PDZ-RhoGEF Research Articles

Solo regulates the localization and activity of PDZ-RhoGEF for actin cytoskeletal remodeling in response to substrate stiffness.

Recent findings indicate that Solo, a RhoGEF, is involved in cellular mechanical stress responses. However, the mechanism of actin cytoskeletal remodeling via Solo remains unclear. Therefore, this study aimed to identify Solo-interacting proteins using the BioID, a proximal-dependent labeling method, and elucidate the molecular mechanisms of function of Solo. We identified PDZ-RhoGEF (PRG) as a Solo-interacting protein. PRG colocalized with Solo in the basal area of cells, depending on Solo localization, and enhanced actin polymerization at the Solo accumulation sites. Additionally, Solo and PRG interaction was necessary for actin cytoskeletal remodeling. Furthermore, the purified Solo itself had little or negligible GEF activity, even its GEF-inactive mutant directly activated the GEF activity of PRG through interaction. Moreover, overexpression of the Solo and PRG binding domains, respectively, had a dominant-negative effect on actin polymerization and actin stress fiber formation in response to substrate stiffness. Therefore, Solo restricts the localization of PRG and regulates actin cytoskeletal remodeling in synergy with PRG in response to the surrounding mechanical environment.

Stabilization of interdomain interactions in G protein α subunits as a determinant of Gαi subtype signaling specificity

Highly homologous members of the Gαi family, Gαi1-3, have distinct tissue distributions and physiological functions, yet their biochemical and functional properties are very similar. We recently identified PDZ-RhoGEF (PRG) as a novel Gαi1 effector that is poorly activated by Gαi2. In a proteomic proximity labeling screen we observed a strong preference for Gαi1 relative to Gαi2 with respect to engagement of a broad range of potential targets. We investigated the mechanistic basis for this selectivity using PRG as a representative target. Substitution of either the helical domain (HD) from Gαi1 into Gαi2 or substitution of a single amino acid, A230 in Gαi2 with the corresponding D in Gαi1, largely rescues PRG activation and interactions with other potential Gαi targets. Molecular dynamics simulations combined with Bayesian network models revealed that in the GTP bound state, separation at the HD-Ras-like domain (RLD) interface is more pronounced in Gαi2 than Gαi1. Mutation of A230 to D in Gαi2 stabilizes HD-RLD interactions via ionic interactions with R145 in the HD which in turn modify the conformation of Switch III. These data support a model where D229 in Gαi1 interacts with R144 and stabilizes a network of interactions between HD and RLD to promote protein target recognition. The corresponding A230 in Gαi2 is unable to stabilize this network leading to an overall lower efficacy with respect to target interactions. This study reveals distinct mechanistic properties that could underly differential biological and physiological consequences of activation of Gαi1 or Gαi2 by GPCRs.

A network of Gα i signaling partners is revealed by proximity labeling proteomics analysis and includes PDZ-RhoGEF

G protein–coupled receptors (GPCRs) that couple to the Gαi family of G proteins are key regulators of cell and tissue physiology. Our previous work has revealed new roles for Gαi in regulating the migration of neutrophils and fibrosarcoma cells downstream of activated chemoattractant receptors. Here, we used an intact cell proximity–based labeling coupled to tandem mass tag (TMT)–based quantitative proteomics analysis to identify proteins that selectively interacted with the GTP-bound form of Gαi1. Multiple targets were identified and validated with a BioID2-tagged, constitutively active Gαi1 mutant, suggesting a network of interactions for activated GαI proteins in intact cells. We showed that active Gαi1, but not Gαi2, stimulated one candidate protein, PDZ-RhoGEF (PRG), despite more than 85% sequence identity between the G proteins. We also demonstrated in primary human neutrophils that active Gαi likely regulated the polarization of phosphorylated myosin light chain, a process critical for migration, through the activation of PRG. The identification and characterization of new targets directly or indirectly regulated by Gαi will aid in the investigation of the functional roles of Gαi-coupled GPCRs in multiple biological processes.

Gα s Directly Drives PDZ‐RhoGEF Signaling to Cdc42

G proteins stimulate RhoGEFs that control cell morphology through actin-cytoskeleton remodeling. Gα12 and Gα13 interact with RH-RhoGEFs (RGS-homology domain RhoGEFs), driving RhoA activation. However, whether additional Gα proteins directly regulate RH-RhoGEFs was not known. To respond this question, we first evaluated the morphological effects of constitutively active DH/PH constructs from p115RhoGEF/ARHGEF1, PDZ-RhoGEF (PRG)/ARHGEF11, and LARG/ARHGEF12. As expected, the three constructs promoted cell contraction and activated RhoA, consistent with their known specificity. Intriguingly, PRG-DH/PH also induced filopodia-like cell protrusions and directly activated Cdc42. Constitutively active Gαs (GαsQ227L) increased the ability of PRG-DH/PH or endogenous PRG to interact with and activate Cdc42. Using a chemogenetic approach, with DREADD receptors, we demonstrated that Gs-coupled receptors, but not those coupled to Gi or Gq, drive PRG activity towards Cdc42. Given that Gαs interacted with PRG DH/PH region, we looked for potential inhibitory effects of the minimal fragment from PRG (linker) that binds GαsQ227L. This construct, the PRG-linker, interfered with GαsQ227L:PRG interaction and PRG:Cdc42 affinity. Besides, it attenuated Gαs signaling by Gs-DREADDs and Gs-coupled prostaglandin receptors (assessed by phosphorylation of CREB and PKA substrates). Altogether, our results demonstrate that Gαs can recognize PRG as a novel effector directing its DH/PH catalytic module to gain affinity for Cdc42.

Identification of PDZ‐RhoGEF (PRG) as a Novel Gα i Target

Cell-migration and adhesion are fundamental biological processes regulated by chemokine G-protein-coupled receptors that couple to PTX-sensitive Gi proteins. The role of Gβγ subunits released from Gi in chemokine signaling has been well characterized. In contrast, Gαi has been thought to have no direct signaling function. Our laboratory recently uncovered a role for Gαi-GTP in regulating cell adhesion. However, a direct effector of Gαi responsible was not identified. To discover novel targets of active Gαi, we used BioID2 proximity-based labeling coupled to TMT quantitative proteomic mass spectrometry. We subsequently compared the abundance ratios of proteins in Gαi1-QL-BioID2 (constitutively active) to Gαi1-WT-BioID2 (inactive) samples. BioID2-CaaX was used as a membrane-targeted control. Experiments were done in HT1080 fibrosarcoma cells where we previously demonstrated Gαi regulation of adhesion. Known binding partners for Gαi subunits including, GPCRs, Gβ, Gγ, adenylate cyclase, and Ric8A were identified by mass spectrometry further validating the method. A candidate Gαi effector identified was PDZ-RhoGEF (PRG), a Rho guanine nucleotide exchange factor that regulates cell adhesion in neutrophils. To determine if PRG can be activated by Gαi; we co-transfected PRG with Gαi1 subunit and an SRE luciferase Rho reporter in A293 cells. Gαi1-QL but not Gαi1-WT strongly activated PRG dependent SRE luciferase, which was confirmed in a rhotekin assay for active RhoA. PRG activation was specific to the Gαi subfamily and required the PRG-RGS domain. A proximal interaction was confirmed in aproximity ligation assay in A293 cells transfected with PRG and Gαi1 cDNAs. Purified Cy5-labeled myr-His-Gαi bound specifically to GST-PRG in a TR-FRET assay. This binding was independent of the nucleotide state of Gαi1 but was inhibited by the addition of Gβγ subunits. In neutrophils, activation of the formyl peptide receptor (FPR) by fMLP, and subsequent PRG activation leads to Rho-dependent accumulation of phosphorylated-myosin light chain (P-MLC) at the trailing edge and has been proposed to result from FPR-dependent Gα13 activation. In our preliminary experiments, stimulation of primary human neutrophils with 10 nM fMLP promoted polarized P-MLC accumulation that was inhibited by PTX. This supports a model where at physiological concentrations of chemoattractant, activation of PRG/Rho is through Gαi-GTP rather than Gα13 in primary human neutrophils. Overall, we identified relatively ubiquitously expressed PRG as a novel Gαi-GTP target. Identification of a novel signaling pathway regulated by Gi-coupled GPCRs will have a significant impact on our understanding of the biology regulated by these ubiquitous and pharmacologically important receptors.

Signaling Specificity of the Gα i G Protein Subfamily8.5.5

G protein-coupled receptors, or GPCRs, are a major class of cell surface receptors that transduce extracellular stimuli to intracellular signaling events via G proteins. GPCRs catalyze nucleotide exchange on the G protein Gα subunit leading to dissociation from Gβγ subunits. The canonical role of one family of G protein alpha subunits, Gαi, is binding and inhibition of the activity of adenylyl cyclase, leading to reduction in the level of 3′,5’-cyclic adenosine monophosphate (cAMP) in cells. Using proximity labeling mass spectrometry, our laboratory has recently discovered a novel role for Gαi in the activation of PDZ-RhoGEF (PRG), a guanine nucleotide exchange factor for the small G protein RhoA. Surprisingly, we found significant differences in the ability of Gαi1, Gαi2, and Gαi3 isoforms to activate PRG in a cell-based SRE-luciferase assay, despite over 85% sequence identity and equivalent inhibition of their common effector adenylyl cyclase. Gαi1 and Gαi3 strongly activate PRG, while Gαi2 shows very weak activation. A majority of the differences in these proteins are located in the helical domain of Gαi, which supports the central hypothesis that members of the Gαi subfamily differentially bind and activate PRG with specificity determined by structural features in the helical domain. To investigate this, the amino acid sequence of Gαi1 was divided into an N-terminal region, the helical domain (HD), and a C-terminal region. Protein chimeras were then generated, substituting one of each of these regions from Gαi1 into Gαi2 to determine if any of these domains are sufficient to confer the ability of the specific Gαi2-Gαi1 chimera to activate PRG in a SRE-luciferase assay in co-transfected HEK293 cells. All chimeras were validated for proper expression in HEK293 cells using western blotting, and all equally inhibited forskolin-stimulated cAMP production. The Gαi2-Gαi1-HD chimera activated PRG to levels similar to Gαi1, while the N-terminal and C-terminal chimeras did not activate PRG. This demonstrates that the helical domain of Gαi1 is sufficient for conferring the ability to activate PRG and structural features within this domain are responsible for the specificity amongst the Gαi1, 2, and 3 subtypes. In an effort to determine the structural features within the HD that confer Gαi-dependent activation of PRG, three subdivisions of the Gαi1 HD were then substituted into Gαi2. None of these subdivisions substituted alone resulted in activation of PRG, indicating that the requirements for PRG activation likely lie in three-dimensional coordination of multiple key residues throughout the helical domain of Gαi. Current and future work will focus on clusters of residue substitutions using three-dimensional structures and sequence alignments as a guide, substituting amino acids common between Gαi1 and Gαi3 but different in Gαi2. Constructs of interest will be examined in cell-based assays as well as purified and examined for in vitro binding to PRG. The resulting data have the potential to change the existing model for signal transfer from Gαi proteins to effectors and provides some of the first evidence for Gαi subtype-effector specificity. 8.5.5

Gαs directly drives PDZ-RhoGEF signaling to Cdc42

Gα proteins promote dynamic adjustments of cell shape directed by actin-cytoskeleton reorganization via their respective RhoGEF effectors. For example, Gα13 binding to the RGS-homology (RH) domains of several RH-RhoGEFs allosterically activates these proteins, causing them to expose their catalytic Dbl-homology (DH)/pleckstrin-homology (PH) regions, which triggers downstream signals. However, whether additional Gα proteins might directly regulate the RH-RhoGEFs was not known. To explore this question, we first examined the morphological effects of expressing shortened RH-RhoGEF DH/PH constructs of p115RhoGEF/ARHGEF1, PDZ-RhoGEF (PRG)/ARHGEF11, and LARG/ARHGEF12. As expected, the three constructs promoted cell contraction and activated RhoA, known to be downstream of Gα13 Intriguingly, PRG DH/PH also induced filopodia-like cell protrusions and activated Cdc42. This pathway was stimulated by constitutively active Gαs (GαsQ227L), which enabled endogenous PRG to gain affinity for Cdc42. A chemogenetic approach revealed that signaling by Gs-coupled receptors, but not by those coupled to Gi or Gq, enabled PRG to bind Cdc42. This receptor-dependent effect, as well as CREB phosphorylation, was blocked by a construct derived from the PRG:Gαs-binding region, PRG-linker. Active Gαs interacted with isolated PRG DH and PH domains and their linker. In addition, this construct interfered with GαsQ227L's ability to guide PRG's interaction with Cdc42. Endogenous Gs-coupled prostaglandin receptors stimulated PRG binding to membrane fractions and activated signaling to PKA, and this canonical endogenous pathway was attenuated by PRG-linker. Altogether, our results demonstrate that active Gαs can recognize PRG as a novel effector directing its DH/PH catalytic module to gain affinity for Cdc42.

P115 RhoGEF activates the Rac1 GTPase signaling cascade in MCP1 chemokine–induced vascular smooth muscle cell migration and proliferation

Although the involvement of Rho proteins in the pathogenesis of vascular diseases is well studied, little is known about the role of their upstream regulators, the Rho guanine nucleotide exchange factors (RhoGEFs). Here, we sought to identify the RhoGEFs involved in monocyte chemotactic protein 1 (MCP1)-induced vascular wall remodeling. We found that, among the RhoGEFs tested, MCP1 induced tyrosine phosphorylation of p115 RhoGEF but not of PDZ RhoGEF or leukemia-associated RhoGEF in human aortic smooth muscle cells (HASMCs). Moreover, p115 RhoGEF inhibition suppressed MCP1-induced HASMC migration and proliferation. Consistent with these observations, balloon injury (BI) induced p115 RhoGEF tyrosine phosphorylation in rat common carotid arteries, and siRNA-mediated down-regulation of its levels substantially attenuated BI-induced smooth muscle cell migration and proliferation, resulting in reduced neointima formation. Furthermore, depletion of p115 RhoGEF levels also abrogated MCP1- or BI-induced Rac1-NFATc1-cyclin D1-CDK6-PKN1-CDK4-PAK1 signaling, which, as we reported previously, is involved in vascular wall remodeling. Our findings also show that protein kinase N1 (PKN1) downstream of Rac1-cyclin D1/CDK6 and upstream of CDK4-PAK1 in the p115 RhoGEF-Rac1-NFATc1-cyclin D1-CDK6-PKN1-CDK4-PAK1 signaling axis is involved in the modulation of vascular wall remodeling. Of note, we also observed that CCR2-Gi/o-Fyn signaling mediates MCP1-induced p115 RhoGEF and Rac1 GTPase activation. These findings suggest that p115 RhoGEF is critical for MCP1-induced HASMC migration and proliferation in vitro and for injury-induced neointima formation in vivo by modulating Rac1-NFATc1-cyclin D1-CDK6-PKN1-CDK4-PAK1 signaling.

Secondary PDZ domain-binding site on class B plexins enhances the affinity for PDZ–RhoGEF

PDZ domains are abundant protein interaction modules and typically recognize a short motif at the C terminus of their ligands, with a few residues in the motif endowing the binding specificity. The sequence-based rules, however, cannot fully account for the specificity between the vast number of PDZ domains and ligands in the cell. Plexins are transmembrane receptors that regulate processes such as axon guidance and angiogenesis. Two related guanine nucleotide exchange factors (GEFs), PDZ-RhoGEF and leukemia-associated RhoGEF (LARG), use their PDZ domains to bind class B plexins and play critical roles in signaling. Here, we present the crystal structure of the full-length cytoplasmic region of PlexinB2 in complex with the PDZ domain of PDZ-RhoGEF. The structure reveals that, in addition to the canonical C-terminal motif/PDZ interaction, the 3D domain of PlexinB2 forms a secondary interface with the PDZ domain. Our biophysical and cell-based assays show that the secondary interface contributes to the specific interaction between plexin and PDZ-RhoGEF and to signaling by plexin in the cell. Formation of secondary interfaces may be a general mechanism for increasing affinity and specificity of modular domain-mediated interactions.

Gα13/PDZ-RhoGEF/RhoA signaling is essential for gastrin-releasing peptide receptor-mediated colon cancer cell migration.

Gastrin-releasing peptide receptor (GRPR) is ectopically expressed in over 60% of colon cancers. GRPR expression has been correlated with increased colon cancer cell migration. However, the signaling pathway by which GRPR activation leads to increased cancer cell migration is not well understood. We set out to molecularly dissect the GRPR signaling pathways that control colon cancer cell migration through regulation of small GTPase RhoA. Our results show that GRP stimulation activates RhoA predominantly through G13 heterotrimeric G-protein signaling. We also demonstrate that postsynaptic density 95/disk-large/ZO-1 (PDZ)-RhoGEF (PRG), a member of regulator of G-protein signaling (RGS)-homology domain (RH) containing guanine nucleotide exchange factors (RH-RhoGEFs), is the predominant activator of RhoA downstream of GRPR. We found that PRG is required for GRP-stimulated colon cancer cell migration, through activation of RhoA-Rho-associated kinase (ROCK) signaling axis. In addition, PRG-RhoA-ROCK pathway also contributes to cyclo-oxygenase isoform 2 (Cox-2) expression. Increased Cox-2 expression is correlated with increased production of prostaglandin-E2 (PGE2), and Cox-2-PGE2 signaling contributes to total GRPR-mediated cancer cell migration. Our analysis reveals that PRG is overexpressed in colon cancer cell lines. Overall, our results have uncovered a key mechanism for GRPR-regulated colon cancer cell migration through the Gα13-PRG-RhoA-ROCK pathway.

Agonist-induced Ca2+ Sensitization in Smooth Muscle: REDUNDANCY OF RHO GUANINE NUCLEOTIDE EXCHANGE FACTORS (RhoGEFs) AND RESPONSE KINETICS, A CAGED COMPOUND STUDY

Many agonists, acting through G-protein-coupled receptors and Gα subunits of the heterotrimeric G-proteins, induce contraction of smooth muscle through an increase of [Ca(2+)]i as well as activation of the RhoA/RhoA-activated kinase pathway that amplifies the contractile force, a phenomenon known as Ca(2+) sensitization. Gα12/13 subunits are known to activate the regulator of G-protein signaling-like family of guanine nucleotide exchange factors (RhoGEFs), which includes PDZ-RhoGEF (PRG) and leukemia-associated RhoGEF (LARG). However, their contributions to Ca(2+)-sensitized force are not well understood. Using permeabilized blood vessels from PRG(-/-) mice and a new method to silence LARG in organ-cultured blood vessels, we show that both RhoGEFs are activated by the physiologically and pathophysiologically important thromboxane A2 and endothelin-1 receptors. The co-activation is the result of direct and independent activation of both RhoGEFs as well as their co-recruitment due to heterodimerization. The isolated recombinant C-terminal domain of PRG, which is responsible for heterodimerization with LARG, strongly inhibited Ca(2+)-sensitized force. We used photolysis of caged phenylephrine, caged guanosine 5'-O-(thiotriphosphate) (GTPγS) in solution, and caged GTPγS or caged GTP loaded on the RhoA·RhoGDI complex to show that the recruitment and activation of RhoGEFs is the cause of a significant time lag between the initial Ca(2+) transient and phasic force components and the onset of Ca(2+)-sensitized force.

PDZ-RhoGEF and LARG Are Essential for Embryonic Development and Provide a Link between Thrombin and LPA Receptors and Rho Activation

G protein-coupled receptors (GPCRs) linked to both members of the Gα12 family of heterotrimeric G proteins α subunits, Gα12 and Gα13, regulate the activation of Rho GTPases, thereby contributing to many key biological processes. Multiple Rho GEFs have been proposed to link Gα12/13 GPCRs to Rho activation, including PDZ-RhoGEF (PRG), leukemia-associated Rho GEF (LARG), p115-RhoGEF (p115), lymphoid blast crisis (Lbc), and Dbl. PRG, LARG, and p115 share the presence of a regulator of G protein signaling homology (RGS) domain. There is limited information on the biological roles of this RGS-containing family of RhoGEFs in vivo. p115-deficient mice are viable with some defects in the immune system and gastrointestinal motor dysfunctions, whereas in an initial study we showed that mice deficient for Larg are viable and resistant to salt-induced hypertension. Here, we generated knock-out mice for Prg and observed that these mice do not display any overt phenotype. However, deficiency in Prg and Larg leads to complex developmental defects and early embryonic lethality. Signaling from Gα11/q-linked GPCRs to Rho was not impaired in mouse embryonic fibroblasts defective in all three RGS-containing RhoGEFs. However, a combined lack of Prg, Larg, and p115 expression abolished signaling through Gα12/13 to Rho and thrombin-induced cell proliferation, directional migration, and nuclear signaling through JNK and p38. These findings provide evidence of an essential role for the RGS-containing RhoGEF family in signaling to Rho by Gα12/13-coupled GPCRs, which may likely play a critical role during embryonic development.

PDZ-RhoGEF is essential for CXCR4-driven breast tumor cell motility through spatial regulation of RhoA

The CXCL12-CXCR4 chemokine signaling pathway is a well-established driver of cancer progression. One key process promoted by CXCR4 stimulation is tumor cell motility; however, the specific signaling pathways leading to migration remain poorly understood. Previously, we have shown that CXCL12 stimulation of migration depends on temporal regulation of RhoA. However, the specific RhoGEF that translates CXCR4 signaling into RhoA activity and cell motility is unknown. We screened the three regulator of G-protein signaling RhoGEFs (LSC, LARG and PRG) and found that PRG selectively regulated the migration and invasion of CXCR4-overexpressing breast tumor cells. Interestingly, we found that PDZ-RhoGEF (PRG) was required for spatial organization of F-actin structures in the center, but not periphery of the cells. The effects on the cytoskeleton were mirrored by the spatial effects on RhoA activity that were dependent upon PRG. Loss of PRG also enhanced adherens junctions in the epithelial-like MCF7-CXCR4 cell line, and inhibited directional persistence and polarity in the more mesenchymal MDA-MB-231 cell line. Thus, PRG is essential for CXCR4-driven tumor cell migration through spatial regulation of RhoA and the subsequent organization of the cytoskeletal structures that support motility. Furthermore, immunohistochemical analysis of human breast tumor tissues shows a significant increase of PRG expression in the invasive areas of the tumors, suggesting that this RhoGEF is associated with breast tumor invasion in vivo.

Activation of p115-RhoGEF Requires Direct Association of Gα13 and the Dbl Homology Domain

RGS-containing RhoGEFs (RGS-RhoGEFs) represent a direct link between the G(12) class of heterotrimeric G proteins and the monomeric GTPases. In addition to the canonical Dbl homology (DH) and pleckstrin homology domains that carry out the guanine nucleotide exchange factor (GEF) activity toward RhoA, these RhoGEFs also possess RGS homology (RH) domains that interact with activated α subunits of G(12) and G(13). Although the GEF activity of p115-RhoGEF (p115), an RGS-RhoGEF, can be stimulated by Gα(13), the exact mechanism of the stimulation has remained unclear. Using combined studies with small angle x-ray scattering, biochemistry, and mutagenesis, we identify an additional binding site for activated Gα(13) in the DH domain of p115. Small angle x-ray scattering reveals that the helical domain of Gα(13) docks onto the DH domain, opposite to the surface of DH that binds RhoA. Mutation of a single tryptophan residue in the α3b helix of DH reduces binding to activated Gα(13) and ablates the stimulation of p115 by Gα(13). Complementary mutations at the predicted DH-binding site in the αB-αC loop of the helical domain of Gα(13) also affect stimulation of p115 by Gα(13). Although the GAP activity of p115 is not required for stimulation by Gα(13), two hydrophobic motifs in RH outside of the consensus RGS box are critical for this process. Therefore, the binding of Gα(13) to the RH domain facilitates direct association of Gα(13) to the DH domain to regulate its exchange activity. This study provides new insight into the mechanism of regulation of the RGS-RhoGEF and broadens our understanding of G protein signaling.

Insights into the Molecular Activation Mechanism of the RhoA-specific Guanine Nucleotide Exchange Factor, PDZRhoGEF

PDZRhoGEF (PRG) belongs to a small family of RhoA-specific nucleotide exchange factors that mediates signaling through select G-protein-coupled receptors via Gα(12/13) and activates RhoA by catalyzing the exchange of GDP to GTP. PRG is a multidomain protein composed of PDZ, regulators of G-protein signaling-like (RGSL), Dbl-homology (DH), and pleckstrin-homology (PH) domains. It is autoinhibited in cytosol and is believed to undergo a conformational rearrangement and translocation to the membrane for full activation, although the molecular details of the regulation mechanism are not clear. It has been shown recently that the main autoregulatory elements of PDZRhoGEF, the autoinhibitory "activation box" and the "GEF switch," which is required for full activation, are located directly upstream of the catalytic DH domain and its RhoA binding surface, emphasizing the functional role of the RGSL-DH linker. Here, using a combination of biophysical and biochemical methods, we show that the mechanism of PRG regulation is yet more complex and may involve an additional autoinhibitory element in the form of a molten globule region within the linker between RGSL and DH domains. We propose a novel, two-tier model of autoinhibition where the activation box and the molten globule region act synergistically to impair the ability of RhoA to bind to the catalytic DH-PH tandem. The molten globule region and the activation box become less ordered in the PRG-RhoA complex and dissociate from the RhoA-binding site, which may constitute a critical step leading to PRG activation.

Mechanistic Insights into Specificity, Activity, and Regulatory Elements of the Regulator of G-protein Signaling (RGS)-containing Rho-specific Guanine Nucleotide Exchange Factors (GEFs) p115, PDZ-RhoGEF (PRG), and Leukemia-associated RhoGEF (LARG)

The multimodular guanine nucleotide exchange factors (GEFs) of the Dbl family mostly share a tandem Dbl homology (DH) and pleckstrin homology (PH) domain organization. The function of these and other domains in the DH-mediated regulation of the GDP/GTP exchange reaction of the Rho proteins is the subject of intensive investigations. This comparative study presents detailed kinetic data on specificity, activity, and regulation of the catalytic DH domains of four GEFs, namely p115, p190, PDZ-RhoGEF (PRG), and leukemia-associated RhoGEF (LARG). We demonstrate that (i) these GEFs are specific guanine nucleotide exchange factors for the Rho isoforms (RhoA, RhoB, and RhoC) and inactive toward other members of the Rho family, including Rac1, Cdc42, and TC10. (ii) The DH domain of LARG exhibits the highest catalytic activity reported for a Dbl protein till now with a maximal acceleration of the nucleotide exchange by 10(7)-fold, which is at least as efficient as reported for GEFs specific for Ran or the bacterial toxin SopE. (iii) A novel regulatory region at the N terminus of the DH domain is involved in its association with GDP-bound RhoA monitored by a fluorescently labeled RhoA. (iv) The tandem PH domains of p115 and PRG efficiently contribute to the DH-mediated nucleotide exchange reaction. (v) In contrast to the isolated DH or DH-PH domains, a p115 fragment encompassing both the regulator of G-protein signaling and the DH domains revealed a significantly reduced GEF activity, supporting the proposed models of an intramolecular autoinhibitory mechanism for p115-like RhoGEFs.

The Application of Hybrid Methods to Study Complex, Multi-Domain Protein - RhoA-Specific Guanidine Nucleotide Exchange Factor - PDZ-RhoGEF

As the focus of structural biology shifts towards studies of increasingly complex, multi-domain proteins and their complexes, the use of hybrid methodologies is becoming a necessity. Here, we present our findings regarding the mechanism of regulation of a multi-domain, RhoA-specific guanine nucleotide exchange factor, PDZ-RhoGEF (PRG). PRG belongs to a small family of RGS GEFs and is an important component of the ‘RhoA cycle’.Activity of RhoA is tightly regulated by a sophisticated interplay between three proteins - guanine nucleotide exchange factor (RhoGEF), GTPase-activating protein (RhoGAP), and guanine nucleotide dissociation inhibitor (RhoGDI). GEFs catalyze the exchange of a nucleotide on RhoA, and consequently cause RhoA activation. Active RhoA transmits signal to its effectors (e.g. Rho-dependent kinase (ROCK)), and consequently triggers a number of physiological responses, including reorganization of actin cytoskeleton, neurite retraction and neuronal morphogenesis, and Ca2+ - sensitization of smooth muscle.The classical paradigm for regulation of multidomain GEFs states that these proteins are autoinhibited by the supramodular arrangement of their domains, which may interfere with the binding of upstream and downstream binding partners, and that a combination of inter-protein interactions initiates a conformational change, leading to the relief of autoinhibition. Using a broad array of biophysical and biochemical techniques, including SAXS, EPR, DLS, CD and others, we are investigating the structure of several multidomain constructs of PRG and we present current data suggesting a functional role for interdomain linkers.

Activated RhoA Binds to the Pleckstrin Homology (PH) Domain of PDZ-RhoGEF, a Potential Site for Autoregulation

Guanine nucleotide exchange factors (GEFs) catalyze exchange of GDP for GTP by stabilizing the nucleotide-free state of the small GTPases through their Dbl homology/pleckstrin homology (DH.PH) domains. Unconventionally, PDZ-RhoGEF (PRG), a member of the RGS-RhoGEFs, binds tightly to both nucleotide-free and activated RhoA (RhoA.GTP). We have characterized the interaction between PRG and activated RhoA and determined the structure of the PRG-DH.PH-RhoA.GTPgammaS (guanosine 5'-O-[gamma-thio]triphosphate) complex. The interface bears striking similarity to a GTPase-effector interface and involves the switch regions in RhoA and a hydrophobic patch in PRG-PH that is conserved among all Lbc RhoGEFs. The two surfaces that bind activated and nucleotide-free RhoA on PRG-DH.PH do not overlap, and a ternary complex of PRG-DH.PH bound to both forms of RhoA can be isolated by size-exclusion chromatography. This novel interaction between activated RhoA and PH could play a key role in regulation of RhoGEF activity in vivo.

Abstract 5074: The Rho Exchange Factor Arhgef1 in Vascular Smooth Muscle Cells is Essential for Angiotensin II-induced Hypertension

Rho exchange factors (Rho-GEFs) are responsible for Rho protein activation by catalyzing the exchange of GDP for GTP. Although over-activation of RhoA and its effector Rho kinase is recognized as a critical component of the pathogenesis of hypertension in experimental models and in human, the molecular mechanisms and the RhoA-GEF(s) involved in this activation of RhoA signaling in vascular smooth muscle cells (VSMCs) are unknown. Among the Rho-GEFs family, Arhgef1 (p115) constitutes with Arhgef11 (PDZ-Rho GEF) and Arhgef12 (Larg) the subfamily of RGS-containing Rho-GEFs that have been suggested to couple G-protein associated receptors to RhoA activation. To establish the role of Arhgef1 in the regulation of vascular tone and hypertension in vivo , we generated mice lacking Arhgef1 specifically in SMCs (SM-Arhgef1-KO). We used Arhgef1 lox/lox mice, which were mated to SMMHC-CreER T2 . SM-Arhgef1-KO mice were then obtained by treatment with tamoxifen (1 mg/day, IP, 5 days). In VSMCs from SM-Arhgef1-KO mice, stimulation with angiotensin II (Ang II, 0.1 μ M) was unable to induce RhoA/Rho kinase activation whereas that produced by U46619, phenylephrine (PhE), and endothelin-1 (ET1) was similar to that recorded in control mice. Ex vivo analysis of the contractile properties showed that, in aortic segments from SM-Arhgef1-KO mice, KCl-, PhE-, U46619- and ET1-induced contractions were similar to those obtained in control mice. In contrast, Ang II-induced contractions were severely reduced. In vivo, acute pressor responses to U46619 (3–100 μ g/kg), ET1 (0.03–1 μ g/kg) and PhE (0.1–100 μ g/kg) were similar in control and SM-Arhgef1-KO mice, but responses to Ang II (0.1–30 μ g/kg) were abolished. Basal blood pressure levels were similar in SM-Arhgef1-KO and control mice. In control mice, chronic infusion of Ang II (1 mg/kg/day) increased blood pressure whereas SM-Arhgef1-KO mice were resistant to the development of Ang II-induced hypertension. We therefore conclude that Arhgef1, as the molecular link between Ang II, RhoA activation and vascular tone regulation, provides an alternative target to reducing elevated vascular tone under hypertensive conditions without affecting normal blood pressure regulation by other vasoactive factors.

On the mechanism of autoinhibition of the RhoA-specific nucleotide exchange factor PDZRhoGEF

BackgroundThe Dbl-family of guanine nucleotide exchange factors (GEFs) activate the cytosolic GTPases of the Rho family by enhancing the rate of exchange of GTP for GDP on the cognate GTPase. This catalytic activity resides in the DH (Dbl-homology) domain, but typically GEFs are multidomain proteins containing other modules. It is believed that GEFs are autoinhibited in the cytosol due to supramodular architecture, and become activated in diverse signaling pathways through conformational change and exposure of the DH domain, as the protein is translocated to the membrane. A small family of RhoA-specific GEFs, containing the RGSL (regulators of G-protein signaling-like) domain, act as effectors of select GPCRs via Gα12/13, although the molecular mechanism by which this pathway operates is not known. These GEFs include p115, LARG and PDZRhoGEF (PRG).ResultsHere we show that the autoinhibition of PRG is caused largely by an interaction of a short negatively charged sequence motif, immediately upstream of the DH-domain and including residues Asp706, Glu708, Glu710 and Asp712, with a patch on the catalytic surface of the DH-domain including Arg867 and Arg868. In the absence of both PDZ and RGSL domains, the DH-PH tandem with additional 21 residues upstream, is 50% autoinhibited. However, within the full-length protein, the PDZ and/or RGSL domains significantly restore autoinhibition.ConclusionOur results suggest a mechanism for autoinhibition of RGSL family of GEFs, in which the RGSL domain and a unique sequence motif upstream of the DH domain, act cooperatively to reduce the ability of the DH domain to bind the nucleotide free RhoA. The activation mechanism is likely to involve two independent steps, i.e. displacement of the RGSL domain and conformational change involving the autoinhibitory sequence motif containing several negatively charged residues.