The coordinated cross-talk from heterotrimeric G proteins to Rho GTPases is essential during a variety of physiological processes. Emerging data suggest that members of the G␣ 12/13 and G␣ q/11 families of heterotrimeric G proteins signal downstream to RhoA via distinct pathways. Although studies have elucidated mechanisms governing G␣ 12/13 -mediated RhoA activation, proteins that functionally couple G␣ q/11 to RhoA activation have remained elusive. Recently, the Dbl-family guanine nucleotide exchange factor (GEF) p63RhoGEF/GEFT has been described as a novel mediator of G␣ q/11 signaling to RhoA based on its ability to synergize with G␣ q/11 resulting in enhanced RhoA signaling in cells. We have used biochemical/biophysical approaches with purified protein components to better understand the mechanism by which activated G␣ q directly engages and stimulates p63RhoGEF. Basally, p63RhoGEF is autoinhibited by the Dbl homology (DH)-associated pleckstrin homology (PH) domain; activated G␣ q relieves this autoinhibition by interacting with a highly conserved C-terminal extension of the PH domain. This unique extension is conserved in the related Dbl-family members Trio and Kalirin and we show that the C-terminal Rhospecific DH-PH cassette of Trio is similarly activated by G␣ q .Rho GTPases are integral regulators of gene transcription and actin cytoskeletal remodeling during many dynamic cellular processes (1, 2). Signal transduction cascades mediated by Rho GTPases originate via the extracellular stimulation of transmembrane receptors such as G protein-coupled receptors (GPCRs), 4 receptor tyrosine kinases, cytokine receptors, and integrins. Of the 22 human Rho family members, RhoA, Rac1, and Cdc42 are the most characterized, stemming from their ability to induce striking changes in cellular morphology upon activation (3). Numerous studies have established that RhoA activation downstream of GPCRs is vital for a multitude of diverse physiological responses including cell migration (4), lipid metabolism (5), vascular smooth muscle cell contraction (6 -8), and cell survival/apoptosis (9 -12). GPCR-mediated activation of RhoA effectively couples signaling pathways mediated by two distinct groups of guanine nucleotide-binding proteins: the heterotrimeric G␣-subunits and the monomeric small GTPases. These two groups of G proteins share a universal mechanism for guanine nucleotide binding, GTP hydrolysis, and conformational switching between two discrete states: a GDPbound inactive state and a GTP-bound active state (13). Guanine nucleotide exchange factors (GEFs) activate G proteins by promoting the release of bound GDP, allowing the subsequent binding of GTP. Active, GTP-bound G proteins can then interact with numerous downstream effector molecules, further propagating the signal initiated at the plasma membrane.GPCRs function as GEFs for heterotrimeric G␣-subunits, whereas Dbl-family GEFs are the major class of exchange factors for Rho GTPases. Dbl-family GEFs are defined by the presence of a Dbl homology domain (DH doma...

SmgGDS is an atypical guanine nucleotide exchange factor (GEF) that promotes both cell proliferation and migration and is up-regulated in several types of cancer. SmgGDS has been previously shown to activate a wide variety of small GTPases, including the Ras family members Rap1a, Rap1b, and K-Ras, as well as the Rho family members Cdc42, Rac1, Rac2, RhoA, and RhoB. In contrast, here we show that SmgGDS exclusively activates RhoA and RhoC among a large panel of purified GTPases. Consistent with the well known properties of GEFs, this activation is catalytic, and SmgGDS preferentially binds to nucleotide-depleted RhoA relative to either GDP-or GTP␥S-bound forms. However, mutational analyses indicate that SmgGDS utilizes a distinct exchange mechanism compared with canonical GEFs and in contrast to known GEFs requires RhoA to retain a polybasic region for activation. A homology model of SmgGDS highlights an electronegative surface patch and a highly conserved binding groove. Mutation of either area ablates the ability of SmgGDS to activate RhoA. Finally, the in vitro specificity of SmgGDS for RhoA and RhoC is retained in cells. Together, these results indicate that SmgGDS is a bona fide GEF that specifically activates RhoA and RhoC through a unique mechanism not used by other Rho family exchange factors.

Dbl family members are guanine nucleotide exchange factors specific for Rho guanosine triphosphatases (GTPases) and invariably possess tandem Dbl (DH) and pleckstrin homology (PH) domains. Dbs, a Dbl family member specific for Cdc42 and RhoA, exhibits transforming activity when overexpressed in NIH 3T3 mouse fibroblasts. In this study, the PH domain of Dbs was mutated to impair selectively either guanine nucleotide exchange or phosphoinositide binding in vitro and resulting physiological alterations were assessed. As anticipated, substitution of residues within the PH domain of Dbs integral to the interface with GTPases reduced nucleotide exchange and eliminated the ability of Dbs to transform NIH 3T3 cells. More interestingly, substitutions within the PH domain that prevent interaction with phosphoinositides yet do not alter in vitro activation of GTPases also do not transform NIH 3T3 cell and fail to activate RhoA in vivo despite proper subcellular localization. Therefore, the PH domain of Dbs serves multiple roles in the activation of GTPases and cannot be viewed as a simple membrane-anchoring device. In particular, the data suggest that binding of phosphoinositides to the PH domain within the context of membrane surfaces may direct orientations or conformations of the linked DH and PH domains to regulate GTPases activation.The Rho family GTPases 1 are an essential subset of the Ras superfamily of small molecular weight GTPases. Like Ras, Rho family GTPases cycle between GDP-and GTP-bound forms. When GDP-bound, Rho proteins are inactive and do not functionally couple to their downstream effectors. However, when GTP-bound, Rho GTPases elicit profound effects on the organization of the actin cytoskeleton, in addition to tightly regulating the activation state of transcription factors such as the serum response factor, c-Jun, and NF-B (1-3). GDP/GTP cycling within Rho proteins is primarily accomplished through the actions of two classes of regulatory proteins. GTPase activating proteins promote the inactive, GDP-bound form of Rho proteins by enhancing their intrinsic GTPase ability to convert bound GTP to GDP. In contrast, the actions of guanine nucleotide exchange factors (GEFs) upon Rho GTPases results in Rho activation by exchanging their bound GDP for GTP.Members of the Dbl family of oncoproteins act as GEFs exclusively for Rho GTPases (RhoGEFs) and, like constitutively activated members of Rho GTPases, can exhibit potent transformation potential within various cell types upon overexpression or constitutive activation (4 -6). Dbl family members invariably contain a Dbl homology (DH) domain in tandem with an adjacent, carboxyl-terminal pleckstrin homology (PH) domain. However, outside of this region, Dbl-related proteins share little sequence conservation and typically possess a variety of protein-signaling domains, presumably reflecting diversity in regulation and cellular function.The invariant positioning of PH domains immediately carboxyl-terminal to DH domains strongly implies a unique functional couplin...

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Guanine nucleotide exchange factors (GEFs) stimulate guanine nucleotide exchange and the subsequent activation of Rhofamily proteins in response to extracellular stimuli acting upon cytokine, tyrosine kinase, adhesion, integrin, and G-proteincoupled receptors (GPCRs). Upon Rho activation, several downstream events occur, such as morphological and cytoskeletal changes, motility, growth, survival, and gene transcription. The leukemia-associated RhoGEF (LARG) is a member of the regulators of G-protein signaling homology domain (RH) family of GEFs originally identified as a result of chromosomal translocation in acute myeloid leukemia. Using a novel fluorescence polarization guanine nucleotide-binding assay using BODIPY-Texas Red-GTPγS (BODIPY-TR-GTPγS), the authors performed a 10,000-compound high-throughput screen for inhibitors of LARG-stimulated RhoA nucleotide binding. Five compounds identified from the high-throughput screen were confirmed in a nonfluorescent radioactive guanine nucleotide-binding assay measuring LARG-stimulated [ 35 S] GTPγS binding. Therefore, these 5 compounds should serve as promising starting points for the development of small-molecule inhibitors of LARG-mediated nucleotide exchange as both pharmacological tools and therapeutics. In addition, the fluorescence polarization guanine nucleotide-binding assay described here should serve as a useful approach for both high-throughput screening and general biological applications.