Liposomes comprising anionic but not neutral phospholipids cause dissociation of Rac(1 or 2) x RhoGDI complexes and support amphiphile-independent NADPH oxidase activation by such complexes

Activation of the phagocyte NADPH oxidase involves the assembly of a membrane-localized cytochrome b559 with the cytosolic components p47(phox), p67(phox), p40(phox), and the GTPase Rac (1 or 2). In resting phagocytes, Rac is found in the cytosol as a prenylated protein in the GDP-bound form, associated with the Rho GDP dissociation inhibitor (RhoGDI). In the process of NADPH oxidase activation, Rac is dissociated from RhoGDI and translocates to the membrane, in concert with the other cytosolic components. The mechanism responsible for dissociation of Rac from RhoGDI is poorly understood. We generated Rac(1 or 2) x RhoGDI complexes in vitro from recombinant Rac(1 or 2), prenylated enzymatically, and recombinant RhoGDI, and purified these by anion exchange chromatography. Exposing Rac(1 or 2)(GDP) x RhoGDI complexes to liposomes containing four different anionic phospholipids caused the dissociation of Rac(1 or 2)(GDP) from RhoGDI and its binding to the anionic liposomes. Rac2(GDP) x RhoGDI complexes were more resistant to dissociation, reflecting the lesser positive charge of Rac2. Liposomes consisting of neutral phospholipid did not cause dissociation of Rac(1 or 2) x RhoGDI complexes. Rac1 exchanged to the hydrolysis-resistant GTP analogue, GMPPNP, associated with RhoGDI with lower affinity than Rac1(GDP) and Rac1(GMPPNP) x RhoGDI complexes were more readily dissociated by anionic liposomes. Rac1(GMPPNP) x RhoGDI complexes elicited NADPH oxidase activation in native phagocyte membrane liposomes in the presence of p67(phox), without the need for an anionic amphiphile, as activator. Both Rac1(GDP) x RhoGDI and Rac1(GMPPNP) x RhoGDI complexes elicited amphiphile-independent, p67(phox)-dependent NADPH oxidase activation in phagocyte membrane liposomes enriched in anionic phospholipids but not in membrane liposomes enriched in neutral phospholipids.     

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.     

Tumor necrosis factor alpha stimulation of Rac1 activity. Role of isoprenylcysteine carboxylmethyltransferase

We have previously demonstrated that both isoprenylcysteine carboxylmethyltransferase (ICMT) and one of its substrates, the RhoGTPase Rac1, are critical for the tumor necrosis factor alpha (TNF alpha) stimulation of vascular cell adhesion molecule-1 expression in endothelial cells (EC). Here, we have shown that ICMT regulates TNF alpha stimulation of Rac1 activity. TNF alpha stimulation of EC increased the membrane association of Rac1, an event that is essential for Rac1 activity. ICMT inhibitor N-acetyl-S-farnesyl-L-cysteine (AFC) blocked the accumulation of Rac1 into the membrane both in resting and TNF alpha-stimulated conditions. Similarly, the membrane-associated Rac1 was lower in Icmt-deficient versus wild-type mouse embryonic fibroblasts (MEFs). TNF alpha also increased the level of GTP-Rac1, the active form of Rac1, in EC. AFC completely suppressed the TNF alpha stimulation of increase in GTP-Rac1 levels. Confocal microscopy revealed resting EC Rac1 was present in the plasma membrane and also in the perinuclear region. AFC mislocalized Rac1, both from the plasma membrane and the perinuclear region. Mislocalization of Rac1 was also observed in Icmt-deficient versus wild-type MEFs. To determine the consequences of ICMT inhibition, we investigated the effect of AFC on p38 mitogen-activated protein (MAP) kinase phosphorylation, which is downstream of Rac1. AFC inhibited the TNF alpha stimulation of p38 MAP kinase phosphorylation in EC. TNF alpha stimulation of p38 MAP kinase phosphorylation was also significantly attenuated in Icmt-deficient versus wild-type MEFs. To understand the mechanism of inhibition of Rac1 activity, we examined the effect of ICMT inhibition on the interaction of Rac1 with its inhibitor, Rho guanine nucleotide dissociation inhibitor (RhoGDI). The association of Rac1 with its inhibitor RhoGDI was dramatically increased in the Icmt-deficient versus wild-type MEFs both in resting as well as in TNF alpha-stimulated conditions, suggesting that RhoGDI was involved in inhibiting Rac1 activity under the conditions of ICMT inhibition. These results suggest that ICMT regulates Rac1 activity by controlling the interaction of Rac1 with RhoGDI. We hypothesize that ICMT regulates the release of Rac1 from RhoGDI.     

Coronin 1A promotes a cytoskeletal-based feedback loop that facilitates Rac1 translocation and activation

The activation of the Rac1 GTPase during cell signalling entails its translocation from the cytosol to membranes, release from sequestering Rho GDP dissociation inhibitors (RhoGDI), and GDP/GTP exchange. In addition to those steps, we show here that optimal Rac1 activation during cell signalling requires the engagement of a downstream, cytoskeletal-based feedback loop nucleated around the cytoskeletal protein coronin 1A and the Rac1 exchange factor ArhGEF7. These two proteins form a cytosolic complex that, upon Rac1-driven F-actin polymerization, translocates to juxtamembrane areas where it expands the pool of activated, membrane-bound Rac1. Such activity requires the formation of an F-actin/ArhGEF7-dependent physical complex of coronin 1A with Pak1 and RhoGDIα that, once assembled, promotes the Pak1-dependent dissociation of Rac1 from the Rac1/RhoGDIα complex and subsequent Rac1 activation. Genetic evidence demonstrates that this relay circuit is essential for generating sustained Rac1 activation levels during cell signalling.     

Structural consequences of site-directed mutagenesis in flexible protein domains: NMR characterization of the L(55,56)S mutant of RhoGDI.

The guanine dissociation inhibitor RhoGDI consists of a folded C-terminal domain and a highly flexible N-terminal region, both of which are essential for biological activity, that is, inhibition of GDP dissociation from Rho GTPases, and regulation of their partitioning between membrane and cytosol. It was shown previously that the double mutation L55S/L56S in the flexible region of RhoGDI drastically decreases its affinity for Rac1. In the present work we study the effect of this double mutation on the conformational and dynamic properties of RhoGDI, and describe the weak interaction of the mutant with Rac1 using chemical shift mapping. We show that the helical content of the region 45-56 of RhoGDI is greatly reduced upon mutation, thus increasing the entropic penalty for the immobilization of the helix, and contributing to the loss of binding. In contrast to wild-type RhoGDI, no interaction with Rac1 could be identified for amino-acid residues of the flexible domain of the mutant RhoGDI and only very weak binding was observed for the folded domain of the mutant. The origins of the effect of the L55S/L56S mutation on the binding constant (decreased by at least three orders of magnitude relative to wild-type) are discussed with particular reference to the flexibility of this part of the protein.     

Cholecystokinin-Mediated RhoGDI Phosphorylation via PKCα Promotes both RhoA and Rac1 Signaling

RhoA and Rac1 have been implicated in the mechanism of CCK-induced amylase secretion from pancreatic acini. In all cell types studied to date, inactive Rho GTPases are present in the cytosol bound to the guanine nucleotide dissociation inhibitor RhoGDI. Here, we identified the switch mechanism regulating RhoGDI1-Rho GTPase dissociation and RhoA translocation upon CCK stimulation in pancreatic acini. We found that both Gα13 and PKC, independently, regulate CCK-induced RhoA translocation and that the PKC isoform involved is PKCα. Both RhoGDI1 and RhoGDI3, but not RhoGDI2, are expressed in pancreatic acini. Cytosolic RhoA and Rac1 are associated with RhoGDI1, and CCK-stimulated PKCα activation releases the complex. Overexpression of RhoGDI1, by binding RhoA, inhibits its activation, and thereby, CCK-induced apical amylase secretion. RhoA translocation is also inhibited by RhoGDI1. Inactive Rac1 influences CCK-induced RhoA activation by preventing RhoGDI1 from binding RhoA. By mutational analysis we found that CCK-induced PKCα phosphorylation on RhoGDI1 at Ser96 releases RhoA and Rac1 from RhoGDI1 to facilitate Rho GTPases signaling.     

ExoS Rho GTPase-activating protein activity stimulates reorganization of the actin cytoskeleton through Rho GTPase guanine nucleotide disassociation inhibitor

ExoS is a bifunctional Type III cytotoxin of Pseudomonas aeruginosa with N-terminal Rho GTPase-activating protein (RhoGAP) and C-terminal ADP-ribosyltransferase domains. Although the ExoS RhoGAP inactivates Cdc42, Rac, and RhoA in vivo, the relationship between ExoS RhoGAP and the eukaryotic regulators of Rho GTPases is not clear. The present study investigated the roles of Rho GTPase guanine nucleotide disassociation inhibitor (RhoGDI) in the reorganization of actin cytoskeleton mediated by ExoS RhoGAP. A green fluorescent protein-RhoGDI fusion protein was engineered and found to elicit actin reorganization through the inactivation of Rho GTPases. Green fluorescent protein-RhoGDI and ExoS RhoGAP cooperatively stimulated actin reorganization and translocation of Cdc42 from membrane to cytosol, and a RhoGDI mutant, RhoGDI(I177D), that is defective in extracting Rho GTPases off the membrane inhibited the actions of RhoGDI and ExoS RhoGAP on the translocation of Cdc42 from membrane to cytosol. A human RhoGDI small interfering RNA was transfected into HeLa cells to knock down 90% of the endogenous RhoGDI expression. HeLa cells with knockdown RhoGDI were resistant to the reorganization of the actin cytoskeleton elicited by type III-delivered ExoS RhoGAP. This indicates that ExoS RhoGAP and RhoGDI function in series to inactivate Rho GTPases, in which RhoGDI extracting GDP-bound Rho GTPases off the membrane and sequestering them in cytosol is the rate-limiting step in Rho GTPase inactivation. A eukaryotic GTPase-activating protein, p50RhoGAP, showed a similar cooperativity with RhoGDI on actin reorganization, suggesting that ExoS RhoGAP functions as a molecular mimic of eukaryotic RhoGAPs to inactivate Rho GTPases through RhoGDI.     

Differential binding of arfaptin 2/POR1 to ADP-ribosylation factors and Rac1

We first identified arfaptin as a protein that bound to GTP-ARFs (especially ARF1). However, a second group reported that POR1, a truncated form of arfaptin, bound to GTP-Rac1. Therefore, we examined the possibility that arfaptin 2/POR1 was a common downstream effector for both ARF1 and Rac1. In this study, we found that constitutively active Rac1 or GTP-Rac1 showed negligible or no binding to arfaptin 2/POR1 in a yeast two-hybrid assay or a GST pull-down assay. However, wild-type or dominant negative Rac1 or Rac1 liganded to GDP showed strong binding. In contrast, constitutively active ARFs1, 5, and 6 showed binding, whereas the wild-type and dominant negative forms did not. Furthermore, the GTP-liganded ARFs bound arfaptin 2, whereas the GDP-bound forms showed little or no binding. Based on these observations, we suggest that arfaptin 2/POR1 is a target protein for GTP-ARFs and for GDP-Rac1, and that it may be involved in interactions between the Rac and ARF signaling pathways.     

How fatty acids of different chain length enter and leave cells by free diffusion

Opposing views exist as to how unesterified fatty acids (FA) enter and leave cells. It is commonly believed that for short- and medium-chain FA free diffusion suffices whereas it is questioned whether proteins are required to facilitate transport of long-chain fatty acid (LCFA). Furthermore, it is unclear whether these proteins facilitate binding to the plasma membrane, trans-membrane movement, dissociation into the cytosol and/or transport in the cytosol. In this mini-review we approach the controversy from a different point of view by focusing on the membrane permeability constant (P) of FA with different chain length. We compare experimentally derived values of the P of short and medium-chain FA with values of apparent permeability coefficients for LCFA calculated from their dissociation rate constant (k(off)), flip-flop rate constant (k(flip)) and partition coefficient (Kp) in phospholipid bilayers. It was found that Overton's rule is valid as long as k(flip)相似文献   

Phospholipase D activity regulates integrin-mediated cell spreading and migration by inducing GTP-Rac translocation to the plasma membrane

Small GTPase Rac is a crucial regulator of actin cytoskeletal rearrangement, and it plays an important role in cell spreading, migration, mitogenesis, phagocytosis, superoxide generation, and axonal growth. It is generally accepted that Rac activity is regulated by the guanosine triphosphate (GTP)/guanosine diphosphate (GDP) cycle. But, it is suggested that in addition to Rac-GTP loading, membrane localization is required for the initiation of downstream effector signaling. However, the molecular mechanisms that control the targeting of GTP-Rac to the plasma membrane remain largely unknown. Here, we have uncovered a signaling pathway linking phospholipase D (PLD) to the localized functions of Rac1. We show that PLD product phosphatidic acid (PA) acts as a membrane anchor of Rac1. The C-terminal polybasic motif of Rac1 is responsible for direct interaction with PA, and Rac1 mutated in this region is incapable of translocating to the plasma membrane and of activating downstream target p21-activated kinase upon integrin activation. Finally, we show that PA induces dissociation of Rho-guanine nucleotide dissociation inhibitor from Rac1 and that PA-mediated Rac1 localization is important for integrin-mediated lamellipodia formation, cell spreading, and migration. These results provide a novel molecular mechanism for the GTP-Rac1 localization through the elevating PLD activity, and they suggest a general mechanism for diverse cellular functions that is required localized Rac activation.     

Diacylglycerol Kinase ζ Regulates Actin Cytoskeleton Reorganization through Dissociation of Rac1 from RhoGDI

Activation of Rac1 GTPase signaling is stimulated by phosphorylation and release of RhoGDI by the effector p21-activated kinase 1 (PAK1), but it is unclear what initiates this potential feed-forward mechanism for regulation of Rac activity. Phosphatidic acid (PA), which is produced from the lipid second messenger diacylglycerol (DAG) by the action of DAG kinases (DGKs), is known to activate PAK1. Here, we investigated whether PA produced by DGKζ initiates RhoGDI release and Rac1 activation. In DGKζ-deficient fibroblasts PAK1 phosphorylation and Rac1–RhoGDI dissociation were attenuated, leading to reduced Rac1 activation after platelet-derived growth factor stimulation. The cells were defective in Rac1-regulated behaviors, including lamellipodia formation, membrane ruffling, migration, and spreading. Wild-type DGKζ, but not a kinase-dead mutant, or addition of exogenous PA rescued Rac activation. DGKζ stably associated with PAK1 and RhoGDI, suggesting these proteins form a complex that functions as a Rac1-selective RhoGDI dissociation factor. These results define a pathway that links diacylglycerol, DGKζ, and PA to the activation of Rac1: the PA generated by DGKζ activates PAK1, which dissociates RhoGDI from Rac1 leading to changes in actin dynamics that facilitate the changes necessary for cell motility.     

Serum-activated assembly and membrane translocation of an endogenous Rac1:effector complex

Rho family GTPases (Cdc42, Rac1, and RhoA) function downstream of Ras [1], and in a variety of cellular processes [2]. Studies to examine these functions have not directly linked endogenous protein interactions with specific in vivo functions of Rho GTPases. Here, we show that endogenous Rac1 and two known binding partners, Rho GDP dissociation inhibitor (RhoGDI) and p21-activated kinase (PAK), fractionate as distinct cytosolic complexes. A Rac1:PAK complex is translocated from the cytosol to ruffling membranes upon cell activation by serum. Overexpression of dominant-negative (T17N) Rac1 does not affect the assembly or distribution of this Rac1:PAK complex. This is the first direct evidence of how a specific function of Rac1 is selected by the assembly and membrane translocation of a distinct Rac1:effector complex.     

Phosphorylation of RhoGDI by Pak1 mediates dissociation of Rac GTPase

Selective activation of Rac GTPase signaling pathways requires the specific release of Rac from RhoGDI complexes. We identified a RhoGDI kinase from bovine brain as p21-activated kinase (Pak). Pak1 binds and phosphorylates RhoGDI both in vitro and in vivo at Ser101 and Ser174. This resulted in dissociation of Rac1-RhoGDI, but not RhoA-RhoGDI, complexes, as determined by in vitro assays of complexation and in vivo by coimmunoprecipitation analysis. We observed that Cdc42-induced Rac1 activation is inhibited by expression of Pak1 autoinhibitory domain. The dissociation of Rac1 from RhoGDI and its subsequent activation stimulated by PDGF or EGF is also attenuated by Pak1 autoinhibitory domain, and this is dependent on the ability of RhoGDI to be phosphorylated at Ser101/174. These results support a role for Pak1-mediated RhoGDI phosphorylation as a mechanism for Cdc42-mediated Rac activation, and suggest the possibility of Rac-induced positive feed-forward regulation of Rac activity.     

Suppression of RhoG activity is mediated by a syndecan 4–synectin–RhoGDI1 complex and is reversed by PKCα in a Rac1 activation pathway

Fibroblast growth factor 2 (FGF2) is a major regulator of developmental, pathological, and therapeutic angiogenesis. Its activity is partially mediated by binding to syndecan 4 (S4), a proteoglycan receptor. Angiogenesis requires polarized activation of the small guanosine triphosphatase Rac1, which involves localized dissociation from RhoGDI1 and association with the plasma membrane. Previous work has shown that genetic deletion of S4 or its adapter, synectin, leads to depolarized Rac activation, decreased endothelial migration, and other physiological defects. In this study, we show that Rac1 activation downstream of S4 is mediated by the RhoG activation pathway. RhoG is maintained in an inactive state by RhoGDI1, which is found in a ternary complex with synectin and S4. Binding of S4 to synectin increases the latter''s binding to RhoGDI1, which in turn enhances RhoGDI1''s affinity for RhoG. S4 clustering activates PKCα, which phosphorylates RhoGDI1 at Ser⁹⁶. This phosphorylation triggers release of RhoG, leading to polarized activation of Rac1. Thus, FGF2-induced Rac1 activation depends on the suppression of RhoG by a previously uncharacterized ternary S4–synectin–RhoGDI1 protein complex and activation via PKCα.     

Protein Tyrosine Phosphatase-PEST and β8 Integrin Regulate Spatiotemporal Patterns of RhoGDI1 Activation in Migrating Cells

Directional cell motility is essential for normal development and physiology, although how motile cells spatiotemporally activate signaling events remains largely unknown. Here, we have characterized an adhesion and signaling unit comprised of protein tyrosine phosphatase (PTP)-PEST and the extracellular matrix (ECM) adhesion receptor β8 integrin that plays essential roles in directional cell motility. β8 integrin and PTP-PEST form protein complexes at the leading edge of migrating cells and balance patterns of Rac1 and Cdc42 signaling by controlling the subcellular localization and phosphorylation status of Rho GDP dissociation inhibitor 1 (RhoGDI1). Translocation of Src-phosphorylated RhoGDI1 to the cell''s leading edge promotes local activation of Rac1 and Cdc42, whereas dephosphorylation of RhoGDI1 by integrin-bound PTP-PEST promotes RhoGDI1 release from the membrane and sequestration of inactive Rac1/Cdc42 in the cytoplasm. Collectively, these data reveal a finely tuned regulatory mechanism for controlling signaling events at the leading edge of directionally migrating cells.     

Mapping the binding site for the GTP-binding protein Rac-1 on its inhibitor RhoGDI-1

BACKGROUND: Members of the Rho family of small GTP-binding proteins, such as Rho, Rac and Cdc42, have a role in a wide range of cell responses. These proteins function as molecular switches by virtue of a conformational change between the GTP-bound (active) and GDP-bound (inactive) forms. In addition, most members of the Rho and Rac subfamilies cycle between the cytosol and membrane. The cytosolic guanine nucleotide dissociation inhibitors, RhoGDIs, regulate both the GDP/GTP exchange cycle and the membrane association/dissociation cycle. RESULTS: We have used NMR spectroscopy and site-directed mutagenesis to identify the regions of human RhoGDI-1 that are involved in binding Rac-1. The results emphasise the importance of the flexible regions of both proteins in the interaction. At least one specific region (residues 46-57) of the flexible N-terminal domain of RhoGDI, which has a tendency to form an amphipathic helix in the free protein, makes a major contribution to the binding energy of the complex. In addition, the primary site of Rac-1 binding on the folded domain of RhoGDI involves the beta4-beta5 and beta6-beta7 loops, with a slight movement of the 3(10) helix accompanying the interaction. This binding site is on the same face of the protein as the binding site for the isoprenyl group of post-translationally modified Rac-1, but is distinct from this site. CONCLUSIONS: Isoprenylated Rac-1 appears to interact with three distinct sites on RhoGDI. The isoprenyl group attached to the C terminus of Rac-1 binds in a pocket in the folded domain of RhoGDI. This is distinct from the major site on this domain occupied by Rac-1 itself, which involves two loops at the opposite end to the isoprenyl-binding site. It is probable that the flexible C-terminal region of Rac-1 extends from the site at which Rac-1 contacts the folded domain of RhoGDI to allow the isoprenyl group to bind in the pocket at the other end of the RhoGDI molecule. Finally, the flexible N terminus of RhoGDI-1, and particularly residues 48-58, makes a specific interaction with Rac-1 which contributes substantially to the binding affinity.     

Disruption of RhoGDI and RhoA regulation by a Rac1 specificity switch mutant

Rho family GTPases are important regulators of the actin cytoskeleton. Activation of these proteins can be promoted by guanine nucleotide exchange factors containing Dbl and Pleckstrin homology domains resulting in membrane insertion of a Rho family member, whereas the inactive GDP-bound form is sequestered primarily in the cytoplasm, bound to the guanosine dissociation inhibitor RhoGDI. Dominant interfering variants of Rac1, but not Cdc42, inhibit beta1 integrin-promoted uptake of Yersinia pseudotuberculosis. Unexpectedly, we found that the Rac1(W56F) guanine nucleotide exchange factors specificity switch mutant blocked invasin-promoted uptake as well as Cdc42-dependent uptake of enteropathogenic Escherichia coli. Fluorescence resonance energy transfer experiments demonstrated that Rac1(W56F) retained the ability to be loaded with GTP, bind a downstream effector, and interact with RhoGDI. Mutational analyses of intragenic suppressors and coexpression studies demonstrated that binding of the Rac1(W56F) mutant to RhoGDI appeared to play a role in the inhibition of uptake. As RhoGDI inhibits RhoA, overactivation of RhoA may account for the uptake interference caused by Rac1(W56F). Consistent with this model, a dominant interfering form of RhoA restored significant uptake in the presence of the Rac1(W56F) mutant but had no effect on another interfering Rac1 form. Furthermore, the cellular GTP-RhoA level was elevated by the presence of Rac1(W56F) mutant protein. These data are consistent with the proposition that Rac1(W56F) blocks invasin-promoted uptake by preventing RhoGDI from inactivating RhoA. We conclude that RhoGDI allows cross-talk between Rho family members that promote potentially antagonistic processes, and disruption of this cross-talk can interfere with invasin-promoted uptake.     

Rho guanine dissociation inhibitors: pivotal molecules in cellular signalling.

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.     

Differential localization of Rho GTPases in live cells: regulation by hypervariable regions and RhoGDI binding

Determinants of membrane targeting of Rho proteins were investigated in live cells with green fluorescent fusion proteins expressed with or without Rho-guanine nucleotide dissociation inhibitor (GDI)alpha. The hypervariable region determined to which membrane compartment each protein was targeted. Targeting was regulated by binding to RhoGDI alpha in the case of RhoA, Rac1, Rac2, and Cdc42hs but not RhoB or TC10. Although RhoB localized to the plasma membrane (PM), Golgi, and motile peri-Golgi vesicles, TC10 localized to PMs and endosomes. Inhibition of palmitoylation mislocalized H-Ras, RhoB, and TC10 to the endoplasmic reticulum. Although overexpressed Cdc42hs and Rac2 were observed predominantly on endomembrane, Rac1 was predominantly at the PM. RhoA was cytosolic even when expressed at levels in vast excess of RhoGDI alpha. Oncogenic Dbl stimulated translocation of green fluorescent protein (GFP)-Rac1, GFP-Cdc42hs, and GFP-RhoA to lamellipodia. RhoGDI binding to GFP-Cdc42hs was not affected by substituting farnesylation for geranylgeranylation. A palmitoylation site inserted into RhoA blocked RhoGDI alpha binding. Mutations that render RhoA, Cdc42hs, or Rac1, either constitutively active or dominant negative abrogated binding to RhoGDI alpha and redirected expression to both PMs and internal membranes. Thus, despite the common essential feature of the CAAX (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) motif, the subcellular localizations of Rho GTPases, like their functions, are diverse and dynamic.