contraction and actin polymerization- based protrusion to control their shape and motility. The small GTPase Rho triggers the formation of contractile stress fibers and focal adhesion complexes (Ridley, A.J., and A. Hall. 1992. Cell. 70:389-399) while a close relative, Rac, induces lamellipodial protrusions and focal complexes in the lamellipodium (Nobes, C.D., and A. Hall. 1995. Cell. 81:53-62; Ridley, A.J., H.F. Paterson, C.L. Johnston, D. Diekmann, and A. Hall. 1992. Cell. 70:401-410); the Rho family of small GTPases may thus play an important role in regulating cell movement. Here we explore the roles of actin polymerization and extracellular matrix in Rho- and Rac-stimulated cytoskeletal changes. To examine the underlying mechanisms through which these GTPases control F-actin assembly, fluorescently labeled monomeric actin, Cy3-actin, was introduced into serum-starved Swiss 3T3 fibroblasts. Incorporation of Cy3- actin into lamellipodial protrusions is concomitant with F-actin assembly after activation of Rac, but Cy3-actin is not incorporated into stress fibers formed immediately after Rho activation. We conclude that Rac induces rapid actin polymerization in ruffles near the plasma membrane, whereas Rho induces stress fiber assembly primarily by the bundling of actin filaments. Activation of Rho or Rac also leads to the formation of integrin adhesion complexes. Integrin clustering is not required for the Rho-induced assembly of actin-myosin filament bundles, or for vinculin association with actin bundles, but is required for stress fiber formation. Integrin-dependent focal complex assembly is not required for the Rac-induced formation of lamellipodia or membrane ruffles. It appears, therefore, that the assembly of large integrin complexes is not required for most of the actin reorganization or cell morphology changes induced by Rac or Rho activation in Swiss 3T3 fibroblasts.

• Courjal F, Chuchana P, Theillet C, Fort P
• Structure and chromosomal assignment to 22q12 and 17qter of the ras-related Rac2 and Rac3 human genes.
• Genomics. 1997; 44: 242-6
• Display abstract

• Members of the Rho/Rac/Cdc42Hs family of GTPases have been shown to participate in many aspects of the signaling of cell growth and differentiation. Although the biochemical properties of these GTPases have been extensively studied, very little is known about the structure of the corresponding genes. To gain insight on the evolution of the Rho family, we were interested in studying the genomic structure of several members. We report here the structure and the localization to 22q12 of the human Rac2 gene, as well as the localization to 17qter of Rac3, a new member closely related to Rac1 and Rac2. Unlike the structure of its closest relative ARH-G gene, which contains a single intron, Rac2 is made of at least 7 exons, spanning over 18 kb of DNA. Comparison of gene structure and exonic borders suggests that the emergence of the whole superfamily took place early during evolution.

• Parish CA, Smrcka AV, Rando RR
• The role of G protein methylation in the function of a geranylgeranylated beta gamma isoform.
• Biochemistry. 1996; 35: 7499-505
• Display abstract

• The gamma subunit of heterotrimeric G proteins is isoprenylated and methylated on its carboxyl terminal cysteine residue. While retinal transducin is farnesylated, all other gamma subunits are modified by geranylgeranylation. An immobilized form of pig liver esterase (iPLE) is able to hydrolyze the methyl ester of a geranylgeranylated beta gamma isoform (beta 1 gamma 2). Since methylation is the only reversible reaction in the isoprenylation pathway, it could be a site of regulation of G protein activity. With both the methylated and demethylated beta 1 gamma 2 now available, the role of methylation for a geranylgeranylated heterotrimeric G protein may be addressed. Here, it is reported that methylation has no effect on the ability of beta gamma to interact with an alpha subunit, as probed by ADP-ribosylation studies with pertussis toxin, and has a small effect (less than 2-fold) on the ability of geranylgeranylated beta gamma to activate phosphatidylinositol-specific phospholipase C (PIPLC) and phosphoinositide 3 kinase (PI3K). In binding studies, demethylation only slightly decreased the ability of beta 1 gamma 2 to adhere to azolectin vesicles. Therefore, methylation of heterotrimeric G proteins appears to have only a minor effect in signal transduction processes which can be correlated to a decrease in hydrophobicity of the beta gamma subunit.

• Schalk I et al.
• Structure and mutational analysis of Rab GDP-dissociation inhibitor.
• Nature. 1996; 381: 42-8
• Display abstract

• The crystal structure of the bovine alpha-isoform of Rab GDP-dissociation inhibitor (GDI), which functions in vesicle-membrane transport to recycle and regulate Rab GTPases, has been determined to a resolution of 1.81 A. GDI is constructed of two main structural units, a large complex multisheet domain I and a smaller alpha-helical domain II. The structural organization of domain I is surprisingly closely related to FAD-containing monooxygenases and oxidases. Sequence-conserved regions common to GDI and the choroideraemia gene product, which delivers Rab to catalytic subunits of Rab geranylgeranyltransferase II, are clustered on one face of the molecule. The two most sequence-conserved regions, which form a compact structure at the apex of GDI, are shown by site-directed mutagenesis to play a critical role in the binding of Rab proteins.

• Fincham VJ, Unlu M, Brunton VG, Pitts JD, Wyke JA, Frame MC
• Translocation of Src kinase to the cell periphery is mediated by the actin cytoskeleton under the control of the Rho family of small G proteins.
• J Cell Biol. 1996; 135: 1551-64
• Display abstract

• We have isolated Swiss 3T3 subclones that are resistant to the mitogenic and morphological transforming effects of v-Src as a consequence of aberrant translocation of the oncoprotein under low serum conditions. In chicken embryo and NIH 3T3 fibroblasts under similar conditions, v-Src rapidly translocates from the perinuclear region to the focal adhesions upon activation of the tyrosine kinase, resulting in downstream activation of activator protein-1 and mitogen-activated protein kinase, which are required for the mitogenic and transforming activity of the oncoprotein. Since serum deprivation induces cytoskeletal disorganization in Swiss 3T3, we examined whether regulators of the cytoskeleton play a role in the translocation of v-Src, and also c-Src, in response to biological stimuli. Actin stress fibers and translocation of active v-Src to focal adhesions in quiescent Swiss 3T3 cells were restored by microinjection of activated Rho A and by serum. Double labeling with anti-Src and phalloidin demonstrated that v-Src localized along the reformed actin filaments in a pattern that would be consistent with trafficking in complexes along the stress fibers to focal adhesions. Furthermore, treatment with the actin-disrupting drug cytochalasin D, but not the microtubule-disrupting drug nocodazole, prevented v-Src translocation. In addition to v-Src, we observed that PDGF-induced, Rac-mediated membrane ruffling was accompanied by translocation of c-Src from the cytoplasm to the plasma membrane, an effect that was also blocked by cytochalasin D. Thus, we conclude that translocation of Src from its site of synthesis to its site of action at the cell membrane requires an intact cytoskeletal network and that the small G proteins of the Rho family may specify the peripheral localization in focal adhesions or along the membrane, mediated by their effects on the cytoskeleton.

• Remmers AE, Neubig RR
• Partial G protein activation by fluorescent guanine nucleotide analogs. Evidence for a triphosphate-bound but inactive state.
• J Biol Chem. 1996; 271: 4791-7
• Display abstract

• N-methyl-3'-O-anthranoyl (MANT) guanine nucleotide analogs are useful environmentally sensitive fluorescent probes for studying G protein mechanisms. Previously, we showed that MANT fluorescence intensity when bound to G protein was related to the degree of G protein activation where MANT-guanosine-5'-O-(3-thiotriphosphate) (mGTP gammaS) had the highest fluorescence followed by mGTP and mGDP, respectively (Remmers, A. E., Posner, R., and Neubig, R. R. (1994) J. Biol. Chem. 269, 13771-13778). To directly examine G protein conformations with nucleotide triphosphates bound, we synthesized several nonhydrolyzable MANT-labeled guanine nucleotides. The relative maximal fluorescence levels observed upon binding to recombinant myristoylated Goalpha (myrGoalpha) and myrGialpha1 were: mGTPgammaS > MANT-5'-guanylyl-imidodiphosphate > MANT-guanylyl-(beta,gamma-methylene)-diphosphonate > MANT-guanosine 5'-O-2-(thio)diphosphate. Using protection against tryptic digestion as a measure of the activated conformation, the ability of the MANT guanine nucleotides to maximally activate myrGo alpha correlated with maximal fluorescence. Biphasic dissociation kinetics were observed for all of the MANT guanine nucleotides. The data were consistent with the following model, [formula: see text] where G protein activation (G*-GXP) is determined by a conformational equilibrium between two triphosphate bound states as well as by the balance between binding and hydrolysis of the nucleotide triphosphate. Compared with myrGialpha1, maximal mGTP fluorescence was only 2-fold higher for the myrGialpha1 Q204L mutant, a mutant with greatly reduced GTPase activity, and only 24% that of mGTPgammaS, indicating that partial activation by mGTP was not just due to hydrolysis of mGTP. These results extend our previous conclusion that GTP analogs do not fully activate G protein.

• Kjeldgaard M, Nyborg J, Clark BF
• The GTP binding motif: variations on a theme.
• FASEB J. 1996; 10: 1347-68
• Display abstract

• GTP binding proteins (G-proteins) have wide-ranging functions in biology, being involved in cell proliferation, signal transduction, protein synthesis, and protein targeting. Common to their functioning is that they are active in the GTP-bound form and inactive in the GDP-bound form. The protein synthesis elongation factor EF-Tu was the first G-protein whose nucleotide binding domain was solved structurally by X-ray crystallography to yield a structural definition of the GDP-bound form, but a still increasing number of new structures of G-proteins are appearing in the literature, in both GDP and GTP bound forms. A common structural core for nucleotide binding is present in all these structures, and this core has long been known to include common consensus sequence elements involved in binding of the nucleotide. Nevertheless, subtle changes in the common sequences reflect functional differences. Therefore, it becomes increasingly important to focus on how these differences are reflected in the structures, and how these structural differences are related to function. The aim of this review is to describe to what extent this structural motif for GDP/GTP binding is common to other known structures of this class of proteins. We first describe the common structural core of the G-proteins. Next, examples are based on information available on the Ras protein superfamily, the targeting protein ARF, elongation factors EF-Tu and EF-G, and the heterotrimeric G-proteins. Finally, we discuss the important structures of complexes between GTP binding proteins and their substrates that have appeared in the literature recently.

• Bussey H
• Cell shape determination: a pivotal role for Rho.
• Science. 1996; 272: 224-5

• Lin HC, Gilman AG
• Regulation of dynamin I GTPase activity by G protein betagamma subunits and phosphatidylinositol 4,5-bisphosphate.
• J Biol Chem. 1996; 271: 27979-82
• Display abstract

• Dynamin I is a 100-kDa GTPase that plays an important role in the recycling of synaptic vesicles. Hydrolysis of GTP by dynamin is thought to be a critical step in fission of coated pits to form coated vesicles. We report that the heterotrimeric G protein betagamma subunit complex (Gbetagamma) and phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P2) are negative and positive regulators of dynamin GTPase activity, respectively. Furthermore, the apparent affinity of dynamin for Gbetagamma is substantially enhanced by PtdIns(4,5)P2. However, the GTPase activity of oligomeric dynamin is unaffected by Gbetagamma. The effects of heterotrimeric G proteins on endocytosis may thus be mediated directly and not involve more remote aspects of their signaling properties.

• Poland BW, Hou Z, Bruns C, Fromm HJ, Honzatko RB
• Refined crystal structures of guanine nucleotide complexes of adenylosuccinate synthetase from Escherichia coli.
• J Biol Chem. 1996; 271: 15407-13
• Display abstract

• Structures of adenylosuccinate synthetase from Escherichia coli complexed with guanosine-5'-(beta,gamma-imido) triphosphate and guanosine-5'-(beta,gamma-methylene)triphosphate in the presence and the absence of Mg2+ have been refined to R-factors below 0.2 against data to a nominal resolution of 2.7 A. Asp333 of the synthetase hydrogen bonds to the exocyclic 2-amino and endocyclic N1 groups of the guanine nucleotide base, whereas the hydroxyl of Ser414 and the backbone amide of Lys331 hydrogen bond to the 6-oxo position. The side chains of Lys331 and Pro417 pack against opposite faces of the guanine nucleotide base. The synthetase recognizes neither the N7 position of guanine nucleotides nor the ribose group. Electron density for the guanine-5'-(beta,gamma-imido) triphosphate complex is consistent with a mixture of the triphosphate nucleoside and its hydrolyzed diphosphate nucleoside bound to the active site. The base, ribose, and alpha-phosphate positions overlap, but the beta-phosphates occupy different binding sites. The binding of guanosine-5'-(beta,gamma-methylene)triphosphate to the active site is comparable with that of guanosine-5'-(beta, gamma-imido)triphosphate. No electron density, however, for the corresponding diphosphate nucleoside is observed. In addition, electron density for bound Mg2+ is absent in these nucleotide complexes. The guanine nucleotide complexes of the synthetase are compared with complexes of other GTP-binding proteins and to a preliminary structure of the complex of GDP, IMP, Mg2+, and succinate with the synthetase. The enzyme, under conditions reported here, does not undergo a conformational change in response to the binding of guanine nucleotides, and minimally IMP and/or Mg2+ must be present in order to facilitate the complete recognition of the guanine nucleotide by the synthetase.

• Hotchin NA, Hall A
• Regulation of the actin cytoskeleton, integrins and cell growth by the Rho family of small GTPases.
• Cancer Surv. 1996; 27: 311-22
• Display abstract

• The Rho family of small GTP binding proteins play a key part in regulating the actin cytoskeleton and cell adhesion through integrin receptors. In addition, these proteins regulate signal transduction pathways essential for normal cell growth. Many of the molecules that regulate Rho have oncogenic activity, suggesting that members of the Rho family may have an important role in tumour formation.

• Clapham DE
• The G-protein nanomachine.
• Nature. 1996; 379: 297-9

• Lambright DG, Sondek J, Bohm A, Skiba NP, Hamm HE, Sigler PB
• The 2.0 A crystal structure of a heterotrimeric G protein.
• Nature. 1996; 379: 311-9
• Display abstract

• The structure of a heterotrimeric G protein reveals the mechanism of the nucleotide-dependent engagement of the alpha and beta gamma subunits that regulates their interaction with receptor and effector molecules. The interaction involves two distinct interfaces and dramatically alters the conformation of the alpha but not of the beta gamma subunits. The location of the known sites for post-translational modification and receptor coupling suggest a plausible orientation with respect to the membrane surface and an activated heptahelical receptor.

• Harhammer R, Gohla A, Schultz G
• Interaction of G protein Gbetagamma dimers with small GTP-binding proteins of the Rho family.
• FEBS Lett. 1996; 399: 211-4
• Display abstract

• Gbetagamma dimers of heterotrimeric G proteins have been shown to be important for the translocation of cytosolic proteins to membranes. The involvement of Gbetagamma in those signaling processes mediated by small GTP-binding proteins of the Rho family was studied using purified proteins. We showed specific binding of bovine brain Gbetagamma to immobilized GST-Rho fusion proteins. In addition, brain Gbetagamma, but not transducin Gbetagamma, was able to inhibit GTPgammaS binding to GST-Rho in a concentration-dependent manner. GTPgammaS binding to GST-Rac was also decreased by brain Gbetagamma whereas nucleotide binding to GST-Cdc42 was not changed. We conclude that Gbetagamma dimers may participate in the process of membrane attachment and/or other regulations of Rho and Rac.

• Skiba NP, Bae H, Hamm HE
• Mapping of effector binding sites of transducin alpha-subunit using G alpha t/G alpha i1 chimeras.
• J Biol Chem. 1996; 271: 413-24
• Display abstract

• The G protein transducin has been an often-used model for biochemical, structural, and mechanistic studies of G protein function. Experimental studies have been limited, however, by the inability to express quantities of mutants in heterologous systems with ease. In this study we have made a series of G alpha t/G alpha i1 chimeras differing at as few as 11 positions from native G alpha t. Ten chimeras are properly folded, contain GDP, can assume an A1F4(-)-induced activated conformation, and interact with beta gamma t and light-activated rhodopsin. They differ dramatically in their affinity for GDP, from Gi-like (initial rates 225 mumol/mol s) to Gt-like (initial rates 4.9 mumol/mol s). We have used these chimeras to define contact sites on G alpha t with the effector enzyme cGMP phosphodiesterase. G alpha t GTP but not G alpha t GDP activates it by removing the phosphodiesterase (PDE) gamma inhibitory subunit. In solution, G alpha t GTP interacts with PDE gamma (Kd 12 nM), while G alpha t GDP binds PDE gamma more weakly (Kd 0.88 microM). The interaction of G alpha i GDP with PDE gamma is undetectable, but G alpha i GDP-A1F4- interacts weakly with PDE gamma (Kd 2.4 microM). Using defined G alpha t/G alpha i chimeras, we have individuated the regions on G alpha t most important for interaction with PDE gamma in the basal and activated states. The G alpha t sequence encompassing alpha helix 3 and the alpha 3/beta 5 loop contributes most binding energy to interaction with PDE gamma. Another composite P gamma interaction site is the conserved switch, through which the GTP-bound G alpha t as well as G alpha i1 interact with P gamma. Competition studies between PDE gamma and truncated regions of PDE gamma provide evidence for the point-to-point interactions between the two proteins. The amino-terminal 1-45 segment containing the central polycationic region binds to G alpha t's alpha 3 helix and alpha 3/beta 5 loop, while the COOH-terminal region of P gamma, 63-87, binds in concert to the conserved switch regions. The first interaction provides specific interaction with both the GDP- and GTP-liganded G alpha t, while the second one is conserved between G alpha t and G alpha i1 and dependent on the activated conformation.

• Lindorfer MA, Sherman NE, Woodfork KA, Fletcher JE, Hunt DF, Garrison JC
• G protein gamma subunits with altered prenylation sequences are properly modified when expressed in Sf9 cells.
• J Biol Chem. 1996; 271: 18582-7
• Display abstract

• The gamma subunits of heterotrimeric G proteins undergo post-translational prenylation and carboxylmethylation after formation of the betagamma dimer, modifications that are essential for alpha-betagamma, betagamma-receptor, and betagamma-effector interactions. We have determined the specific prenyl group present on the beta1gamma1, beta1gamma2, and beta1gamma3 dimers purified from baculovirus-infected Sf9 cells by specific binding to G protein alpha subunits immobilized on agarose. These recombinant dimers undergo the same post-translational modifications determined for gamma1 and gamma2 isolated from mammalian tissues. Furthermore, infection of Sf9 cells with a recombinant baculovirus encoding an alteration of the gamma1 CaaX sequence (gamma1-S74L) resulted in geranylgeranylation of the resulting gamma1 subunit, and alteration of the gamma2 CaaX sequence to CAIS (gamma2-L71S) resulted in farnesylation. Both of these altered gamma subunits were able to associate stably with beta1, and the resulting betagamma dimer bound tightly to alpha-agarose and eluted specifically with aluminum fluoride. These results indicate that Sf9 insect cells properly process the CaaX motif in G protein gamma subunits and are a useful model system to study the role of prenylation in the protein-protein interactions in which the betagamma subunits participate.

• Wittinghofer A
• Deciphering the alphabet of G proteins: the structure of the alpha, beta, gamma heterotrimer.
• Structure. 1996; 4: 357-61
• Display abstract

• The recent independent structure elucidations of two heterotrimeric G proteins represent a milestone in our understanding of the regulation of this important class of signal switch molecules. The results show how the introduction of GTP into the heterotrimer produces two signalling molecules: the G alpha-GTP and G beta, gamma subunits.

• Boivin D, Bilodeau D, Beliveau R
• Regulation of cytoskeletal functions by Rho small GTP-binding proteins in normal and cancer cells.
• Can J Physiol Pharmacol. 1996; 74: 801-10
• Display abstract

• The actin cytoskeleton is involved in numerous cellular functions such as cell motility, mitogenesis, morphology, muscle contraction, cytokinesis, and establishment of cell polarity. The members of the Rho subfamily of small GTP-binding proteins emerge as key regulators of cytokeleton organization. Rho, Rac, and CDC42 are implicated in the regulation of actin microfilament organization of different cell structures, such as stress fibers linked to focal adhesions and membrane ruffles induced by extracellular stimuli. Rho proteins also regulate the activity of several enzymes involved in the formation of phospholipid derivatives, which could mediate their effect on the cytoskeleton. The activity of Rho proteins is regulated by many nucleotide exchange factors and GTPase-activating proteins, some of which are oncogene products, and other disease-associated proteins. The potential role of these small GTP-binding proteins in carcinogenesis is suggested by the actin reorganization seen in transforming cells and by the need for functional Rho proteins in Ras mitogenic activation.

• Neer EJ, Smith TF
• G protein heterodimers: new structures propel new questions.
• Cell. 1996; 84: 175-8

• Del Villar K et al.
• C-terminal motifs found in Ras-superfamily G-proteins: CAAX and C-seven motifs.
• Biochem Soc Trans. 1996; 24: 709-13

• Symons M
• Rho family GTPases: the cytoskeleton and beyond.
• Trends Biochem Sci. 1996; 21: 178-81
• Display abstract

• Until recently, members of the Rho family of GTPases were considered primarily to be regulators of the distinct structures making up the actin cytoskeleton. Several Rho GTPases have now also been shown to play an important role in cell transformation. In addition, Cdc42, Rac and Rho activate transcription, providing a possible mechanism for their role in growth control.

• Parsons JT
• Integrin-mediated signalling: regulation by protein tyrosine kinases and small GTP-binding proteins.
• Curr Opin Cell Biol. 1996; 8: 146-52
• Display abstract

• Integrin signalling requires the activation of protein tyrosine kinases and members of the Rho family of small GTP-binding proteins. Recent evidence shows that coordinated regulation of these signalling molecules is central to the control of cell adhesion, formation of the actin cytoskeleton and activation of intracellular signalling cascades.

• Wu SK, Zeng K, Wilson IA, Balch WE
• Structural insights into the function of the Rab GDI superfamily.
• Trends Biochem Sci. 1996; 21: 472-6
• Display abstract

• The 1.81 A crystal structure of Rab GDP-dissociation inhibitor (GDI), a protein that plays a critical role in the recycling of Rab GTPases involved in membrane vesicular transport, has been recently determined. Biochemical studies implicate a highly conserved region involved in Rab binding, which is common to both GDI and the evolutionarily-related choroideremia gene product (CHM/REP) required for Rab prenylation. Here, we summarize the mechanisms by which members of the GDI superfamily might function to coordinate events leading to membrane fusion, and we discuss the unexpected, yet striking structural homology of GDI to FAD-binding proteins.

• Nolte C, Moller T, Walter T, Kettenmann H
• Complement 5a controls motility of murine microglial cells in vitro via activation of an inhibitory G-protein and the rearrangement of the actin cytoskeleton.
• Neuroscience. 1996; 73: 1091-107
• Display abstract

• Microglial cells respond to most pathological events by rapid transformation from a quiescent to an activated phenotype characterized by increased cytotoxicity and motile activity. To investigate the regulation of microglial motility by different inflammatory mediators, we studied cultured murine microglia by time-lapse video microscopy and a computer-based motility assay. Microglial cells exhibited a high resting motility. The acute application of complement 5a (C5a) immediately induced intense ruffling of microglial membranes followed by lamellipodia extension within few seconds, while formyl-Met-Leu-Phe-OH, bacterial endotoxin (lipopolysaccharide) or inflammatory cytokines did not increase motility. This process was accompanied by a rapid rearrangement of the actin cytoskeleton as demonstrated by labelling with fluorescein isothiocyanate-phalloidin and could be inhibited by cytochalasin B. A GTP-binding protein was involved in the signal cascade, since pertussis toxin inhibited motility and actin assembly in response to C5a. Chemotactic migration in a gradient of C5a was also completely blocked by pertussis toxin and cytochalasin B. The C5a-induced motility reaction was accompanied by an increase in intracellular calcium ([Ca2+]i) as measured by a Fluo-3 based imaging system. Ca2+ transients were, however, not a prerequisite for triggering the increase in motility; motility could be repeatedly evoked by C5a in nominally Ca(2+)-free solution, while Ca2+ signals occurred only upon the first stimulation. Moreover, conditions mimicking intracellular Ca2+ transients, like incubation with thapsigargin or Ca2+ ionophore A23187, were not able to induce any motility reaction, suggesting that Ca2+ transients are not necessary for, but are associated with, microglial motility. Motile activity was shown to be restricted to a defined concentration range of [Ca2+]i as revealed by lowering [Ca2+]i with BAPTA-AM or increasing [Ca2+]i with A23187. Since complement factors are released at pathological sites, this signal cascade could serve to increase motility and to direct microglial cells to the lesioned or damaged area by means of a G-protein-dependent pathway and via the rearrangement of the actin cytoskeleton.

• Lamaze C, Chuang TH, Terlecky LJ, Bokoch GM, Schmid SL
• Regulation of receptor-mediated endocytosis by Rho and Rac.
• Nature. 1996; 382: 177-9
• Display abstract

• Pinocytosis and membrane ruffling are among the earliest and most dramatic cellular responses to stimulation by growth factors or other mitogens. The small Ras-related G proteins Rho and Rac have a regulatory role in membrane ruffling and activated Rho has been shown to stimulate pinocytosis when microinjected into Xenopus oocytes. In contrast to these well established effects of Rho and Rac on plasma membrane morphology and bulk pinocytosis, there has been no evidence for their involvement in the regulation of receptor-mediated endocytosis in clathrin-coated pits. Here we show that activated Rho and Rac inhibit transferrin-receptor-mediated endocytosis when expressed in intact cells. Furthermore, we have reconstituted these effects in a cell-free system and established that Rho and Rac can regulate clathrin-coated vesicle formation.

• Hoffenberg S et al.
• Specific and effective interaction of a guanine nucleotide analogue with small G proteins.
• Mol Pharmacol. 1996; 49: 156-64
• Display abstract

• G proteins are molecular switches that use a cycle of GTP binding and hydrolysis to regulate a wide variety of cellular biochemical processes. Because the functional state of these proteins is allosterically determined by bound guanine nucleotides, a nucleotide analogue with protein specificity might have pharmacological or biochemical value. The binding of [alpha-32P]GTP to four small G proteins immobilized on nitrocellulose was competed by a series of analogues with modifications at multiple sites. One analogue, N2-(p-n-butylphenyl)guanosine 5'-(beta,gamma-difluoromethylene)triphosphate, had a approximately 40-fold higher affinity for one small G protein than for two of the others. Systematic analysis of each modification in the synthetic nucleotide revealed that specificity was conferred by the carbon substitution in the beta,gamma-phosphoanhydride bond. These observations were then extended to purified proteins of known sequence in solution by filtration binding studies with H-ras and rab5. Ras was 9-fold more discriminant between guanosine-5'-(beta,gamma-difluoromethylene)triphosphate and guanosine-5'-O-(3-thiotriphosphate) than was rab5, and the Q79L GTPase-defective mutant of rab5 was 6-fold more discriminant than wild-type rab5. Guanosine-5'-(beta,gamma-difluoromethylene)triphosphate protected a 20-kDa fragment of rab5 from tryptic proteolysis with greater efficacy than guanosine-5'-O-(3-thiotriphosphate) or guanosine-5'-(beta,gamma-imido)triphosphate despite its lower affinity, and GMP stabilized a conformation indistinguishable from apo-rab5. These results identify a synthetic guanine nucleotide analogue with differential affinity for closely related G proteins, determine the atomic substitution in the analogue that confers specificity, demonstrate discrimination by the analogue between wild-type and a point-mutant G protein, and establish efficacy of the analogue in inducing conformational change of a target protein disproportionate to the affinity of the interaction.

• Jilkina O, Bhullar RP
• Generation of antibodies specific for the RalA and RalB GTP-binding proteins and determination of their concentration and distribution in human platelets.
• Biochim Biophys Acta. 1996; 1314: 157-66
• Display abstract

• Peptide specific polyclonal antibodies directed against C-termini of ras p21 related GTP-binding proteins, ralA and ralB, were generated. To assess antibody specificity, cDNAs coding for full length ralA and ralB were expressed in Escherichia coli as GST fusion proteins. Western blotting analysis using enhanced chemiluminescence technique confirmed that ralA and ralB antibodies were specific for their respective protein. To determine the concentration and distribution, varying amounts of GST-ralA and GST-ralB and, human platelet particulate and cytosolic proteins were loaded during Western blotting. The amount of ralA and ralB proteins in the platelet particulate fraction was determined to be 0.16 +/- 0.017 microgram/mg protein (n = 3) and 0.15 +/- 0.009 microgram/mg protein (n = 3) respectively. In the cytosol, only ralB protein was detected and its concentration was estimated to be 0.03 +/- 0.009 microgram/mg protein (n = 3). Both ralA and ralB proteins were isoprenylated in the presence of [3H] mevalonolactone plus rabbit reticulocyte lysate although radioactivity incorporated into ralA was three times higher than that associated with the ralB protein. Addition of geranylgeranyl pyrophosphate to the reaction mixture inhibited incorporation of radioactivity into ralA and ralB but not cH-ras suggesting that both ralA and ralB proteins are geranylgeranylated. Differential distribution of ralA and ralB GTP-binding proteins in human platelets suggests a distinct role for each of these proteins in platelet function.

• Touhara K
• [Structure and function of beta gamma subunits of heterotrimeric G proteins]
• Seikagaku. 1996; 68: 210-4

• Tilly BC et al.
• Activation of the osmo-sensitive chloride conductance involves P21rho and is accompanied by a transient reorganization of the F-actin cytoskeleton.
• Mol Biol Cell. 1996; 7: 1419-27
• Display abstract

• Hypo-osmotic stimulation of human Intestine 407 cells rapidly activated compensatory CL- and K+ conductances that limited excessive cell swelling and, finally, restored the original cell volume. Osmotic cell swelling was accompanied by a rapid and transient reorganization of the F-actin cytoskeleton, affecting both stress fibers as well as apical ruffles. In addition, an increase in total cellular F-actin was observed. Pretreatment of the cells with recombinant Clostridium botulinum C3 exoenzyme, but not with mutant enzyme (C3-E173Q) devoid of ADP-ribosyltransferase activity, greatly reduced the activation of the osmo-sensitive anion efflux, suggesting a role for the ras-related GTPase p21rho. In contrast, introducing dominant negative N17-p21rac into the cells did not affect the volume-sensitive efflux. Cell swelling-induced reorganization of F-actin coincided with a transient, C3 exoenzyme-sensitive tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK) as well as with an increase in phosphatidylinositol-3-kinase (PtdIns-3-kinase) activity. Pretreatment of the cells with wortmannin, a specific inhibitor of PtdIns-3-kinase, largely inhibited the volume-sensitive ion efflux. Taken together, our results indicate the involvement of a p21rho signaling cascade and actin filaments in the activation of volume-sensitive chloride channels.

• Norman JC, Price LS, Ridley AJ, Koffer A
• The small GTP-binding proteins, Rac and Rho, regulate cytoskeletal organization and exocytosis in mast cells by parallel pathways.
• Mol Biol Cell. 1996; 7: 1429-42
• Display abstract

• In mast cells, activation of GTP-binding proteins induces centripetal reorganization of actin filaments. This effect is due to disassembly, relocalization, and polymerization of F-actin and is dependent on two small GTPases, Rac and Rho. Activities of Rac and Rho are also essential for the secretory function of mast cells. In response to GTP-gamma-S and/or calcium, only a proportion of permeabilized mast cells is capable of secretory response. Here, we have compared actin organization of secreting and nonsecreting cell populations. We show that the cytoskeletal and secretory responses are strongly correlated, indicating a common upstream regulator of the two functions. The secreting cell population preferentially displays both relocalization and polymerization of actin. However, when actin relocalization or polymerization is inhibited by phalloidin or cytochalasin, respectively, secretion is unaffected. Moreover, the ability of the constitutively active mutants of Rac and Rho to enhance secretion is also unaffected in the presence of cytochalasin. Therefore, Rac and Rho control these two functions by divergent, parallel signaling pathways. Cortical actin disassembly occurs in both secreting and nonsecreting populations and does not, by itself, induce exocytosis. A model for the control of exocytosis is proposed that includes at least four GTP-binding proteins and suggests the presence of both shared and divergent signaling pathways from Rac and Rho.

• Glotzer M, Hyman AA
• Cell polarity. The importance of being polar.
• Curr Biol. 1995; 5: 1102-5
• Display abstract

• Cell polarization is often accompanied by cytoskeletal rearrangements. Two signalling proteins, a GTPase and a kinase, are required for both actin and microtubule rearrangements. Are these two systems coupled?

• Takai Y, Sasaki T, Tanaka K, Nakanishi H
• Rho as a regulator of the cytoskeleton.
• Trends Biochem Sci. 1995; 20: 227-31
• Display abstract

• Many cell functions, including maintenance of morphology, aggregation, motility, membrane ruffling, smooth-muscle contraction, cytokinesis in mammals and bud formation in yeast, are regulated through the dynamic reorganization of actin filaments. Although it has long been known that Ca2+ is a key regulator of the cytoskeleton, evidence is now accumulating that Rho, a Ras-related small GTP-binding protein, is another important regulator of these actin-dependent cell functions.

• Mixon MB, Lee E, Coleman DE, Berghuis AM, Gilman AG, Sprang SR
• Tertiary and quaternary structural changes in Gi alpha 1 induced by GTP hydrolysis.
• Science. 1995; 270: 954-60
• Display abstract

• Crystallographic analysis of 2.2 angstrom resolution shows that guanosine triphosphate (GTP) hydrolysis triggers conformational changes in the heterotrimeric G-protein alpha subunit, Gi alpha 1. The switch II and switch III segments become disordered, and linker II connecting the Ras and alpha helical domains moves, thus altering the structures of potential effector and beta gamma binding regions. Contacts between the alpha-helical and Ras domains are weakened, possibly facilitating the release of guanosine diphosphate (GDP). The amino and carboxyl termini, which contain receptor and beta gamma binding determinants, are disordered in the complex with GTP, but are organized into a compact microdomain on GDP hydrolysis. The amino terminus also forms extensive quaternary contacts with neighboring alpha subunits in the lattice, suggesting that multimers of alpha subunits or heterotrimers may play a role in signal transduction.

• Ridley AJ
• Rho-related proteins: actin cytoskeleton and cell cycle.
• Curr Opin Genet Dev. 1995; 5: 24-30
• Display abstract

• The past year has produced an abundance of data on the function and regulation of Rho-related GTP-binding proteins. In mammalian cells, it has been shown that Rho is required for contractile ring assembly at cell division, as well as for regulating extracellular factor induced actin reorganization. In addition, many new regulators and/or potential targets for Rho, Rac and Cdc42 have been characterized, including several oncogene products, protein kinases and signal transducing proteins in mammalian cells, and genes defined by cell cycle or bud emergence mutations in yeast. These provide further connections between Rho-related proteins, signal transduction pathways and changes in actin organization during cell cycle entry and progression.

• Hotchin NA, Hall A
• The assembly of integrin adhesion complexes requires both extracellular matrix and intracellular rho/rac GTPases.
• J Cell Biol. 1995; 131: 1857-65
• Display abstract

• Interaction of cells with extracellular matrix via integrin adhesion receptors plays an important role in a wide range of cellular: functions, for example cell growth, movement, and differentiation. Upon interaction with substrate, integrins cluster and associate with a variety of cytoplasmic proteins to form focal complexes and with the actin cytoskeleton. Although the intracellular signals induced by integrins are at present undefined, it is thought that they are mediated by proteins recruited to the focal complexes. It has been suggested, for example, that after recruitment to focal adhesions p125FAK can activate the ERK1/2 MAP kinase cascade. We have previously reported that members of the rho family of small GTPases can trigger the assembly of focal complexes when activated in cells. Using microinjection techniques, we have now examined the role of the extracellular matrix and of the two GTP-binding proteins, rac and rho, in the assembly of integrin complexes in both mouse and human fibroblasts. We find that the interaction of integrins with extracellular matrix alone is not sufficient to induce integrin clustering and focal complex formation. Similarly, activation of rho or rac by extracellular growth factors does not lead to focal complex formation in the absence of matrix. Focal complexes are only assembled in the presence of both matrix and functionally active members of the rho family. In agreement with this, the interaction of integrins with matrix in the absence of rho/rac activity is unable to activate the ERK1/2 kinases in Swiss 3T3 cells. In fact, ERK1/2 can be activated fully by growth factors in the absence of matrix and it seems unlikely, therefore, that the adhesion dependence of fibroblast growth is mediated through the ras/MAP kinase pathway. We conclude that extracellular matrix is not sufficient to trigger focal complex assembly and subsequent integrin-dependent signal transduction in the absence of functionally active members of the rho family of GTPases.

• Eaton S, Auvinen P, Luo L, Jan YN, Simons K
• CDC42 and Rac1 control different actin-dependent processes in the Drosophila wing disc epithelium.
• J Cell Biol. 1995; 131: 151-64
• Display abstract

• Cdc42 and Rac1 are members of the rho family of small guanosinetriphosphatases and are required for a diverse set of cytoskeleton-membrane interactions in different cell types. Here we show that these two proteins contribute differently to the organization of epithelial cells in the Drosophila wing imaginal disc. Drac1 is required to assemble actin at adherens junctions. Failure of adherens junction actin assembly in Drac1 dominant-negative mutants is associated with increased cell death. Dcdc42, on the other hand, is required for processes that involve polarized cell shape changes during both pupal and larval development. In the third larval instar, Dcdc42 is required for apico-basal epithelial elongation. Whereas normal wing disc epithelial cells increase in height more than twofold during the third instar, cells that express a dominant-negative version of Dcdc42 remain short and are abnormally shaped. Dcdc42 localizes to both apical and basal regions of the cell during these events, and mediates elongation, at least in part, by effecting a reorganization of the basal actin cytoskeleton. These observations suggest that a common cdc42-based mechanism may govern polarized cell shape changes in a wide variety of cell types.

• Wall MA et al.
• The structure of the G protein heterotrimer Gi alpha 1 beta 1 gamma 2.
• Cell. 1995; 83: 1047-58
• Display abstract

• The crystallographic structure of the G protein heterotrimer Gi alpha 1(GDP)beta 1 gamma 2 (at 2.3 A) reveals two nonoverlapping regions of contact between alpha and beta, an extended interface between beta and nearly all of gamma, and limited interaction of alpha with gamma. The major alpha/beta interface covers switch II of alpha, and GTP-induced rearrangement of switch II causes subunit dissociation during signaling. Alterations in GDP binding in the heterotrimer (compared with alpha-GDP) explain stabilization of the inactive conformation of alpha by beta gamma. Repeated WD motifs in beta form a circularized sevenfold beta propeller. The conserved cores of these motifs are a scaffold for display of their more variable linkers on the exterior face of each propeller blade.

• Manser E, Leung T, Lim L
• Identification of GTPase-activating proteins by nitrocellulose overlay assay.
• Methods Enzymol. 1995; 256: 130-9

• Mackay DJ, Nobes CD, Hall A
• The Rho's progress: a potential role during neuritogenesis for the Rho family of GTPases.
• Trends Neurosci. 1995; 18: 496-501
• Display abstract

• Growth cones navigate by coupling extracellular guidance cues to directed outgrowth of the actin cytoskeleton through cyclical extension of filopodia and lamellipodia, but the biochemical basis of this coupling is at present unknown. Recent studies have shown that members of the Rho family of small GTPases regulate the formation of filopodia, lamellipodia and stress fibres in fibroblasts, and there are striking morphological similarities between spreading fibroblasts and advancing growth cones. This resemblance suggests that the Rho family of proteins could be the link between incoming signals and the regulation of both actin dynamics and cell-substratum adhesion in the neuronal growth cone.

• Masuda T, Tanaka K, Nonaka H, Yamochi W, Maeda A, Takai Y
• Molecular cloning and characterization of yeast rho GDP dissociation inhibitor.
• J Biol Chem. 1994; 269: 19713-8
• Display abstract

• We have previously isolated rho GDP dissociation inhibitor (rho DGI) from bovine brain and characterized it. Bovine rho GDI is a protein of a M(r) of 23,421 with 204 amino acids. rho GDI inhibits the GDP/GTP exchange reaction of post-translationally lipid-modified small GTP-binding proteins (G proteins) of the rho family, including the rho, rac, and cdc42 subfamilies, and keeps them in the GDP-bound inactive form. In the present study, we first purified rho GDI from the cytosol fraction of the yeast Saccharomyces cerevisiae and isolated its gene. Yeast rho GDI gene had an open reading frame without introns encoding a protein of a M(r) of 23,138 with 202 amino acids. Yeast rho GDI protein was 36% identical with bovine rho GDI. Yeast rho GDI expressed in Escherichia coli was active not only on yeast rho1 but also on mammalian rho family members which were post-translationally modified. Disruption of rho GDI did not induce apparent phenotypes, whereas overexpression of yeast or bovine rho GDI resulted in the inhibition of cell growth. These results indicate that rho GDI exists and regulates the function of the rho family members in yeast.

• Hall A
• Small GTP-binding proteins and the regulation of the actin cytoskeleton.
• Annu Rev Cell Biol. 1994; 10: 31-54

• Norman JC, Price LS, Ridley AJ, Hall A, Koffer A
• Actin filament organization in activated mast cells is regulated by heterotrimeric and small GTP-binding proteins.
• J Cell Biol. 1994; 126: 1005-15
• Display abstract

• Rat peritoneal mast cells, both intact and permeabilized, have been used widely as model secretory cells. GTP-binding proteins and calcium play a major role in controlling their secretory response. Here we have examined changes in the organization of actin filaments in intact mast cells after activation by compound 48/80, and in permeabilized cells after direct activation of GTP-binding proteins by GTP-gamma-S. In both cases, a centripetal redistribution of cellular F-actin was observed: the content of F-actin was reduced in the cortical region and increased in the cell interior. The overall F-actin content was increased. Using permeabilized cells, we show that AIF4-, an activator of heterotrimeric G proteins, induces the disassembly of F-actin at the cortex, while the appearance of actin filaments in the interior of the cell is dependent on two small GTPases, rho and rac. Rho was found to be responsible for de novo actin polymerization, presumably from a membrane-bound monomeric pool, while rac was required for an entrapment of the released cortical filaments. Thus, a heterotrimeric G-protein and the small GTPases, rho and rac, participate in affecting the changes in the actin cytoskeleton observed after activation of mast cells.

• Chong LD, Traynor-Kaplan A, Bokoch GM, Schwartz MA
• The small GTP-binding protein Rho regulates a phosphatidylinositol 4-phosphate 5-kinase in mammalian cells.
• Cell. 1994; 79: 507-13
• Display abstract

• Integrin-mediated adhesion is known to stimulate production of phosphatidylinositol 4,5-bisphosphate (4,5-PIP2) and increase 4,5-PIP2 hydrolysis in response to platelet-derived growth factor (PDGF). We now show that treatment of cells with lovastatin, which inhibits modification of small GTP-binding proteins, reduced PIP2 levels and decreased calcium mobilization in response to PDGF and thrombin. In cell lysates, GTP gamma S stimulated PIP 5-kinase activity, and this effect was blocked by botulinum C3 exoenzyme, suggesting that Rho was responsible. GTP-bound recombinant Rho stimulated PIP 5-kinase activity, whereas GDP-Rho was much less potent and GTP-bound Rac was ineffective. Microinjected botulinum C3 exoenzyme caused diminished calcium mobilization in response to PDGF or thrombin. Conversely, microinjection of activated Rho reversed the decrease in calcium mobilization normally seen in nonadherent cells. These data demonstrate that Rho regulates 4,5-PIP2 synthesis and, indirectly, 4,5-PIP2 hydrolysis. They also raise the possibility that PIP2 synthesis could mediate the effects of Rho on the actin cytoskeleton.

• Yaku H, Sasaki T, Takai Y
• The Dbl oncogene product as a GDP/GTP exchange protein for the Rho family: its properties in comparison with those of Smg GDS.
• Biochem Biophys Res Commun. 1994; 198: 811-7
• Display abstract

• The dbl oncogene product (Dbl) showed the GDP/GTP exchange protein (GEP) activity on all the rho family small GTP-binding protein (G protein) members including RhoA and Rac1 as well as mCdc42. Dbl was active on both the lipid-modified and -unmodified forms of these small G proteins, but was much more active on the former form than on the latter form. In the presence of Rho GDI, an inhibitory GEP for the rho family members, the GEP activity of Dbl was markedly reduced. These properties of Dbl were partly different from those of Smg GDS, another GEP which is active not only on the rho family members but also on Ki-Ras and Rap1 and is active only on their lipid-modified form.

• Takai Y, Kaibuchi K, Sasaki T, Tanaka K, Shirataki H, Nakanishi H
• Rho small G protein and cytoskeletal control.
• Princess Takamatsu Symp. 1994; 24: 338-50
• Display abstract

• The Rho family belongs to the Ras-related small GTP-binding protein (G protein) superfamily and regulates various cell functions in which the actomyosin system is involved, including cell morphology, membrane ruffling, cell motility, cell aggregation, cytokinesis, smooth muscle contraction, and yeast budding. Three GDP/GTP exchange proteins (GEPs), named Smg GDS, Dbl, and Rho GDI, and two GTPase activating proteins (GAPs), named Rho GAP and p190 associated with Ras GAP, have been identified. The Rho activity is likely to be regulated by protein kinase C which is linked through phospholipase C to the tyrosine kinase-type membrane receptors and the heterotrimeric G protein-linked receptors. It is likely that both Ras and Rho receive signals from the membrane receptors through different pathways and transduce signals to genes and cytoskeleton, respectively. In carcinogenesis, mutational activation of any component in the Ras signaling pathway may cause abnormal cell proliferation, whereas mutational activation of any component in the Rho signaling pathway may cause invasiveness and metastasis of carcinoma cells.

• Crawford LE, Tucker RW, Heldman AW, Goldschmidt-Clermont PJ
• Actin regulation and surface catalysis.
• Adv Exp Med Biol. 1994; 358: 105-12

• Lamarche N, Hall A
• GAPs for rho-related GTPases.
• Trends Genet. 1994; 10: 436-40
• Display abstract

• Ras-related GTP-binding proteins (GTPases) of the rho subfamily play important roles in regulating the organization of the actin cytoskeleton. A large number of multifunctional proteins that can stimulate their intrinsic GTPase activity have been identified. Here, we discuss the nature of such GTPase-activating proteins (GAPs) and their potential importance for cell signalling.

• Coleman DE, Berghuis AM, Lee E, Linder ME, Gilman AG, Sprang SR
• Structures of active conformations of Gi alpha 1 and the mechanism of GTP hydrolysis.
• Science. 1994; 265: 1405-12
• Display abstract

• Mechanisms of guanosine triphosphate (GTP) hydrolysis by members of the G protein alpha subunit-p21ras superfamily of guanosine triphosphatases have been studied extensively but have not been well understood. High-resolution x-ray structures of the GTP gamma S and GDP.AlF4- complexes formed by the G protein Gi alpha 1 demonstrate specific roles in transition-state stabilization for two highly conserved residues. Glutamine204 (Gln61 in p21ras) stabilizes and orients the hydrolytic water in the trigonal-bipyramidal transition state. Arginine 178 stabilizes the negative charge at the equatorial oxygen atoms of the pentacoordinate phosphate intermediate. Conserved only in the G alpha family, this residue may account for the higher hydrolytic rate of G alpha proteins relative to those of the p21ras family members. The fold of Gi alpha 1 differs from that of the homologous Gt alpha subunit in the conformation of a helix-loop sequence located in the alpha-helical domain that is characteristic of these proteins; this site may participate in effector binding. The amino-terminal 33 residues are disordered in GTP gamma S-Gi alpha 1, suggesting a mechanism that may promote release of the beta gamma subunit complex when the alpha subunit is activated by GTP.

• Fawcett JW
• Growth-cone collapse: too much of a good thing?
• Trends Neurosci. 1993; 16: 165-7

• Stouten PF, Sander C, Wittinghofer A, Valencia A
• How does the switch II region of G-domains work?
• FEBS Lett. 1993; 320: 1-6
• Display abstract

• The transition of guanine nucleotide binding proteins between the 'on' (GTP-bound) and 'off' (GDP-bound) states has become a paradigm of molecular switching after a chemical reaction. The mechanism by which the switch signal is transmitted to the downstream recipients in the intracellular signal pathway has been extensively studied by biochemical, biophysical and genetic methods, but a clear picture of this process has yet to emerge. Based on the similarities of ras-p21 and elongation factor Tu we propose here a model of the GDP state of ras-p21 that is in agreement with all relevant experimental evidence. The model provides important clues about: (1) a possible molecular mechanism for signal transmission from the site of GTP hydrolysis to downstream effectors; (2) a major conformational change during signal generation and a key residue involved in this process (Tyr-64); and (3) regions in ras-p21 that can be differentially recognized by binding to external partners in a GTP/GDP state dependent fashion, most notably residues D69, Q70, R73, T74, R102, K104, D105 at the end of the alpha-helices 2 and 3.

• Heldman AW, Goldschmidt-Clermont PJ
• Cell signalling and motile activity.
• Symp Soc Exp Biol. 1993; 47: 317-24
• Display abstract

• The actin cytoskeleton is a remarkably dynamic structure in non-muscle cells which responds to cell stimulation by a variety of cytokines. A paradigm for such cytokine-induced reorganization of the actin superstructure is the actin response to growth factor of the EGF/PDGF family. This paper reviews the role of the polyphosphoinositide-metabolism, small GTP-binding proteins and profilin in the reorganization of the actin cytoskeleton in cells activated by growth factors.

• Dratz EA et al.
• NMR structure of a receptor-bound G-protein peptide.
• Nature. 1993; 363: 276-81
• Display abstract

• Heterotrimeric GTP-binding proteins (G proteins) regulate cellular activity by coupling to hormone or sensory receptors. Stimulated receptors catalyse the release of GDP from G protein alpha-subunits and GTP bound to the empty alpha-subunits provides signals that control effectors such as adenylyl cyclases, phosphodiesterases, phospholipases and ion channels. Three cytoplasmic loops of the activated receptor are thought to interact with three sites on the heterotrimeric G protein to provide high-affinity interaction and catalyse G-protein activation. The carboxyl terminus of the alpha-subunit is particularly important for interaction with the receptor. Here we study the structure of part of the active interface between the photon receptor rhodopsin and the G protein transducin, or Gt, using nuclear magnetic resonance. An 11-amino-acid peptide from the C terminus of the alpha-subunit of Gt (alpha t (340-350)) binds to rhodopsin and mimics the G protein in stabilizing its active form, metarhodopsin II. The peptide alpha t (340-350) binds to both excited and unexcited rhodopsin and conformational differences between the two bound forms suggest a mechanism for activation of G proteins by agonist-stimulated receptors. Insight into receptor-catalysed GDP release will have broad application because the GTP/GDP exchange and the intrinsic GTPase activity of GTP-binding proteins constitute a widespread regulatory mechanism.

• Leffers H et al.
• Identification of two human Rho GDP dissociation inhibitor proteins whose overexpression leads to disruption of the actin cytoskeleton.
• Exp Cell Res. 1993; 209: 165-74
• Display abstract

• Proteins (IEF's 1120, 8118, 8120) sharing similarity to the bovine Rho GDP dissociation inhibitor (GDI) have been identified in the human two-dimensional-gel database of keratinocyte proteins. Molecular cloning of the corresponding cDNAs showed that IEF 8118 is the human homolog of bovine GDI while IEF 8120 is a distinct although related protein. All available information indicates the IEF 1120 is a derivative of IEF 8120. The cDNAs coding for IEF's 8118 and 8120 were recombined into vaccinia virus and expressed in differentiated human keratinocytes and their effect on the actin cytoskeleton was assessed by immunofluorescence using TRITC-phalloidin. The results showed that overexpression of both GDI proteins leads to rounding up of the cells and loss of stress fibers and focal contact sites. In addition, the cell to cell adhesion belts gradually disappeared, an effect that was particularly pronounced in infected cells overexpressing IEF 8120. Taken together, the results imply that Rho GDI's play a role in modulating the activity of the Rho proteins as their overexpression mimics phenotypic changes associated with the inactivation of these proteins.

• Menard L, Snyderman R
• Role of phosphate-magnesium-binding regions in the high GTPase activity of rac1 protein.
• Biochemistry. 1993; 32: 13357-61
• Display abstract

• rac1, a member of the low molecular mass GTP-binding protein family, has a 20-fold higher GTPase activity than H-ras, but the structural motifs responsible for this property do not appear to reside within the conserved amino acids of the consensus GTP-binding domains [Menard, L., Tomhave, E., Casey, P.J., Uhing, R.J., Snyderman, R., & Disbury, J.R. (1992) Eur. J. Biochem 206, 537-546]. In this study, we determined the contribution, to the GTPase activity, of additional amino acids found in the phosphate-magnesium-binding (PM) regions of rac1. rac1 has three different amino acids from H-ras in each of the first two PM regions, while the third PM region is identical to that of H-ras. Mutation of the amino acids in the first PM region (aa 10-17) to the corresponding amino acids found in H-ras showed that modification of one of them, Asp11, resulted in a 50% decrease of the GTPase in rac1, whereas Gly13 and Ser17 had no effect. In the second PM region (aa 29-35), modification of the Pro29-Gly30 pair also reduced GTPase activity by 50% in rac1. rac1 mutated at positions 11 and 29 as well as 30 (P1-P2 mutant) had a 3-4-fold reduced GTPase activity compared to native rac1 (190 vs 552 pmol of GTP hydrolyzed/nmol of GTP gamma S-binding protein/min at 37 degrees C), suggesting a cooperative (but nonadditive) interaction between both domains. All mutants with reduced GTPase activity also had reduced affinity for GDP, shown by both equilibrium binding and GDP dissociation measurements.(ABSTRACT TRUNCATED AT 250 WORDS)

• Antonny B, Sukumar M, Bigay J, Chabre M, Higashijima T
• The mechanism of aluminum-independent G-protein activation by fluoride and magnesium. 31P NMR spectroscopy and fluorescence kinetic studies.
• J Biol Chem. 1993; 268: 2393-402
• Display abstract

• With magnesium present, fluoride and aluminum ions activate heterotrimeric G-proteins by forming AlFx complexes that mimic the gamma phosphate of a GTP. We report compelling evidence for a newly proposed process of G-protein activation by fluoride and magnesium, without Al3+. With millimolar Mg2+ and F-, Gs and Gt activate adenylylcyclase and cGMP-phosphodiesterase, respectively. In 31P NMR, addition of magnesium to Gi1 alpha GDP or Gt alpha GDP solutions containing fluoride, but no Al3+, modifies the chemical shift of the GDP beta phosphorus, suggesting that magnesium interacts with the beta phosphate. Titration of this effect indicates that two Mg2+ are bound per G alpha. Biphasic activation kinetics, monitored by G alpha tryptophan fluorescence, suggests the rapid binding of one Mg2+ to G alpha GDP and the slow association of another Mg2+, in correlation with fluoride binding and G alpha activation. The deactivation rate upon fluoride dilution shows a second order dependence with respect to the residual F- concentration, suggesting the sequential release of at least three F-/G alpha. Thus, in millimolar Mg2+ and F-, and without Al3+, two Mg2+ and three F- bind sequentially to G alpha GDP and induce the switch to an active G alpha (GDP-MgF3)Mg state, which is structurally analogous to G alpha (GDP-AlFx)Mg and to G alpha (GTP)Mg.

• Bush J, Franek K, Cardelli J
• Cloning and characterization of seven novel Dictyostelium discoideum rac-related genes belonging to the rho family of GTPases.
• Gene. 1993; 136: 61-8
• Display abstract

• Cellular processes including proliferation, organization of the actin cytoskeleton, vesicular traffic and secretion of proteins comprising the lysosomal/endosomal system are regulated by low-molecular-weight GTP-binding proteins of the Ras superfamily. However, to date only three Dictyostelium discoideum ras-like genes and two ypt-1/sec4-like genes have been identified and characterized. We report here the identification (using an oligodeoxyribonucleotide probe) of seven additional cDNAs coding for members highly related to the Rac proteins (Ras-related-C3 botulinum toxin substrate) which belong to the Rho (Ras homologous) family of GTPases. Three of these rac-related genes (rac1A, rac1B and rac1C) predict proteins with > 90% amino acid (aa) sequence identity with each other and > 80% identity to the human rac1 gene product, whereas the other members (racA, racB, racC and racD) predict proteins with 46-74% identity to the rac1 and rhoA gene products and to each other. The D. discoideum proteins were entirely conserved over the four regions known to be important for GTP binding and all contained the C-terminal CAAX aa motifs shared by other Rho proteins. Interestingly, the D. discoideum rac-related genes revealed unique patterns of expression during growth and development. For instance, the steady-state level of rac1 mRNA, encoded by three highly related genes, increased transiently during aggregation and then rapidly decreased. In contrast, the cellular abundance of mRNAs encoded by the other rac-like genes decreased at different rates and to different levels during development from the peak levels observed during growth. This suggests that the GTP-binding proteins encoded by these genes may play unique roles during the different stages of the D. discoideum life cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

• Lang P et al.
• Characterization of a monoclonal antibody specific for the Ras-related GTP-binding protein Rho A.
• Biochem Biophys Res Commun. 1993; 196: 1522-8
• Display abstract

• The Rho family of small GTP-binding proteins is one of the three subgroups which, together with the Ras and Rab families, constitute the Ras-related superfamily. The Rho subgroup contains at least seven highly homologous members including 4 Rho proteins (RhoA, RhoC, RhoB, and RhoG), the Rac1 and Rac2 proteins, and CDC42Hs, which are involved in various aspects of cytoskeleton organisation and cell polarity. We have raised antibodies to individual members of the Rho family, and we report here the characterization of a monoclonal antibody (26C4) specific for RhoA. When used in western blot experiments, the 26C4 antibody recognizes the recombinant RhoA protein but not the almost identical RhoC or the RhoG, Rac and CDC42Hs proteins. Furthermore the 26C4 antibody identifies the natural RhoA protein in human lymphocyte cell extracts and was used to study the level of RhoA expression in several lymphoblastoid cell lines, and its association with the cell membrane.

• Ridley AJ, Hall A
• Distinct patterns of actin organization regulated by the small GTP-binding proteins Rac and Rho.
• Cold Spring Harb Symp Quant Biol. 1992; 57: 661-71

• Hart MJ, Maru Y, Leonard D, Witte ON, Evans T, Cerione RA
• A GDP dissociation inhibitor that serves as a GTPase inhibitor for the Ras-like protein CDC42Hs.
• Science. 1992; 258: 812-5
• Display abstract

• Members of the family of Ras-related guanosine triphosphate (GTP) binding proteins appear to take part in the regulation of a number of biological processes, including cell growth and differentiation. Three different classes of proteins that regulate the GTP binding and GTP hydrolytic activities of the Ras family members have been identified. These different regulatory proteins inhibit guanosine diphosphate (GDP) dissociation (designated as GDIs), stimulate GDP dissociation and GDP-GTP exchange (designated as GDSs), or stimulate GTP hydrolysis (designated as GAPs). In the case of the Ras-like protein CDC42Hs, which is the human homolog of a Saccharomyces cerevisiae cell division cycle protein, the GDI protein also inhibited both the intrinsic and GAP-stimulated hydrolysis of GTP. These findings establish an additional role for the GDI protein--namely, as a guanosine triphosphatase (GTPase) inhibitory protein for a Ras-like GTP binding protein.

• Hall A
• Ras-related GTPases and the cytoskeleton.
• Mol Biol Cell. 1992; 3: 475-9
• Display abstract

• Incorporation of the available data on rac in neutrophils, CDC42 in yeast, and rho in fibroblasts suggests a general model for the function of rho-like GTPase (Figure 1). Conversion of an inactive cytoplasmic rho-related p21GDP/GDI complex to active p21. GTP occurs by inhibition of GAP and/or stimulation of exchange factors in response to cell signals. p21.GTP is then able to interact with its target at the plasma membrane. This could result in a conformational change in the target, enabling it to bind cytosolic protein(s). Alternatively, p21.GTP could be actively involved in transporting cytosolic protein(s) to the target. A GAP protein, perhaps intrinsic to the complex, would stimulate GTP hydrolysis allowing p21.GDP to dissociate. Solubilization of p21GDP by interaction with GDI would complete a cycle. What about the nature of the final complex? The rac-regulated NADPH oxidase complex in neutrophils is currently the best understood and most amenable to further biochemical analysis. Two plasma-membrane bound subunits encode the catalytic function necessary for producing superoxide, but the two cytosolic proteins, p47 and p67, are essential for activity. Why the complexity? Production of superoxide is tightly coordinated with phagocytosis, a membrane process driven by rearrangement of cortical actin. This is not unrelated to the membrane ruffling and macropinocytosis that we observe in fibroblasts microinjected with p21rac. It is tempting to speculate, therefore, that in neutrophils rac is involved not only in promoting the assembly of the NADPH oxidase but also in the coordinate reorganization of cortical actin leading to phagocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)

• Hart MJ, Shinjo K, Hall A, Evans T, Cerione RA
• Identification of the human platelet GTPase activating protein for the CDC42Hs protein.
• J Biol Chem. 1991; 266: 20840-8
• Display abstract

• The CDC42Hs protein appears to be an isoform of the ras-related GTP-binding protein G25K and is an apparent human homolog of the Saccharomyces cerevisiae cell-division-cycle protein, CDC42Sc. In this study, we report the identification of a GTPase-activating protein (GAP) for CDC42Hs from human platelets (designated from here on as CDC42Hs-GAP). The CDC42Hs-GAP activity was solubilized from platelet membranes, recovered through successive chromatography steps (the final step being Mono-Q chromatography), and purified approximately 3500-fold. The CDC42Hs-GAP activity appeared to correspond to a polypeptide with an apparent Mr of approximately 25,000. The GTPase activities of the purified human platelet CDC42Hs, the Escherichia coli-recombinant CDC42Hs, and the Spodoptera frugiperda-recombinant GTP-binding proteins are all stimulated by the CDC42Hs-GAP to identical extents, which indicates that the recombinant CDC42Hs proteins are as effective as the native human platelet protein in coupling to the GAP. However, a mutant form of the E. coli-recombinant CDC42Hs which contains a valine residue at position 12 (CDC42HsVal-12) has a significantly reduced intrinsic GTPase activity (relative to the wild type CDC42HsGly-12) which is not stimulated by the CDC42Hs-GAP. The CDC42Hs-GAP also does not stimulate the GTPase activities of the ras or rap GTP-binding proteins; however, it is capable of a weak stimulation of the GTPase activity of mammalian rho. Based on the apparent similarities in the molecular size of the CDC42Hs- and rho-GAPs (i.e. 25-30 kDa), and the cross-reactivity of rho with the CDC42Hs-GAP, it seems likely that the CDC42Hs- and rho-GAPs will constitute a specific subclass of the ras-related GAP superfamily.

• Masters SB, Landis CA, Bourne HR
• Mutational analysis of the structure and function of GTP-binding proteins.
• Adv Enzyme Regul. 1990; 30: 75-87
• Display abstract

• Structural, biochemical and molecular genetic studies of EF-Tu, p21ras and alpha s have begun to reveal the inner workings of the molecular machine used by these and other GTP-binding proteins. Further understanding of this molecular machine will ultimately come from crystal structures of the G protein alpha chains as well as from crystal structures of the GTP-bound forms of p21ras and EF-Tu. Mutational analysis will continue to add meaning to the static pictures provided by these crystal structures. Aside from their intrinsic biological interest, other reasons motivate our exploration of the GTP-dependent molecular machine used by GTP-binding proteins. Mutations or bacterial toxins cause disease by inhibiting the GTPase function of p21ras and alpha s. Other G protein alpha chains carry signals that regulate important cell functions, including proliferation. Malfunctions of these other G proteins are highly likely to cause disease. Applying our knowledge of p21ras and alpha s to these additional proteins may turn out to have significant practical consequences.

© 2021 EMBL | Privacy Policy