RalGDS family members couple Ras to Ral signalling and that's not all

Ras proteins function as molecular switches that are activated in response to signalling pathways initiated by various extracellular stimuli and subsequently bind to numerous effector proteins leading to the activation of several signalling cascades within the cell. Ras and Ras-related proteins belong to a large superfamily of small GTPases characterized by significant sequence and function similarities. Several evidence indicate the existence of complex signalling networks that link Ras with its relatives in the family. A key role in this cross-talk is played by guanine nucleotide exchange factors (GEFs) that serve both as regulators and as effectors of Ras family proteins. The members of the RalGDS family, RalGDS, RGL, RGL2/Rlf and RGL3, can interact with activated Ras through their Ras Binding Domain (RBD), but may function as effectors for other Ras family members. They possess a REM-CDC25 homology region like RasGEFs, but specifically activate only RalA and RalB and not Ras or other Ras-related small GTPases. In this review we provide an update on this recently discovered family of GEFs, highlighting their crucial role in coupling activated Ras to activation of Ral, thus regulating several fundamental cell processes, and also discussing some evidence supporting Ras-independent additional functions of RalGDS proteins.     

The Rho Guanine Nucleotide Exchange Factors Intersectin 1L and β-Pix Control Calcium-Regulated Exocytosis in Neuroendocrine PC12 Cells

GTPases of the Rho family are molecular switches that play an important role in a wide range of membrane-trafficking processes including neurotransmission and hormone release. We have previously demonstrated that RhoA and Cdc42 regulate calcium-dependent exocytosis in chromaffin cells by controlling actin dynamics, whereas Rac1 regulates lipid organisation. These findings raised the question of the upstream mechanism activating these GTPases during exocytosis. The guanine nucleotide exchange factors (GEFs) that catalyse the exchange of GDP for GTP are crucial elements regulating Rho signalling. Using an RNA interference approach, we have recently demonstrated that the GEFs Intersectin-1L and β-Pix, play essential roles in neuroendocrine exocytosis by controlling the activity of Cdc42 and Rac1, respectively. This review summarizes these results and discusses the functional importance of Rho GEFs in the exocytotic machinery in neuroendocrine cells.     

The Rac1- and RhoG-specific GEF domain of Trio targets filamin to remodel cytoskeletal actin

Rho GTPases control actin reorganization and many other cellular functions. Guanine nucleotide-exchange factors (GEFs) activate Rho GTPases by promoting their exchange of GDP for GTP. Trio is a unique Rho GEF, because it has separate GEF domains, GEFD1 and GEFD2, that control the GTPases RhoG/Rac1 and RhoA, respectively. Dbl-homology (DH) domains that are common to GEFs catalyse nucleotide exchange, and pleckstrin-homology (PH) domains localize Rho GEFs near their downstream targets. Here we show that Trio GEFD1 interacts through its PH domain with the actin-filament-crosslinking protein filamin, and localizes with endogenous filamin in HeLa cells. Trio GEFD1 induces actin-based ruffling in filamin-expressing, but not filamin-deficient, cells and in cells transfected with a filamin construct that lacks the Trio-binding domain. In addition, Trio GEFD1 exchange activity is not affected by filamin binding. Our results indicate that filamin, as a molecular target of Trio, may be a scaffold for the spatial organization of Rho-GTPase-mediated signalling pathways.     

Rho-GTPase signaling drives melanoma cell plasticity

To investigate mechanisms that underlie different modes of tumor cell movement we have studied how regulation of the activity of the Rho family GTPases determines the mode of tumor cell movement. Guanine nucleotide exchange factors (GEFs) and GTPase accelerating proteins (GAPs) are key regulators of the activity of small GTPases with GEFs promoting activation to the GTP bound state and GAPs promoting inactivation by stimulating GTP hydrolysis. We identified two important signaling pathways regulating amoeboid and mesenchymal types of motility in melanoma. Here, we discuss our findings in the context of how specificity of Rho signaling is achieved by GEFs and GAPs.     

Rho GAPs and GEFs: Controling switches in endothelial cell adhesion

Within blood vessels, endothelial cell–cell and cell–matrix adhesions are crucial to preserve barrier function, and these adhesions are tightly controlled during vascular development, angiogenesis, and transendothelial migration of inflammatory cells. Endothelial cellular signaling that occurs via the family of Rho GTPases coordinates these cell adhesion structures through cytoskeletal remodelling. In turn, Rho GTPases are regulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). To understand how endothelial cells initiate changes in the activity of Rho GTPases, and thereby regulate cell adhesion, we will discuss the role of Rho GAPs and GEFs in vascular biology. Many potentially important Rho regulators have not been studied in detail in endothelial cells. We therefore will first overview which GAPs and GEFs are highly expressed in endothelium, based on comparative gene expression analysis of human endothelial cells compared with other tissue cell types. Subsequently, we discuss the relevance of Rho GAPs and GEFs for endothelial cell adhesion in vascular homeostasis and disease.     

Detection of Rho GEF and GAP activity through a sensitive split luciferase assay system

Rho GTPases regulate the assembly of cellular actin structures and are activated by GEFs (guanine-nucleotide-exchange factors) and rendered inactive by GAPs (GTPase-activating proteins). Using the Rho GTPases Cdc42, Rac1 and RhoA, and the GTPase-binding portions of the effector proteins p21-activated kinase and Rhophilin1, we have developed split luciferase assays for detecting both GEF and GAP regulation of these GTPases. The system relies on purifying split luciferase fusion proteins of the GTPases and effectors from bacteria, and our results show that the assays replicate GEF and GAP specificities at nanomolar concentrations for several previously characterized Rho family GEFs (Dbl, Vav2, Trio and Asef) and GAPs [p190, Cdc42 GAP and PTPL1-associated RhoGAP]. The assay detected activities associated with purified recombinant GEFs and GAPs, cell lysates expressing exogenous proteins, and immunoprecipitates of endogenous Vav1 and p190. The results demonstrate that the split luciferase system provides an effective sensitive alternative to radioactivity-based assays for detecting GTPase regulatory protein activities and is adaptable to a variety of assay conditions.     

Structural basis for the selective activation of Rho GTPases by Dbl exchange factors

Activation of Rho-family GTPases involves the removal of bound GDP and the subsequent loading of GTP, all catalyzed by guanine nucleotide exchange factors (GEFs) of the Dbl-family. Despite high sequence conservation among Rho GTPases, Dbl proteins possess a wide spectrum of discriminatory potentials for Rho-family members. To rationalize this specificity, we have determined crystal structures of the conserved, catalytic fragments (Dbl and pleckstrin homology domains) of the exchange factors intersectin and Dbs in complex with their cognate GTPases, Cdc42 and RhoA, respectively. Structure-based mutagenesis of intersectin and Dbs reveals the key determinants responsible for promoting exchange activity in Cdc42, Rac1 and RhoA. These findings provide critical insight into the structural features necessary for the proper pairing of Dbl-exchange factors with Rho GTPases and now allow for the detailed manipulation of signaling pathways mediated by these oncoproteins in vivo.     

Vav GEFs regulate macrophage morphology and adhesion-induced Rac and Rho activation

The Vav family of proteins have the potential to act as both signalling adapters and GEFs for Rho GTPases. They have therefore been proposed as regulators of the cytoskeleton in various cell types. We have used macrophages from mice deficient in all three Vav isoforms to determine how their function affects cell morphology and migration. Macrophages lacking Vav proteins adopt an elongated morphology and have enhanced migratory persistence in culture. To investigate the pathways through which Vav proteins exert their effects we analysed the responses of macrophages to the chemoattractant CSF-1 and to adhesion. We found that morphological and signalling responses of macrophages to CSF-1 did not require Vav proteins. In contrast, adhesion-induced cell spreading, RhoA and Rac1 activation and cell signalling were all dependent on Vav proteins. We propose that Vav proteins affect macrophage morphology and motile behaviour by coupling adhesion receptors to Rac1 and RhoA activity and regulating adhesion signalling events such as paxillin and ERK1/2 phosphorylation by acting as adapters.     

Rho GAPs and GEFs

Within blood vessels, endothelial cell–cell and cell–matrix adhesions are crucial to preserve barrier function, and these adhesions are tightly controlled during vascular development, angiogenesis, and transendothelial migration of inflammatory cells. Endothelial cellular signaling that occurs via the family of Rho GTPases coordinates these cell adhesion structures through cytoskeletal remodelling. In turn, Rho GTPases are regulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). To understand how endothelial cells initiate changes in the activity of Rho GTPases, and thereby regulate cell adhesion, we will discuss the role of Rho GAPs and GEFs in vascular biology. Many potentially important Rho regulators have not been studied in detail in endothelial cells. We therefore will first overview which GAPs and GEFs are highly expressed in endothelium, based on comparative gene expression analysis of human endothelial cells compared with other tissue cell types. Subsequently, we discuss the relevance of Rho GAPs and GEFs for endothelial cell adhesion in vascular homeostasis and disease.     

Myosin II directly binds and inhibits Dbl family guanine nucleotide exchange factors: a possible link to Rho family GTPases

Cell migration requires the coordinated spatiotemporal regulation of actomyosin contraction and cell protrusion/adhesion. Nonmuscle myosin II (MII) controls Rac1 and Cdc42 activation, and cell protrusion and focal complex formation in migrating cells. However, these mechanisms are poorly understood. Here, we show that MII interacts specifically with multiple Dbl family guanine nucleotide exchange factors (GEFs). Binding is mediated by the conserved tandem Dbl homology–pleckstrin homology module, the catalytic site of these GEFs, with dissociation constants of ∼0.3 µM. Binding to the GEFs required assembly of the MII into filaments and actin-stimulated ATPase activity. Binding of MII suppressed GEF activity. Accordingly, inhibition of MII ATPase activity caused release of GEFs and activation of Rho GTPases. Depletion of βPIX GEF in migrating NIH3T3 fibroblasts suppressed lamellipodial protrusions and focal complex formation induced by MII inhibition. The results elucidate a functional link between MII and Rac1/Cdc42 GTPases, which may regulate protrusion/adhesion dynamics in migrating cells.     

GEF what? Dock180 and related proteins help Rac to polarize cells in new ways

Rho GTPase activation, which is mediated by guanine nucleotide exchange factors (GEFs), is tightly regulated in time and space. Although Rho GTPases have a significant role in many biological events, they are best known for their ability to restructure the actin cytoskeleton profoundly through the activation of specific downstream effectors. Two distinct families of GEFs for Rho GTPases have been reported so far, based on the features of their catalytic domains: firstly, the classical GEFs, which contain a Dbl homology-pleckstrin homology domain module with GEF activity, and secondly, the Dock180-related GEFs, which contain a Dock homology region-2 domain that catalyzes guanine nucleotide exchange on Rho GTPases. Recent exciting data suggest key roles for the DHR-2 domain-containing GEFs in a wide variety of fundamentally important biological functions, including cell migration, phagocytosis of apoptotic cells, myoblast fusion and neuronal polarization.     

VAV proteins as signal integrators for multi-subunit immune-recognition receptors

In recent years, substantial progress has been made towards the identification of intracellular signalling molecules that couple multi-subunit immune-recognition receptors (MIRRs) to their various effector functions. Among these, the VAV proteins have been observed to have a crucial role in regulating some of the earliest events in receptor signalling. VAV proteins function, in part, as guanine-nucleotide exchange factors (GEFs) for the RHO/RAC family of GTPases. This review focuses on the role of VAV proteins in the regulation of lymphocyte development and function, and emphasizes the regulatory roles that these proteins have through both GEF-dependent and -independent mechanisms.     

RasGRF suppresses Cdc42-mediated tumour cell movement, cytoskeletal dynamics and transformation

Individual tumour cells move in three-dimensional environments with either a rounded or an elongated 'mesenchymal' morphology. These two modes of movement are tightly regulated by Rho family GTPases: elongated movement requires activation of Rac1, whereas rounded/amoeboid movement engages specific Cdc42 and Rho signalling pathways. In siRNA screens targeting the genes encoding guanine nucleotide exchange factors (GEFs), we found that the Ras GEF RasGRF2 regulates conversion between elongated- and rounded-type movement. RasGRF2 suppresses rounded movement by inhibiting the activation of Cdc42 independently of its capacity to activate Ras. RasGRF2 and RasGRF1 directly bind to Cdc42, outcompeting Cdc42 GEFs, thereby preventing Cdc42 activation. By this mechanism, RasGRFs regulate other Cdc42-mediated cellular processes such as the formation of actin spikes, transformation and invasion in vitro and in vivo. These results demonstrate a role for RasGRF GEFs as negative regulators of Cdc42 activation.     

Myoblasts and macrophages share molecular components that contribute to cell-cell fusion

Cell-cell fusion is critical to the normal development of certain tissues, yet the nature and degree of conservation of the underlying molecular components remains largely unknown. Here we show that the two guanine-nucleotide exchange factors Brag2 and Dock180 have evolutionarily conserved functions in the fusion of mammalian myoblasts. Their effects on muscle cell formation are distinct and are a result of the activation of the GTPases ARF6 and Rac, respectively. Inhibition of ARF6 activity results in a lack of physical association between paxillin and beta(1)-integrin, and disruption of paxillin transport to sites of focal adhesion. We show that fusion machinery is conserved among distinct cell types because Dock180 deficiency prevented fusion of macrophages and the formation of multinucleated giant cells. Our results are the first to demonstrate a role for a single protein in the fusion of two different cell types, and provide novel mechanistic insight into the function of GEFs in the morphological maturation of multinucleated cells.     

Regulation of small GTPases at epithelial cell-cell junctions

Small GTPases of the Rho family (RhoA, Rac1, and Cdc42) and the Ras family GTPase Rap1 are essential for the assembly and function of epithelial cell-cell junctions. Through their downstream effectors, small GTPases modulate junction formation and stability, primarily by orchestrating the polymerization and contractility of the actomyosin cytoskeleton. The major upstream regulators of small GTPases are guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). Several GEFs and a few GAPs have been localized at epithelial junctions, and bind to specific junctional proteins. Thus, junctional proteins can regulate small GTPases at junctions, through their interactions with GEFs and GAPs. Here we review the current knowledge about the mechanisms of regulation of small GTPases by junctional proteins. Understanding these mechanisms will help to clarify at the molecular level how small GTPases control the morphogenesis and physiology of epithelial tissues, and how they are disregulated in disease.     

Structure of Shigella IpgB2 in Complex with Human RhoA: IMPLICATIONS FOR THE MECHANISM OF BACTERIAL GUANINE NUCLEOTIDE EXCHANGE FACTOR MIMICRY*

A common theme in bacterial pathogenesis is the manipulation of eukaryotic cells by targeting the cytoskeleton. This is in most cases achieved either by modifying actin, or indirectly via activation of key regulators controlling actin dynamics such as Rho-GTPases. A novel group of bacterial virulence factors termed the WXXXE family has emerged as guanine nucleotide exchange factors (GEFs) for these GTPases. The precise mechanism of nucleotide exchange, however, has remained unclear. Here we report the structure of the WXXXE-protein IpgB2 from Shigella flexneri and its complex with human RhoA. We unambiguously identify IpgB2 as a bacterial RhoA-GEF and dissect the molecular mechanism of GDP release, an essential prerequisite for GTP binding. Our observations uncover that IpgB2 induces conformational changes on RhoA mimicking DbI- but not DOCK family GEFs. We also show that dissociation of the GDP·Mg²⁺ complex is preceded by the displacement of the metal ion to the α-phosphate of the nucleotide, diminishing its affinity to the GTPase. These data refine our understanding of the mode of action not only of WXXXE GEFs but also of mammalian GEFs of the DH/PH family.     

Small-GTPase-Associated Signaling by the Guanine Nucleotide Exchange Factors CpDock180 and CpCdc24, the GTPase Effector CpSte20, and the Scaffold Protein CpBem1 in Claviceps purpurea

Monomeric GTPases of the Rho subfamily are important mediators of polar growth and NADPH (Nox) signaling in a variety of organisms. These pathways influence the ability of Claviceps purpurea to infect host plants. GTPase regulators contribute to the nucleotide loading cycle that is essential for proper functionality of the GTPases. Scaffold proteins gather GTPase complexes to facilitate proper function. The guanine nucleotide exchange factors (GEFs) CpCdc24 and CpDock180 activate GTPase signaling by triggering nucleotide exchange of the GTPases. Here we show that CpCdc24 harbors nucleotide exchange activity for both Rac and Cdc42 homologues. The GEFs partly share the cellular distribution of the GTPases and interact with the putative upstream GTPase CpRas1. Interaction studies show the formation of higher-order protein complexes, mediated by the scaffold protein CpBem1. Besides the GTPases and GEFs, these complexes also contain the GTPase effectors CpSte20 and CpCla4, as well as the regulatory protein CpNoxR. Functional characterizations suggest a role of CpCdc24 mainly in polarity, whereas CpDock180 is involved in stress tolerance mechanisms. These findings indicate the dynamic formation of small GTPase complexes and improve the model for GTPase-associated signaling in C. purpurea.     

GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors

Guanine nucleotide-exchange factors (GEFs) are directly responsible for the activation of Rho-family GTPases in response to diverse extracellular stimuli, and ultimately regulate numerous cellular responses such as proliferation, differentiation and movement. With 69 distinct homologues, Dbl-related GEFs represent the largest family of direct activators of Rho GTPases in humans, and they activate Rho GTPases within particular spatio-temporal contexts. The failure to do so can have significant consequences and is reflected in the aberrant function of Dbl-family GEFs in some human diseases.