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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 Gα12/13 and Gαq/11 families of heterotrimeric G proteins signal downstream to RhoA via distinct pathways. Although studies have elucidated mechanisms governing Gα12/13-mediated RhoA activation, proteins that functionally couple Gαq/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 Gαq/11 signaling to RhoA based on its ability to synergize with Gαq/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 Gαq directly engages and stimulates p63RhoGEF. Basally, p63RhoGEF is autoinhibited by the Dbl homology (DH)-associated pleckstrin homology (PH) domain; activated Gαq 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 Rho-specific DH-PH cassette of Trio is similarly activated by Gαq.
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 Gα-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 GDP-bound 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 Gα-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 domain), which is almost invariantly followed by a pleckstrin homology domain (PH domain) (14). The catalytic guanine nucleotide exchange activity resides entirely within the DH domain, although recent evidence indicates that the PH domain can function to fine-tune this exchange activity (15, 16). Previous studies have focused on the DH-associated PH domain as a simple membrane targeting device, by virtue of its ability to bind phosphoinositides. However, emerging evidence suggests that PH domains may also play important regulatory roles by serving as protein-protein interaction modules (17).
The coordinated cross-talk from GPCR stimulation to RhoA activation is mediated by Dbl-family GEFs that are responsive to activated Gα-subunits. A growing body of literature implicates both Gα12/13 and Gαq/11 family members as upstream activators of RhoA (18–20). Moreover, members of theGα12/13 and Gαq/11 families utilize distinct pathways to signal downstream to RhoA (21). RhoA activation downstream of the Gα12/13 family is mediated by the p115 family members, which consists of p115-RhoGEF, PDZ-RhoGEF, and leukemia-associated RhoGEF. The p115 family members are directly activated by Gα12/13 via a protein-protein interaction mediated by a highly divergent regulator of G protein signaling (RGS) domain, but are not activated by Gαq/11 family members (22–25). Gαq/11-coupled GPCRs can signal downstream to RhoA via a pathway distinct from Gα12/13 and independent of the classically described Gαq/11 effector phospholipase C-β (21, 26–29). However, whereas numerous studies have elucidated mechanisms underlying Gα12/13-mediated RhoA activation, the signaling pathways that couple Gαq/11 to RhoA activation have remained elusive.
Recently, the Dbl-family member p63RhoGEF/GEFT has been described as a novel mediator of Gαq/11 signaling to RhoA based on its ability to synergize with Gαq/11 resulting in enhanced RhoA signaling (30). Using cell model systems, the authors clearly demonstrate thatGαq/11-coupled GPCR activation or overexpression of activated mutants of Gαq/11 enhance the ability for overexpressed p63RhoGEF to activate serum response factor-dependent gene reporters. Furthermore, using co-immunoprecipitation studies, the authors deduced that activated Gαq/11 associates with the C-terminal half of p63RhoGEF, which contains the PH domain. However, the mechanistic aspects underlying Gαq/11-mediated p63RhoGEF activation remain unclear. In particular, the previous co-immunoprecipitation studies do not rule out the indirect association ofGαq/11 with p63RhoGEF through ancillary proteins. Furthermore, it is necessary to determine whether Gαq/11 can directly modulate the guanine nucleotide exchange activity of p63RhoGEF using a defined in vitro system. Here we use biochemical/ biophysical approaches with highly purified protein components to show that p63RhoGEF directly and specifically associates with activated Gαq to enhance robustly the catalyzed guanine nucleotide exchange of RhoA, RhoB, and RhoC. Therefore, p63RhoGEF is a bona fide effector of Gαq. Furthermore, these studies strongly implicate p63RhoGEF, together with the related Dbl-family members, Trio and Kalirin, as a major nexus for the activation of RhoA downstream of Gαq/11.
Truncation mutant constructs of human p63RhoGEF were PCR amplified from full-length human p63RhoGEF (GenBank accession number BC012860, kindly provided by T. Wieland) resulting in the following constructs: DH-Ct (residues 155–580), DH-Ext (residues 155– 493), DH-PH (residues 155–472), and DH (residues 155–347). PCR products were then subcloned into a modified pET-21a vector (Novagen) using a previously published ligation-independent cloning strategy (31). The bacterial expression vector, pLiC-His-TEV, which encodes an N-terminal His6 tag followed by a tobacco etch virus (TEV) cleavage site, was used to generate vectors for the DH-Ct, DH-Ext, and DH-PH His6-tagged p63RhoGEF constructs. The p63RhoGEF DH construct was cloned into a His6-tagged, TEV-cleavable, maltose-binding protein (MBP) fusion vector (pLiC-His-MBP-TEV) for improved expression and solubility. N-terminal glutathione S-transferase (GST)-tagged constructs for p63RhoGEF DH-Ext and DH-PH were cloned into a GST fusion vector using a similar strategy. Point mutant constructs of p63RhoGEF (F471A, L472A, N473A, L474A, Q476A, S477A, P478A, I479A, E480A, Y481A, Q482A, R483A) were generated in the context of the His6-tagged DH-Ext using the Quik Change site-directed mutagenesis kit (Stratagene) followed by automated sequencing to confirm each mutation. The coding region for the C-terminal DH-Ext region of Trio (Trio-C DH-Ext, residues 1291–2299) was PCR amplified from full-length human Trio (Gen Bank accession number NM_007118, kindly provided by M. Strueli) and introduced into the pLiC-His-TEV bacterial expression vector as above. Baculovirus for the new Gαi/q chimera was constructed with the N-terminal His6 tag followed by the N-terminal sequence ofGαi1 (1–28), TEV cleavage site, and Gαq sequence starting at Ala8. Baculovirus for the Gα13 chimera was described in Ref. 32.
All p63RhoGEF and Trio-C recombinant protein expression constructs were expressed in the BL21 (DE3) Escherichia coli strain. Cells were grown up at 37 °C in LB media containing 0.1 mg/ml ampicillin until an A600 of ~0.6, then induced with 0.1 mm isopropyl β-d-thiogalactopyranoside and grown up at 18 °C for ~18 h. Cells containing His6-tagged proteins were harvested and soluble recombinant proteins were purified using standard Ni2+-affinity chromatography followed by size-exclusion chromatography. Prior to size-exclusion chromatography, some His6-tagged proteins were treated with TEV to remove the His6 tag. Additionally, treatment with TEV allowed for removal of the N-terminal MBP fusion of the p63RhoGEFDHconstruct. E. coli cells containing GST fusion p63RhoGEF proteins (GST-DH-Ext, GST-DH-PH) were also harvested and recombinant proteins purified using standard glutathione-affinity chromatography followed by size-exclusion chromatography. Chimeric fusion constructs of the heterotrimeric G proteins Gαq andGα13 were purified using a baculovirus-based expression system (Invitrogen) in High-5 insect cells based on methods previously described (32, 33). Purified protein samples for the heterotrimeric G proteins Gαi, Gαo, Gαt, and Gαs were generously provided by C. Johnston and D. Siderovski (34, 35). Heterotrimeric Gα-subunits were confirmed active using several independent methods including AlF4-dependent binding to effectors proteins. Additionally, Dbs DH-PH (residues 623–967), Tiam1 DH-PH (residues 1022–1406), Rac1 (residues 1–189 C189S), Cdc42 (residues 1–189 C189S), RhoA (residues 1–190 C190S), RhoB (residues 1–190, C190S), and RhoC (residues 1–191, C191S) were expressed in E. coli and purified essentially as previously described (15, 36, 37). Size-exclusion chromatography was used for all recombinant protein preparations to ensure samples eluted as monodispersed species of correct molecular weight. All recombinant protein concentrations were determined using the A280 method with extinction coefficients calculated using the ProtParam tool (ExPASy Molecular Biology Server (38)), analyzed by SDS-PAGE to confirm concentration and ensure purity, and subsequently stored at −80 °C.
The guanine nucleotide exchange activity of purified RhoGEFs was determined using a kinetic, fluorescence-based assay with Rho GTPases (RhoA, RhoB, RhoC, Rac1, Cdc42) that were preloaded with BODIPY FL-conjugated GDP (BODIPY-GDP, Molecular Probes) essentially as previously described (39). All exchange assays were performed using an LS-55 fluorescence spectrometer (PerkinElmer) with wavelengths set at λex = 500 nm (slits = 5 nm),λem = 511 nm (slits = 5 nm), and quartz cuvettes thermostatted at 20 °C while constantly stirred. Reactions were carried out in exchange buffer consisting of 20 mm Tris, pH 7.5, 200mm NaCl, 10mm MgCl2, 5% (v/v) glycerol, and 10µm GDP. For each exchange assay, BODIPY-GDP-preloaded Rho GTPases (200 nm) were allowed to equilibrate in exchange buffer. Then 30µm AlF4 (30µm AlCl3+10mm NaF) and/or heterotrimeric G proteins at the indicated concentrations were added. The presence of AlF4 had no impact on the spontaneous exchange rate of Rho GTPases and was used to selectively activate heterotrimeric G proteins. Finally, the guanine nucleotide exchange reaction was initiated by the manual addition of the RhoGEF at the indicated concentrations and the exchange reaction was monitored in real time until completion. The observed exchange rates (kobs) were then calculated for each condition by fitting the change in relative fluorescence intensity over time for a given condition to a single-phase exponential decay using Prizm data analysis software (GraphPad). Exchange data depicted in bar graphs are the mean ± S.D. for each condition, conducted in triplicate. Representative real time kinetic exchange data depicted in curves are normalized as follows: relative fluorescence units prior to addition of RhoGEF (100% BODIPY-GDP bound) and relative fluorescence units at reaction completion (0% BODIPY-GDP bound).
All surface plasmon resonance (SPR) studies were performed using a Biacore 3000 instrument (GE Healthcare). An anti-GST antibody was covalently coupled to a CM5 Biacore chip per the manufacturer’s protocol. Binding studies were performed in SPR buffer consisting of 20 mm HEPES, pH 7.5, 150 mm NaCl, 10 mm MgCl2, 0.05% (v/v) Nonidet P-40, 100 µm GDP, and 30 µm AlF4 (30 µm AlCl3 + 10 mm NaF). GST fusion binding surfaces were subsequently generated for individual flow cells by the application of GST only, or the GST-tagged p63RhoGEF constructs GST-DHExt and GST-DH-PH. To generate SPR-based binding isotherms, an analyte consisting of 10 µm Gαq in AlF4-containing SPR buffer was flowed over each surface; background binding to the GST only surface was subsequently subtracted from each condition and the corresponding relative units were plotted.
Apeptide spanning the conserved PH domain extension of p63RhoGEF was synthesized and high pressure liquid chromatography purified by the Tufts University peptide core facility. This peptide consisted of an N-terminal fluorescein moiety followed by a β-alanine linker and residues 467–493 of human p63RhoGEF followed by a C-terminal amide group. Gαq or Gαi were added at varying concentrations to a 96-well plate containing 5 nm peptide in buffer consisting of 20 mm Tris, pH 7.5, 200 mm NaCl, 20 mm MgCl2, 0.05% (v/v) Nonidet P-40, 30 µm GDP, and 30 µm AlF4 (30 AlCl3+10mm NaF) with a total volume of 200µl. Each condition was allowed to equilibrate at 25 °C for ~15 min before polarization was determined using a PHERAstar fluorescence microplate reader (BMG Labtech) using the polarization mode. The excitation laser (λex = 485 nm) was vertically polarized and the subsequent fluorescence emission intensity (λem= 520 nm) was observed through a polarizer orientated parallel or perpendicular to the excitation vector. Polarization (P) was then calculated using the formula: p=(I‖−I)/(I‖+I), where I‖is the intensity of the parallel component and I is the intensity of the perpendicular component of the emitted light (40). Peptide in the absence of heterotrimeric G protein was used to adjust the gain prior to data collection.
Unlike the majority of the 69 human Dbl-family GEFs, p63RhoGEF lacks any additional signaling domains outside of the canonical DH-PH cassette that defines this family. To identify conserved regions that may impart signaling properties or suggest modes of regulation for p63RhoGEF, we generated a multiple sequence alignment using Clustal-X (41) for eight representative p63RhoGEF orthologs and projected the sequence conservation for each residue onto the predicted domain architecture (Fig. 1A). The three-dimensional structure of PH domains is well characterized and takes on a β-sandwich fold capped on one side by a C-terminal α-helix, termed αC (17). Interestingly, the predicted αC helix of p63RhoGEF has a highly conserved extension, which is predicted to be unstructured and is not considered an integral part of the PH domain based on sequence analysis (Fig. 1A). The strict conservation of this region and its proximity to the PH domain led us to hypothesize that this unique extension may be essential for regulating the exchange activity of p63RhoGEF. Based on these sequence analysis studies, we generated several p63RhoGEF truncation mutant constructs (Fig. 1A) and purified recombinant protein components to near homogeneity for use in our subsequent biochemical/biophysical analyses (Fig. 1B).
We tested p63RhoGEF truncation mutants for their ability to promote guanine nucleotide exchange using RhoA as a substrate GTPase to investigate the mechanism of autoregulation. The exchange activities of p63RhoGEF constructs encompassing the DH-Ct, DH-Ext, and DH-PH were similarly activating toward RhoA, yielding an ~2–3-fold increase in the exchange rate over the spontaneous exchange rate of RhoA alone (Fig. 2). Full-length p63RhoGEF was similar in its activation of RhoA (data not shown). These results rule out possible regulation by inhibitory sequences, which have been well characterized for Vav and more recently, Tim-family RhoGEFs (42). However, under identical conditions, the DH construct was ~22-fold more active than the spontaneous exchange rate of RhoA alone (Fig. 2), implicating the PH domain as a negative regulator of p63RhoGEF exchange activity. To generate a soluble p63RhoGEFDHfragment, we used a TEV-cleavable MBP fusion at the N terminus; both MBP fusion and TEV-treated DH constructs retained similar activity toward RhoA. Whereas there is conflicting literature regarding the regulatory role of the PH domain of p63RhoGEF, our results are in accordance with previous studies that suggest an autoinhibitory role (43, 44).
Whereas Lutz and colleagues (30) elegantly demonstrated that Gαq/11 synergizes with p63RhoGEF to activate RhoA signaling pathways in cells, the authors did not explore the underlying mechanism. Therefore, we used previously published methods (33) to generate soluble recombinant Gαq protein to test the hypothesis that Gαq directly stimulates p63RhoGEF activity. The new Gαi/q chimera contains the N-terminal α-helix of Gαi1 followed by a TEV cleavage site fused to the N terminus of Gαq-(8–359); treatment with TEV-generated recombinant soluble Gαq protein with amino acid sequence from Ala8 to the end and was purified with high purity (Fig. 1B). In contrast to the previous Gαq chimera (33), this new chimera demonstrated phospholipase C-β stimulating activity in vitro.5 Subsequently, we established that purified Gαq directly stimulates the exchange activity of autoinhibited p63RhoGEF (Fig. 3, A and B). The exchange activity of each p63RhoGEF truncation mutant construct in the presence of inactive GDP-bound Gαq or AlF4 alone was comparable with the control exchange rate, comprising a 2–3-fold activation over the spontaneous exchange rate of RhoA alone. However, the DH-Ct and DH-Ext constructs of p63RhoGEF were robustly stimulated by AlF4-activated Gαq by~26-fold over the spontaneous exchange rate of RhoA alone. Additionally, full-length p63RhoGEF was similarly activated by AlF4-activated Gαq; however, the purity of the full-length construct was diminished due to N-terminal degradation (data not shown). Interestingly, the DH-PH construct lacking the conserved extension of the PH domain was not stimulated by AlF4-activated Gαq. This lack of Gαq-mediated stimulation of the DH-PH fragment was not simply due to misfolding as the basal activity closely resembled that of the DH-Ct and DH-Ext constructs. Additionally, the DH construct lacking the autoinhibitory PH domain was not further stimulated by addition of AlF4-activated Gαq (Fig. 3C). Most likely, the DH construct represents constitutively active p63RhoGEF. The exchange rates catalyzed by p63RhoGEF (DH-Ext) in the presence of increasing amount of AlF4-activated Gαq (Fig. 4A), were used to generate a dose-response curve that yielded an EC50 of ~951 nm for the activation of p63RhoGEF by AlF4-activated Gαq (Fig. 4B). Based on these results, the DH-Ext construct comprises the minimal region of p63RhoGEF that is both basally autoinhibited and activated by AlF4-activated Gαq; subsequent experiments utilized this DH-Ext construct.
Next, we identified key residues within p63RhoGEF essential for Gαq-mediated activation using site-directed mutagenesis of the conserved PH domain extension (residues 466–483, alanine 474 was not mutated). In particular, alanine substitutions in the context of DH-Ext at Phe471, Leu472, Leu475, Pro478, and Ile479 substantially diminished the capacity for AlF4-activated Gαq to stimulate the exchange activity of p63RhoGEF compared with wild-type (Fig. 5A). The basal exchange activity of these point mutants (i.e. in the absence of activated Gαq) was comparable with the basal exchange activity of wild-type p63RhoGEF and proteins eluted as mono-dispersed species of correct molecular weight when analyzed by size-exclusion chromatography and SDS-PAGE (Fig. 5C), indicating proper folding and protein integrity. Interestingly, mutations most deleterious to Gαq-mediated stimulation of p63RhoGEF display helical periodicity and appear to encompass the single face of an α-helix when analyzed using bioinformatics-based methods (ExPASy Molecular Biology Server (38)) (Fig. 5B). This was an unexpected finding as this region is predicted to be unstructured based on secondary structure prediction methods. We hypothesize that this normally disordered extension undergoes a conformational change to an α-helix upon binding Gαq.
We then performed binding assays to determine whether this conserved extension was important for directly engaging activated Gαq or merely contributed to allosteric activation of the DH domain. We used both analytical size-exclusion chromatography (Fig. 6A) as well as SPR analysis (Fig. 6B) to show that the p63RhoGEF DH-Ext binds AlF4-activated Gαq with a high affinity, whereas the DH-PH construct lacking the extension motif does not interact with activated Gαq. Further analysis of the SPR-generated binding isotherms indicate that the DH-Ext construct bound activated Gαq with an association rate constant of 0.12 and a dissociation rate constant of 0.039. Whereas these results indicate that the C-terminal extension was necessary for binding activated Gαq, they do not address whether it was sufficient for binding. Therefore we generated a peptide corresponding to the C-terminal extension of p63RhoGEF (residues 467– 493) and showed that this peptide was sufficient to bind AlF4-activated Gαq using a polarization/anisotropy-based binding assay, whereas AlF4-activated Gαi did not bind this peptide (Fig. 6C). However, the relatively low affinity for this interaction suggests to us that additional regions outside of this minimal peptide are required for full engagement of activated Gαq by p63RhoGEF.
To determine the full spectrum of heterotrimeric G proteins specific for p63RhoGEF, we tested a panel of highly purified recombinant Gα-subunits for their ability to directly stimulate the exchange activity of p63RhoGEF. As expected, the exchange activity of p63RhoGEF (DH-Ext) was not affected by the addition of high concentrations of AlF4-activated Gαi, Gαo, Gαs, Gαt, and Gα13 (Fig. 7A). The resulting exchange rates were comparable with that of the control exchange rate in the absence of heterotrimeric G proteins, comprising a 2–3-fold activation over the spontaneous exchange rate of RhoA alone. Only Gαq robustly stimulated the guanine nucleotide exchange activity in an AlF4-dependent manner. Secondary studies, including AlF4-dependent binding of effector proteins, were used to confirm the activity of each heterotrimeric G protein used (34, 35); the purity and concentration of each heterotrimeric G protein used was additionally confirmed using SDS-PAGE analysis (Fig. 7B). Currently, we are developing baculoviral expression constructs to probe additional Gαq family members (e.g. Gα11 and Gα14) for activation of p63RhoGEF.
The substrate Rho GTPase specificity for p63RhoGEF has not been well characterized. Therefore, we investigated the substrate Rho GTPase specificities for Gαq-activated p63RhoGEF. High concentrations of AlF4-activated Gαq in combination with p63RhoGEF (DH-Ext) did not promote guanine nucleotide exchange on BODIPY-GDP-preloaded Rac1 (Fig. 7C) or Cdc42 (Fig. 7D). The activity of both Rac1 and Cdc42 were confirmed using the RhoGEFs Tiam1 and Dbs, respectively (Fig. 7, C and D). Additionally, we demonstrated that Gαq-activated p63RhoGEF catalyzes guanine nucleotide exchange on Rho isozymes RhoB and RhoC (Fig. 7E). This activation of RhoB and RhoC by p63RhoGEF was comparable with that observed with RhoA, implicating p63RhoGEF as a Rho isoform-specific exchange factor.
Because there is a large precedent for RhoA signaling downstream of Gαq we hypothesized additional Dbl-family members related to p63RhoGEF may also be directly by activated Gαq. Therefore, we used the basic local alignment search tool (45) to identify additional proteins homologous to p63RhoGEF that may also interact directly with activated Gαq. We identified the Dbl-family proteins Trio and Kalirin as the closest paralogs to p63RhoGEF. More importantly, Trio and Kalirin were the only other proteins that contain the highly conserved C-terminal extension of the PH domain (residues 471– 483), which is required for direct engagement of activated Gαq by p63RhoGEF. Trio and Kalirin are unique in that they are the only Dbl-family members that contain two independent DH-PH cassettes (14). The N-terminal DH-PH cassette is Rac1/RhoG-specific, whereas the C-terminal DH-PH cassette is RhoA-specific (46, 47). Only the C-terminal RhoA-specific and not the N-terminal Rac1/RhoG-specific DH-PH cassette of Trio and Kalirin bear significant homology to p63RhoGEF (Fig. 8A). Interestingly, residues within the PH domain extension that were essential for p63RhoGEF activation by Gαq (Phe471, Leu472, Leu475, Pro478, and Ile479) are 100% conserved in Trio and Kalirin. We subsequently determined that AlF4-activated Gαq can directly stimulate the RhoA-specific guanine nucleotide exchange activity of the C-terminal DH-PH cassette of Trio (Trio-C DH-Ext) by ~2-fold over inactive GDP-bound Gαq or AlF4 alone (Fig. 8, B and C). This stimulation by Gαq was not nearly as robust as that seen for p63RhoGEF, suggesting that other mechanisms may facilitate the interaction of activated Gαq with Trio. Alternatively, Trio-C DH-Ext possesses a higher capacity to catalyze guanine nucleotide exchange upon RhoA relative to the equivalent fragment of p63RhoGEF. Therefore, AlF4-activated Gαq is stimulating a form of Trio that is already highly exchange-competent such that the measured enhancement by Gαq belies the full potential of Gαq to activate full-length and, presumably, fully autoinhibited Trio.
There is considerable evidence suggesting that Gα12/13 and Gαq/11 family members independently activate RhoA signaling in response to extracellular stimuli (18, 19, 21, 26–29). Whereas the Gαq/11-specific pathway has remained poorly understood, numerous studies indicate that Gα12/13 engage the RGS domain of the p115 family members and directly stimulate their RhoA-specific exchange activity (23). The p115 family RhoGEFs are the only Dbl-family members that contain an RGS domain; however, previous efforts to implicate p115 family members as Gαq/11-responsive RhoGEFs have been largely unsuccessful. 6 The recent finding that Gαq/11 synergizes with p63RhoGEF to promote Rho signaling in cells was significant given that Gαq-responsive RhoGEFs have remained elusive. However, given that p63RhoGEF lacks any semblance of an effector-binding site for activated heterotrimeric G proteins, such as an RGS domain, there was no precedent for a direct mode of regulation. Here we provide evidence that Gαq directly engages and stimulates the Dbl-family member p63RhoGEF via novel mechanisms distinct from that previously described for the RGS containing RhoGEFs of the p115 family.
Like numerous Dbl-family members before it, p63RhoGEF was first identified during an oncogenic screen based on its ability to robustly transform NIH3T3 cells (48). An N-terminal truncation of p63RhoGEF that most likely arises by alternative splicing has been previously described in the literature as GEFT. GEFT lacks the first 106 amino acids, but is nevertheless, considered functionally redundant with the full-length protein, p63RhoGEF. Previous studies have implicated p63RhoGEF/GEFT in muscle regeneration and myogenesis (43), regulation of cardiac sarcomeric actin (49), cell proliferation and migration (50), dendritic spine formation (51), and neurite outgrowth (52). Collectively, these suggest p63RhoGEF is an important regulator of actin in excitatory tissues such as muscle and neurons. Interestingly, Gαq-mediated activation of RhoA is also implicated in the pathophysiology of myocardial hypertrophy (6–8). Additional studies are needed to explore the contribution of p63RhoGEF to these and other physiological responses.
Our results support previous reports suggesting an autoinhibitory role for the PH domain of p63RhoGEF. Previous studies have demonstrated that the DH domain of p63RhoGEF activated serum response factor-dependent gene transcription more robustly than full-length protein (44). Previous work has also shown that the PH domain functioned in trans as a dominant- negative by reducing serum response factor-dependent gene transcription mediated by full-length (43) or isolated DH domains (44). However, conflicting reports also suggest the PH domain is essential for induction of stress fibers (49); additional studies may be needed to explore the membrane-targeting capacity of the PH domain and other associated in vivo roles.
We hypothesize that p63RhoGEF is autoinhibited in a manner analogous to that described for Sos1. The x-ray crystal structure of the Sos1 DH-PH cassette indicates that the PH domain folds back onto the DH domain, thereby occluding access to the Rho GTPase binding site and inhibiting activity (53). An extended linker region that joins the adjacent DH and PH domains facilitates this intramolecular interaction within Sos1. However, p63RhoGEF bears no significant homology to the regions of Sos1 responsible for intramolecular binding.
Our results clarify conflicting literature regarding the Rho GTPase substrate specificity of p63RhoGEF. Previous reports suggest that p63RhoGEF is specific for RhoA in REF52 fibroblasts (49), H9C2 cardiomyocytes (49), J82 epithelial cells (44), and HEK-293 cells (30, 44). Yet, other studies also characterized p63RhoGEF as specific for Rac1/Cdc42 in COS-7 and HeLa cells (50) or promiscuous for RhoA/Rac1/Cdc42 in C2C12 muscle cells (43) and N2A neuroblastoma cells (51, 52). A high degree of cross-talk within the Rho subfamily typically complicates the interpretation of these cell-based specificity studies. Furthermore, previous in vitro analysis of p63RhoGEF specificity have relied on suboptimal methodology and have produced results suggesting either RhoA (44, 49) or Rac1/Cdc42 specificity (50). Therefore, we performed in vitro characterization of substrate Rho GTPases using highly purified components with a robust real-time assay (39) to demonstrate that p63RhoGEF specifically activates the Rho isozymes RhoA, RhoB, and RhoC with similar potency. This result is in accordance with studies demonstrating the specific activation of RhoA, and not Rac1 or Cdc42, downstream of Gαq/11 (18, 19, 21, 26–29). Whereas the three Rho isozymes are highly homologous, recent evidence suggests they are not functionally redundant (54); additional studies are required to determine the functional relevance of RhoB/RhoC activation downstream of Gαq/11 and p63RhoGEF. Interestingly, the p63RhoGEF gene, GEFT, bears the official moniker RAC/CDC42 exchange factor; our studies indicate this is a misnomer.
Based on sequence similarity, strict conservation of the PH domain extension, and evidence that Trio-C is directly stimulated by activated Gαq we hypothesize that p63RhoGEF, Trio, and Kalirin represent a novel subset of Dbl-family Rho-GEFs regulated by Gαq/11. Trio and Kalirin share remarkable similarity with each other in their domain architecture and are both essential regulators of axon guidance and neuronal cell migration during neuronal development. A majority of studies on Trio and Kalirin have focused on the N-terminal Rac1/RhoG-specific DH-PH cassette; little is known about the C-terminal RhoA-specific DH-PH cassette. Interestingly, Trio-like proteins have been highly conserved throughout evolution. For example, the Trio orthologs in Caenorhabditis elegans, UNC-73, and Drosophila, d Trio, are essential for proper neuronal/axonal development (55, 56). Whereas no current evidence directly implicates Trio and Kalirin in Gαq/11-mediated signaling pathways, future studies in model organisms such as Drosophila and C. elegans may lend credence to this intriguing notion.
In summary, the studies presented here uncover a novel mode of regulation for Dbl-family GEFs by heterotrimeric G proteins and suggests that, in addition to p63RhoGEF, Kalirin and Trio may also signal downstream of Gαq/11. Now, p63RhoGEF joins a small group of Dbl-family members that have been shown to be directly activated by heterotrimeric signaling components. Ongoing crystallographic studies within our group should soon uncover the molecular details underlying p63RhoGEF activation by Gαq at atomic resolution.
We are grateful to G. Waldo and A. Kimple for technical assistance, as well as C. Johnston and D. Siderovski for providing purified heterotrimeric G protein components. Additionally we are appreciative of plasmid DNA gifts from T. Weiland and M. Strueli.
1Supported by United States Army Medical Research and Materiel Command, Breast Cancer Research Program Grant DAMD17-03-1-0646.
2Supported by National Institutes of Health Grant R01 GM6145408.
3Supported by National Institutes of Health Grants P01-GM65533 and R01-GM62299.
4The abbreviations used are: GPCR, G protein-coupled receptor; GEF, guanine nucleotide exchange factor; DH, Dbl homology; PH, pleckstrin homology; RGS, regulator of G protein signaling; TEV, tobacco etch virus; MBP, maltose-binding protein; GST, glutathione S-transferase; SPR, surface plasmon resonance.
5T. Kawano and T. Kozasa, unpublished results.
6R. J. Rojas and J. Sondek, unpublished results.