Liposome Reconstitution and Modulation of Recombinant Prenylated Human Rac1 by GEFs,GDI1 and Pak1

Small Rho GTPases are well known to regulate a variety of cellular processes by acting as molecular switches. The regulatory function of Rho GTPases is critically dependent on their posttranslational modification at the carboxyl terminus by isoprenylation and association with proper cellular membranes. Despite numerous studies, the mechanisms of recycling and functional integration of Rho GTPases at the biological membranes are largely unclear. In this study, prenylated human Rac1, a prominent member of the Rho family, was purified in large amount from baculovirus-infected Spodoptera frugiperda insect cells using a systematic detergent screening. In contrast to non-prenylated human Rac1 purified from Escherichia coli, prenylated Rac1 from insect cells was able to associate with synthetic liposomes and to bind Rho-specific guanine nucleotide dissociation inhibitor 1 (GDI1). Subsequent liposome reconstitution experiments revealed that GDI1 efficiently extracts Rac1 from liposomes preferentially in the inactive GDP-bound state. The extraction was prevented when Rac1 was activated to its GTP-bound state by Rac-specific guanine nucleotide exchange factors (GEFs), such as Vav2, Dbl, Tiam1, P-Rex1 and TrioN, and bound by the downstream effector Pak1. We found that dissociation of Rac1-GDP from its complex with GDI1 strongly correlated with two distinct activities of especially Dbl and Tiam1, including liposome association and the GDP/GTP exchange. Taken together, our results provided first detailed insights into the advantages of the in vitro liposome-based reconstitution system to study both the integration of the signal transducing protein complexes and the mechanisms of regulation and signaling of small GTPases at biological membranes.     

Structural mimicry of DH domains by Arfaptin suggests a model for the recognition of Rac-GDP by its guanine nucleotide exchange factors.

Small G proteins cycle between an inactive form bound to GDP, and an active form bound to GTP. The two forms have different conformations and interact specifically with different partners, hence, the ability of G proteins to function as molecular switches. This view has been challenged by recent structural and biochemical studies of the Arfaptin/Por protein, which interacts equally well with the GDP- and GTP-bound forms of the G protein Rac. Here it is shown that the dimeric helical domain of Arfaptin superimposes with a monomeric helical domain from the Dbl homology domain of Tiam, a guanine nucleotide exchange factor (GEF) for Rac, in their respective complexes with Rac. This unexpected structural mimicry suggests that the Rac-GDP-Arfaptin complex resembles the low-affinity Rac-GDP-GEF complex that initiates the exchange reaction. This provides a model for the exchange mechanism where DH domains first dock onto Rac-GDP at the switch 2 before they undergo domain closure to catalyze GDP dissociation.     

Activity of the Bcr GTPase-activating domain is regulated through direct protein/protein interaction with the Rho guanine nucleotide dissociation inhibitor

The cycling of Rac GTPases, alternating between an active GTP- and an inactive GDP-bound state, is controlled by guanine nucleotide exchange factors, GTPase-activating proteins (GAPs), and guanine nucleotide dissociation inhibitors (GDIs). Little is known about how these controlling activities are coordinated. Studies using null mutant mice have demonstrated that Bcr and Abr are two physiologically important GAPs for Rac. Here, we report that in the presence of RhoGDIalpha, Bcr is unable to convert Rac-GTP to Rac-GDP because RhoGDI forms a direct protein complex with Bcr. Interestingly, RhoGDIalpha binds to the GAP domain in Bcr and Abr, a domain that also binds to Rac-GTP and catalyzes conversion of the bound GTP to GDP on Rac. The presence of activated Rac diminished the Bcr/RhoGDIalpha interaction. Moreover, a Bcr mutant that lacks the ability to promote hydrolysis of Rac-GTP bound to its GAP domain did not bind to RhoGDIalpha in cell lysates, indicating that binding of RhoGDIalpha and Rac-GTP to the Bcr GAP domain is mutually exclusive. Our results provide the first identification of a protein that regulates BcrGAP activity.     

Regulation by GDI of RhoA/Rho-kinase-induced Ca2+ sensitization of smooth muscle myosin II

We characterized the role of guanine nucleotide dissociation inhibitor (GDI) in RhoA/Rho-kinase-mediated Ca2+ sensitization of smooth muscle. Endogenous contents (approximately 2-4 microM) of RhoA and RhoGDI were near stoichiometric, whereas a supraphysiological GDI concentration was required to relax Ca2+ sensitization of force by GTP and guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). GDI also inhibited Ca2+ sensitization by GTP. G14V RhoA, by alpha-adrenergic and muscarinic agonists, and extracted RhoA from membranes. GTPgammaS translocated Rho-kinase to a Triton X-114-extractable membrane fraction. GTP. G14V RhoA complexed with GDI also induced Ca2+ sensitization, probably through in vivo dissociation of GTP. RhoA from the complex, because it was reversed by addition of excess GDI. GDI did not inhibit Ca2+ sensitization by phorbol ester. Constitutively active Cdc42 and Rac1 inhibited Ca2+ sensitization by GTP. G14V RhoA. We conclude that 1) the most likely in vivo function of GDI is to prevent perpetual "recycling" of GDP. RhoA to GTP. RhoA; 2) nucleotide exchange (GTP for GDP) on complexed GDP. RhoA/GDI can precede translocation of RhoA to the membrane; 3) activation of Rho-kinase exposes a hydrophobic domain; and 4) Cdc42 and Rac1 can inhibit Ca2+ sensitization by activated GTP. RhoA.     

The N-terminal DH-PH domain of Trio induces cell spreading and migration by regulating lamellipodia dynamics in a Rac1-dependent fashion

The guanine-nucleotide exchange factor Trio encodes two DH-PH domains that catalyze nucleotide exchange on Rac1, RhoG and RhoA. The N-terminal DH-PH domain is known to activate Rac1 and RhoG, whereas the C-terminal DH-PH domain can activate RhoA. The current study shows that the N-terminal DH-PH domain, upon expression in HeLa cells, activates Rac1 and RhoG independently from each other. In addition, we show that the flanking SH3 domain binds to the proline-rich region of the C-terminus of Rac1, but not of RhoG. However, this SH3 domain is not required for Rac1 or RhoG GDP-GTP exchange. Rescue experiments in Trio-shRNA-expressing cells showed that the N-terminal DH-PH domain of Trio, but not the C-terminal DH-PH domain, restored fibronectin-mediated cell spreading and migration defects that are observed in Trio-silenced cells. Kymograph analysis revealed that the N-terminal DH-PH domain, independent of its SH3 domain, controls the dynamics of lamellipodia. Using siRNA against Rac1 or RhoG, we found that Trio-D1-induced lamellipodia formation required Rac1 but not RhoG expression. Together, we conclude that the GEF Trio is responsible for lamellipodia formation through its N-terminal DH-PH domain in a Rac1-dependent manner during fibronectin-mediated spreading and migration.     

Quantitative analysis of the effect of phosphoinositide interactions on the function of Dbl family proteins

Normally, Rho GTPases are activated by the removal of bound GDP and the concomitant loading of GTP catalyzed by members of the Dbl family of guanine nucleotide exchange factors (GEFs). This family of GEFs invariantly contain a Dbl homology (DH) domain adjacent to a pleckstrin homology (PH) domain, and while the DH domain usually is sufficient to catalyze nucleotide exchange, possible roles for the conserved PH domain remain ambiguous. Here we demonstrate that the conserved PH domains of three distinct Dbl family proteins, intersectin, Dbs, and Tiam1, selectively bind lipid vesicles only when phosphoinositides are present. While the PH domains of intersectin and Dbs promiscuously bind several multiphosphorylated phosphoinositides, Tiam1 selectively interacts with phosphatidylinositol 3-phosphate (K(D) approximately 5-10 microm). In addition, and in contrast to recent reports, catalysis of nucleotide exchange on nonprenylated Rac1 provided by various extended portions of Tiam1 is not influenced by (a) soluble phosphoinositide head groups, (b) dibutyl versions of phosphoinositides, or (c) lipid vesicles containing phosphoinositides. Likewise, GEF activity afforded by DH/PH fragments of intersectin and Dbs are also not altered by phosphoinositide interactions. These results strongly suggest that unless all relevant components are localized to a lipid membrane surface, Dbl family GEFs generally are not intrinsically modulated by binding phosphoinositides.     

Dissociation of Rac1(GDP).RhoGDI complexes by the cooperative action of anionic liposomes containing phosphatidylinositol 3,4,5-trisphosphate, Rac guanine nucleotide exchange factor, and GTP

Rac plays a pivotal role in the assembly of the superoxide-generating NADPH oxidase of phagocytes. In resting cells, Rac is found in the cytosol in complex with Rho GDP dissociation inhibitor (RhoGDI). NADPH oxidase assembly involves dissociation of the Rac.RhoGDI complex and translocation of Rac to the membrane. We reported that liposomes containing high concentrations of monovalent anionic phospholipids cause Rac.RhoGDI complex dissociation ( Ugolev, Y., Molshanski-Mor, S., Weinbaum, C., and Pick, E. (2006) J. Biol. Chem. 281, 19204-19219 ). We now designed an in vitro model mimicking membrane phospholipid remodeling during phagocyte stimulation in vivo. We showed that liposomes of "resting cell membrane" composition (less than 20 mol % monovalent anionic phospholipids), supplemented with 1 mol % of polyvalent anionic phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) in conjunction with constitutively active forms of the guanine nucleotide exchange factors (GEFs) for Rac, Trio, or Tiam1 and a non-hydrolyzable GTP analogue, cause dissociation of Rac1(GDP).RhoGDI complexes, GDP to GTP exchange on Rac1, and binding of Rac1(GTP) to the liposomes. Complexes were not dissociated in the absence of GEF and GTP, and optimal dissociation required the presence of PtdIns(3,4,5)P(3) in the liposomes. Dissociation of Rac1(GDP).RhoGDI complexes was correlated with the affinity of particular GEF constructs, via the N-terminal pleckstrin homology domain, for PtdIns(3,4,5)P(3) and involved GEF-mediated GDP to GTP exchange on Rac1. Phagocyte membranes enriched in PtdIns(3,4,5)P(3) responded by NADPH oxidase activation upon exposure in vitro to Rac1(GDP).RhoGDI complexes, p67(phox), GTP, and Rac GEF constructs with affinity for PtdIns(3,4,5)P(3) at a level superior to that of native membranes.     

Ankyrin-Tiam1 interaction promotes Rac1 signaling and metastatic breast tumor cell invasion and migration

Tiam1 (T-lymphoma invasion and metastasis 1) is one of the known guanine nucleotide (GDP/GTP) exchange factors (GEFs) for Rho GTPases (e.g., Rac1) and is expressed in breast tumor cells (e.g., SP-1 cell line). Immunoprecipitation and immunoblot analyses indicate that Tiam1 and the cytoskeletal protein, ankyrin, are physically associated as a complex in vivo. In particular, the ankyrin repeat domain (ARD) of ankyrin is responsible for Tiam1 binding. Biochemical studies and deletion mutation analyses indicate that the 11-amino acid sequence between amino acids 717 and 727 of Tiam1 ((717)GEGTDAVKRS(727)L) is the ankyrin-binding domain. Most importantly, ankyrin binding to Tiam1 activates GDP/GTP exchange on Rho GTPases (e.g., Rac1).Using an Escherichia coli-derived calmodulin-binding peptide (CBP)-tagged recombinant Tiam1 (amino acids 393-728) fragment that contains the ankyrin-binding domain, we have detected a specific binding interaction between the Tiam1 (amino acids 393-738) fragment and ankyrin in vitro. This Tiam1 fragment also acts as a potent competitive inhibitor for Tiam1 binding to ankyrin. Transfection of SP-1 cell with Tiam1 cDNAs stimulates all of the following: (1) Tiam1-ankyrin association in the membrane projection; (2) Rac1 activation; and (3) breast tumor cell invasion and migration. Cotransfection of SP1 cells with green fluorescent protein (GFP)-tagged Tiam1 fragment cDNA and Tiam1 cDNA effectively blocks Tiam1-ankyrin colocalization in the cell membrane, and inhibits GDP/GTP exchange on Rac1 by ankyrin-associated Tiam1 and tumor-specific phenotypes. These findings suggest that ankyrin-Tiam1 interaction plays a pivotal role in regulating Rac1 signaling and cytoskeleton function required for oncogenic signaling and metastatic breast tumor cell progression.     

The Rac activator Tiam1 controls tight junction biogenesis in keratinocytes through binding to and activation of the Par polarity complex

The GTPases Rac and Cdc42 play a pivotal role in the establishment of cell polarity by stimulating biogenesis of tight junctions (TJs). In this study, we show that the Rac-specific guanine nucleotide exchange factor Tiam1 (T-lymphoma invasion and metastasis) controls the cell polarity of epidermal keratinocytes. Similar to wild-type (WT) keratinocytes, Tiam1-deficient cells establish primordial E-cadherin-based adhesions, but subsequent junction maturation and membrane sealing are severely impaired. Tiam1 and V12Rac1 can rescue the TJ maturation defect in Tiam1-deficient cells, indicating that this defect is the result of impaired Tiam1-Rac signaling. Tiam1 interacts with Par3 and aPKCzeta, which are two components of the conserved Par3-Par6-aPKC polarity complex, and triggers biogenesis of the TJ through the activation of Rac and aPKCzeta, which is independent of Cdc42. Rac is activated upon the formation of primordial adhesions (PAs) in WT but not in Tiam1-deficient cells. Our data indicate that Tiam1-mediated activation of Rac in PAs controls TJ biogenesis and polarity in epithelial cells by association with and activation of the Par3-Par6-aPKC polarity complex.     

Yersinia virulence depends on mimicry of host Rho-family nucleotide dissociation inhibitors

Yersinia spp. cause gastroenteritis and the plague, representing historically devastating pathogens that are currently an important biodefense and antibiotic resistance concern. A critical virulence determinant is the Yersinia protein kinase A, or YpkA, a multidomain protein that disrupts the eukaryotic actin cytoskeleton. Here we solve the crystal structure of a YpkA-Rac1 complex and find that YpkA possesses a Rac1 binding domain that mimics host guanidine nucleotide dissociation inhibitors (GDIs) of the Rho GTPases. YpkA inhibits nucleotide exchange in Rac1 and RhoA, and mutations that disrupt the YpkA-GTPase interface abolish this activity in vitro and impair in vivo YpkA-induced cytoskeletal disruption. In cell culture experiments, the kinase and the GDI domains of YpkA act synergistically to promote cytoskeletal disruption, and a Y. pseudotuberculosis mutant lacking YpkA GDI activity shows attenuated virulence in a mouse infection assay. We conclude that virulence in Yersinia depends strongly upon mimicry of host GDI proteins by YpkA.     

Molecular basis for Rac1 recognition by guanine nucleotide exchange factors.

Rho GTPases are activated by a family of guanine nucleotide exchange factors (GEFs) known as Dbl family proteins. The structural basis for how GEFs recognize and activate Rho GTPases is presently ill defined. Here, we utilized the crystal structure of the DH/PH domains of the Rac-specific GEF Tiam1 in complex with Rac1 to determine the structural elements of Rac1 that regulate the specificity of this interaction. We show that residues in the Rac1 beta2-beta3 region are critical for Tiam1 recognition. Additionally, we determined that a single Rac1-to-Cdc42 mutation (W56F) was sufficient to abolish Rac1 sensitivity to Tiam1 and allow recognition by the Cdc42-specific DH/PH domains of Intersectin while not impairing Rac1 downstream activities. Our findings identified unique GEF specificity determinants in Rac1 and provide important insights into the mechanism of DH/PH selection of GTPase targets.     

Rac1 function is required for Src-induced transformation. Evidence of a role for Tiam1 and Vav2 in Rac activation by Src

The proto-oncogene c-Src has been implicated in the development and progression of a number of human cancers including those of colon and breast. Accumulating evidence indicates that activated alleles of Src may induce cell transformation through Ras-ERK-dependent and -independent pathways. Here we show that Rac1 activity is strongly elevated in Src-transformed cells and that this small G protein is a critical component of the pathway connecting oncogenic Src with cell transformation. We further show that Vav2 and the ubiquitously expressed Rac1 guanine nucleotide exchange factor Tiam1 are phosphorylated in tyrosine residues in cells transfected with active and oncogenic Src. Moreover, phosphorylation of Tiam1 in cells treated with pervanadate, a potent inhibitor of tyrosine phosphatases, was partially inhibited by the Src inhibitor SU6656. Using truncated mutants of Tiam1, we demonstrate that multiple sites can be tyrosine-phosphorylated by Src. Furthermore, Tiam1 cooperated with Src to induce activation of Rac1 in vivo and the formation of membrane ruffles. Similarly, activation of JNK and the c-jun promoter by Src were also potently increased by Tiam1. Together, these results suggest that Vav2 and Tiam1 may act as downstream effectors of Src, thereby regulating Rac1-dependent pathways that participate in Src-induced cell transformation.     

Structural insights into the dual nucleotide exchange and GDI displacement activity of SidM/DrrA

GDP‐bound prenylated Rabs, sequestered by GDI (GDP dissociation inhibitor) in the cytosol, are delivered to destined sub‐cellular compartment and subsequently activated by GEFs (guanine nucleotide exchange factors) catalysing GDP‐to‐GTP exchange. The dissociation of GDI from Rabs is believed to require a GDF (GDI displacement factor). Only two RabGDFs, human PRA‐1 and Legionella pneumophila SidM/DrrA, have been identified so far and the molecular mechanism of GDF is elusive. Here, we present the structure of a SidM/DrrA fragment possessing dual GEF and GDF activity in complex with Rab1. SidM/DrrA reconfigures the Switch regions of the GTPase domain of Rab1, as eukaryotic GEFs do toward cognate Rabs. Structure‐based mutational analyses show that the surface of SidM/DrrA, catalysing nucleotide exchange, is involved in GDI1 displacement from prenylated Rab1:GDP. In comparison with an eukaryotic GEF TRAPP I, this bacterial GEF/GDF exhibits high binding affinity for Rab1 with GDP retained at the active site, which appears as the key feature for the GDF activity of the protein.     

Structural basis for the reversible activation of a Rho protein by the bacterial toxin SopE

The bacterial enteropathogen Salmonella typhimurium employs a type III secretion system to inject bacterial toxins into the host cell cytosol. These toxins transiently activate Rho family GTP-binding protein-dependent signaling cascades to induce cytoskeletal rearrangements. One of these translocated Salmonella toxins, SopE, can activate Cdc42 in a Dbl-like fashion despite its lack of sequence similarity to Dbl-like proteins, the Rho-specific eukaryotic guanine nucleotide exchange factors. To elucidate the mechanism of SopE-mediated guanine nucleotide exchange, we have analyzed the structure of the complex between a catalytic fragment of SopE and Cdc42. SopE binds to and locks the switch I and switch II regions of Cdc42 in a conformation that promotes guanine nucleotide release. This conformation is strikingly similar to that of Rac1 in complex with the eukaryotic Dbl-like exchange factor Tiam1. However, the catalytic domain of SopE has an entirely different architecture from that of Tiam1 and interacts with the switch regions via different amino acids. Therefore, SopE represents the first example of a non-Dbl-like protein capable of inducing guanine nucleotide exchange in Rho family proteins.     

Regulation of p70 S6 kinase by complex formation between the Rac guanine nucleotide exchange factor (Rac-GEF) Tiam1 and the scaffold spinophilin

Tiam1 is a ubiquitous guanine nucleotide exchange factor (GEF) that activates the Rac GTPase. We have shown previously that the N terminus of Tiam1 contributes to the signaling specificity of its downstream target Rac via association with IB2, a scaffold that promotes Rac activation of a p38 kinase cascade. Here we show that the N terminus of Tiam1 can influence Rac signaling specificity in a different way by interaction with spinophilin, a scaffold that binds to p70 S6 kinase, another protein regulated by Rac. In particular, spinophilin binding promotes the plasma membrane localization of Tiam1 and enhances the ability of Tiam1 to activate p70 S6 kinase. In contrast, spinophilin binding suppresses the ability of Tiam to activate Pak1, a different Rac effector. Finally, a mutant spinophilin that cannot bind to Tiam1 suppresses serum-induced p70 S6 kinase activation in cells, suggesting that a Tiam1/spinophilin complex contributes to p70 S6 kinase regulation by extracellular signals. These findings add to a growing body of evidence supporting the concept that some Rac-GEFs not only activate Rac GTPases but also participate in the selection of Rac effector by binding to particular scaffolds that complex with components of specific Rac effector pathways.     

Phosphoinositide-dependent activation of Rho A involves partial opening of the RhoA/Rho-GDI complex.

Rho GTPases have two interconvertible forms and two cellular localizations. In their GTP-bound conformation, they bind to the cell membrane and are activated. In the inactive GDP-bound conformation, they associate with a cytosolic protein called GDP dissociation inhibitor (GDI). We previously reported that the RhoA component of the RhoA/Rho-GDI complex was not accessible to the Clostridium botulinum C3 ADP-ribosyl transferase, unless the complex had been incubated with phosphoinositides. We show here that PtdIns, PtdIns4P, PtdIns3,4P2, PtdIns4,5P2 and PtdInsP3 enhance not only the C3-dependent ADP-ribosylation, but also the GDP/GTP exchange in the RhoA component of the prenylated RhoA/Rho-GDI complex. In contrast, in the nonprenylated RhoA/Rho-GDI complex, the levels of ADP-ribosylation and GDP/GTP exchange are of the same order as those measured on free RhoA and are not modified by phosphoinositides. In both cases, phosphoinositides partially opened, but did not fully dissociate the complex. Upon treatment of the prenylated RhoA/Rho-GDI complex with phosphoinositides, a GTP-dependent transfer to neutrophil membranes was evidenced. Using an overlay assay with the prenylated RhoA/Rho-GDI complex pretreated with PtdIns4P and labeled with [alpha32P]GTP, three membrane proteins with molecular masses between 26 and 32 kDa were radiolabeled. We conclude that in the presence of phosphoinositides, the prenylated RhoA/Rho-GDI complex partially opens, which allows RhoA to exchange GDP for GTP. The opened GTP-RhoA/Rho-GDI complex acquires the capacity to target specific membrane proteins.     

Recognition and activation of Rho GTPases by Vav1 and Vav2 guanine nucleotide exchange factors

Vav proteins are Rho GTPase-specific guanine nucleotide exchange factors (GEFs) that are distinguished by the tandem arrangement of Dbl homology (DH), Pleckstrin homology (PH), and cysteine rich domains (CRD). Whereas the tandem DH-PH arrangement is conserved among Rho GEFs, the presence of the CRD is unique to Vav family members and is required for efficient nucleotide exchange. We provide evidence that Vav2-mediated nucleotide exchange of Rho GTPases follows the Theorell-Chance mechanism in which the Vav2.Rho GTPase complex is the major species during the exchange process and the Vav2.GDP-Mg(2+).Rho GTPase ternary complex is present only transiently. The GTPase specificity for the DH-PH-CRD Vav2 in vitro follows this order: Rac1 > Cdc42 > RhoA. Results obtained from fluorescence anisotropy and NMR chemical shift mapping experiments indicate that the isolated Vav1 CRD is capable of directly associating with Rac1, and residues K116 and S83 that are in the proximity of the P-loop and the guanine base either are part of this binding interface or undergo a conformational change in response to CRD binding. The NMR studies are supported by kinetic measurements on Rac1 mutants S83A, K116A, and K116Q and Vav2 CRD mutant K533A in that these mutants affect both the initial binding event of Vav2 with Rac1 (k(on)) and the rate-limiting dissociation of Vav2 from the Vav2.Rac1 binary complex (thereby influencing the enzyme turnover number, k(cat)). The results suggest that the CRD domain in Vav proteins plays an active role, affecting both the k(on) and the k(cat) for Vav-mediated nucleotide exchange on Rho GTPases.     

Rac activation by lysophosphatidic acid LPA1 receptors through the guanine nucleotide exchange factor Tiam1

Lysophosphatidic acid (LPA) is a serum-borne phospholipid that activates its own G protein-coupled receptors present in numerous cell types. In addition to stimulating cell proliferation, LPA also induces cytoskeletal changes and promotes cell migration in a RhoA- and Rac-dependent manner. Whereas RhoA is activated via Galpha(12/13)-linked Rho-specific guanine nucleotide exchange factors, it is unknown how LPA receptors may signal to Rac. Here we report that the prototypic LPA(1) receptor (previously named Edg2), when expressed in B103 neuroblastoma cells, mediates transient activation of RhoA and robust, prolonged activation of Rac leading to cell spreading, lamellipodia formation, and stimulation of cell migration. LPA-induced Rac activation is inhibited by pertussis toxin and requires phosphoinositide 3-kinase activity. Strikingly, LPA fails to activate Rac in cell types that lack the Rac-specific exchange factor Tiam1; however, enforced expression of Tiam1 restores LPA-induced Rac activation in those cells. Tiam1-deficient cells show enhanced RhoA activation, stress fiber formation, and cell rounding in response to LPA, consistent with Tiam1/Rac counteracting RhoA. We conclude that LPA(1) receptors couple to a G(i)-phosphoinositide 3-kinase-Tiam1 pathway to activate Rac, with consequent suppression of RhoA activity, and thereby stimulate cell spreading and motility.     

Binding of Rab3A to synaptic vesicles

Prenylated Rab GTPases cycle between membrane-bound and soluble forms. Membrane-bound GDP-Rabs interact with GDP dissociation inhibitor (GDI), resulting in the dissociation of a Rab.GDI complex, which in turn serves as a precursor for the membrane re-association of Rabs. We have now characterized the binding of Rab3A to synaptic vesicles in vitro using either purified complexes or rat brain cytosol as source for GDI.Rab3A. Binding of Rab3A results in the immediate release of GDI from the membrane. Furthermore, binding does not require the presence of additional guanine nucleotides (GDP or GTP) or of cytosolic factors. Although nucleotide exchange follows binding, binding is initially reversible, suggesting that binding of GDP-Rab3A and nucleotide exchange are separate and independent events. Comparison with the binding of Rab1B revealed that both Rab proteins bind preferentially to their respective resident membranes although some promiscuity was observable. Binding is saturable and involves a protease-sensitive binding site that is tightly associated with the vesicle membrane.