Receptor-dependent RhoA Activation in G12/G13-deficient Cells

The small GTPase RhoA is involved in the regulation of various cellular functions like the remodeling of the actin cytoskeleton and the induction of transcriptional activity. G-protein-coupled receptors (GPCRs), which are able to activate Gq/G11 and G12/G13 are major upstream regulators of RhoA activity, and G12/G13 have been shown to couple GPCRs to the activation of Rho by regulating the activity of a subfamily of RhoGEF proteins. However, the possible contribution of Gq/G11 to the regulation of RhoA activity via GPCRs is controversial. We have used a genetic approach to study the role of heterotrimeric G-proteins in the activation of RhoA via endogenous GPCRs. In pertussis toxin-treated Gα12/Gα13-deficient as well as in Gαq/Gα11-deficient mouse embryonic fibroblasts (MEFs), in which coupling of receptors is restricted to Gq/G11 and G12/G13, respectively, receptor activation results in Rho activation. Rho activation induced by receptor agonists via Gq/G11 occurs with lower potency than Rho activation via G12/G13. Activation of RhoA via Gq/G11 is not affected by the phospholipase-C blocker U73122 or the Ca2+-chelator BAPTA, but can be blocked by a dominant-negative mutant of the RhoGEF protein LARG. Our data clearly show that G12/G13 as well as Gq/G11 alone can couple GPCRs to the rapid activation of RhoA. Gq/G11-mediated RhoA activation occurs independently of phospholipase C-β and appears to involve LARG. The small GTPase RhoA is involved in the regulation of various cellular functions like the remodeling of the actin cytoskeleton and the induction of transcriptional activity. G-protein-coupled receptors (GPCRs), which are able to activate Gq/G11 and G12/G13 are major upstream regulators of RhoA activity, and G12/G13 have been shown to couple GPCRs to the activation of Rho by regulating the activity of a subfamily of RhoGEF proteins. However, the possible contribution of Gq/G11 to the regulation of RhoA activity via GPCRs is controversial. We have used a genetic approach to study the role of heterotrimeric G-proteins in the activation of RhoA via endogenous GPCRs. In pertussis toxin-treated Gα12/Gα13-deficient as well as in Gαq/Gα11-deficient mouse embryonic fibroblasts (MEFs), in which coupling of receptors is restricted to Gq/G11 and G12/G13, respectively, receptor activation results in Rho activation. Rho activation induced by receptor agonists via Gq/G11 occurs with lower potency than Rho activation via G12/G13. Activation of RhoA via Gq/G11 is not affected by the phospholipase-C blocker U73122 or the Ca2+-chelator BAPTA, but can be blocked by a dominant-negative mutant of the RhoGEF protein LARG. Our data clearly show that G12/G13 as well as Gq/G11 alone can couple GPCRs to the rapid activation of RhoA. Gq/G11-mediated RhoA activation occurs independently of phospholipase C-β and appears to involve LARG. The small GTPase RhoA plays a central role in the organization of the cellular actin cytoskeleton through its ability to stimulate the formation of actomyosin-based structures and to regulate their contractility (1Etienne-Manneville S Hall A. Nature. 2002; 420: 629-635Crossref PubMed Scopus (3821) Google Scholar). In addition to its role in the regulation of the actin cytoskeleton, RhoA has also been involved in various other cellular processes like the regulation of microtubule dynamics or transcriptional activity (1Etienne-Manneville S Hall A. Nature. 2002; 420: 629-635Crossref PubMed Scopus (3821) Google Scholar, 2Ridley A.J. Trends Cell Biol. 2001; 11: 471-477Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar). Analogous to other regulatory guanine nucleotide-binding proteins Rho functions as a molecular switch by cycling between an inactive GDP-bound form and an active GTP-bound form. In the active state RhoA relays extracellular signals to a number of downstream effectors. These include protein kinases like Rho kinase or citron kinase, lipid kinases like phospholipase D, or phosphatidylinositol 4-phosphate 5-kinase as well as non-kinase proteins like rhothekin, rhophilin, or diaphanous (3Aspenstrom P. Curr. Opin. Cell Biol. 1999; 11: 95-102Crossref PubMed Scopus (285) Google Scholar). RhoA is activated through various receptors including those coupled to heterotrimeric G-proteins (4Kjøller L. Hall A. Exp. Cell Res. 1999; 253: 166-179Crossref PubMed Scopus (341) Google Scholar, 5Seasholtz T.M. Majumdar M. Brown J.H. Mol. Pharmacol. 1999; 55: 949-956Crossref PubMed Scopus (204) Google Scholar). Activation of RhoA through G-protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCR, G-protein-coupled receptors; GST, glutathione-S-transferase; RBD, Rho-binding domain; PTX, pertussis toxin; LPA, lysophosphatidic acid; S1P, sphingosine 1-phosphate; PLC, phospholipase C; RhoGEF, Rho guanine nucleotide exchange factor; SRE, serum response element; MEF, mouse embryonic fibroblast; RGS, regulator of G-protein signaling; GST, glutathione S-transferase.1The abbreviations used are: GPCR, G-protein-coupled receptors; GST, glutathione-S-transferase; RBD, Rho-binding domain; PTX, pertussis toxin; LPA, lysophosphatidic acid; S1P, sphingosine 1-phosphate; PLC, phospholipase C; RhoGEF, Rho guanine nucleotide exchange factor; SRE, serum response element; MEF, mouse embryonic fibroblast; RGS, regulator of G-protein signaling; GST, glutathione S-transferase. is involved in a variety of physiological regulatory processes (6Sah V.P. Seasholtz T.M. Sagi S.A. Brown J.H. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 459-489Crossref PubMed Scopus (298) Google Scholar). One of the best described cellular paradigms for GPCR-mediated RhoA activation is the RhoA-dependent actin stress fiber formation in fibroblasts activated by various GPCR agonists like lysophosphatidic acid or thrombin. However, a GPCR/Rho-mediated regulation of actin-based structures has also been shown to occur in many other eukaryotic cells. For instance, in neuronal cells activation of Rho through lysophosphatidic acid or thrombin receptors leads to the formation of contractile actomyosin filaments thereby inducing neurite retraction and cell rounding (7Jalink K. van Corven E.J. Hengeveld T. Morii N. Narumiya S. Moolenaar W.H. J. Cell Biol. 1994; 126: 801-810Crossref PubMed Scopus (575) Google Scholar, 8Katoh H. Aoki J. Yamaguchi Y. Kitano Y. Ichikawa A. Negishi M. J. Biol. Chem. 1998; 273: 28700-28707Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). In vascular smooth muscle cells, the Rho-mediated pathway has been shown to contribute to the vasoconstrictor-induced actomyosin-based cell contraction (9Fukata Y. Amano M. Kaibuchi K. Trends Pharmacol. Sci. 2001; 22: 32-39Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar, 10Gong M.C. Iizuka K. Nixon G. Browne J.P. Hall A. Eccleston J.F. Sugai M. Kobayashi S. Somlyo A.V. Somlyo A.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1340-1345Crossref PubMed Scopus (266) Google Scholar), and the same pathway appears to be centrally involved in the platelet shape change response (11Klages B. Brandt U. Simon M.I. Schultz G. Offermanns S. J. Cell Biol. 1999; 144: 745-754Crossref PubMed Scopus (305) Google Scholar). It is well established that G-proteins of the G12-family, G12 and G13, can couple GPCRs to the activation of RhoA. Constitutively active mutants of Gα12 and Gα13 have been shown to induce actin stress fiber formation as well as other RhoA-dependent cellular effects (Ref. 12Buhl A.M. Johnson N.L. Dhanasekaran N. Johnson G.L. J. Biol. Chem. 1995; 270: 24631-24634Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar; for review see Ref. 6Sah V.P. Seasholtz T.M. Sagi S.A. Brown J.H. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 459-489Crossref PubMed Scopus (298) Google Scholar). Recent studies in reconstituted or co-transfected systems have demonstrated that a group of RhoGEF proteins, consisting of p115 RhoGEF, PDZ-RhoGEF, and LARG, interact with Gα12 and Gα13 through their RGS domains, thereby stimulating RhoA activity (13Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (672) Google Scholar, 14Kozasa T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. Gilman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Crossref PubMed Scopus (736) Google Scholar, 15Fukuhara S. Chikum i H. Gutkind J.S. Oncogene. 2001; 20: 1661-1668Crossref PubMed Scopus (193) Google Scholar, 16Suzuki N. Nakamura S. Mano H. Kozasa T. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 733-738Crossref PubMed Scopus (173) Google Scholar). Receptors, which activate G12/G13 also couple to Gq and G11. It has been a controversial issue, whether Gq/G11-mediated signaling contributes to the activation of RhoA via GPCRs. While various reports show Rho-dependent effects of constitutively active Gαq mutants (Refs. 8Katoh H. Aoki J. Yamaguchi Y. Kitano Y. Ichikawa A. Negishi M. J. Biol. Chem. 1998; 273: 28700-28707Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar and 18Mao J. Yuan H. Xie W. Simon M.I. Wu D. J. Biol. Chem. 1998; 273: 27118-27123Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar; for review see Ref. 6Sah V.P. Seasholtz T.M. Sagi S.A. Brown J.H. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 459-489Crossref PubMed Scopus (298) Google Scholar) other studies demonstrated that active mutants of Gαq are not able to induce Rho-mediated processes (12Buhl A.M. Johnson N.L. Dhanasekaran N. Johnson G.L. J. Biol. Chem. 1995; 270: 24631-24634Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 17Fromm C. Coso O.A. Montaner S. Xu N. Gutkind J.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10098-10103Crossref PubMed Scopus (195) Google Scholar). The recent development of methods for the precipitation of the activated form of RhoA from cell lysates allowed to directly determine the effects of different G-protein α-subunits on Rho activity. It could be confirmed that constitutively active mutants of Gα12/Gα13 can induce RhoA activation (19Kranenburg O. Poland M. van Horck F.P. Drechsel D. Hall A. Moolenaar W.H. Mol. Biol. Cell. 1999; 10: 1851-1857Crossref PubMed Scopus (273) Google Scholar, 20Chikumi H. Fukuhara S. Gutkind J.S. J. Biol. Chem. 2002; 277: 12463-12473Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). However, again, conflicting data exist with regard to the potential role of Gq/G11 in GPCR-mediated RhoA activation. In NIH3T3 and HEK293T cells, expression of constitutively active Gαq-family members results in an increased level of active RhoA (21Booden M.A. Siderovski D.P. Der C.J. Mol. Cell. Biol. 2002; 22: 4053-4061Crossref PubMed Scopus (148) Google Scholar, 22Dutt P. Kjolle L. Giel M. Hall A. Toksoz D. FEBS Lett. 2002; 531: 565-569Crossref PubMed Scopus (40) Google Scholar, 23Chikumi H. Vazquez Prado J. Servitja J.M. Miyazaki H. Gutkind J.S. J. Biol. Chem. 2002; 277: 27130-27134Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). In contrast, expression of mutant Gαq in COS-7 cells does not induce activation of RhoA (24Sagi S.A. Seasholtz T.M. Kobiashvili M. Wilson B.A. Toksoz D. Brown J.H. J. Biol. Chem. 2001; 276: 15445-15452Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). To study the role of different G-proteins under more physiological conditions we used Gαq/Gα11- and Gα12/Gα13-double deficient embryonic fibroblasts (MEFs) to determine their role in the activation of Rho via endogenous receptors. Our data clearly show that Gq/G11 can couple GPCRs to the rapid activation of RhoA. This process occurs in a phospholipase C-β-independent manner and appears to involve the RhoGEF protein LARG. Reagents—Thrombin (T3399), LPA (L7260), bradykinin (B3259), and U73122 were from Sigma-Aldrich (Dreieich, Germany). Pertussis toxin, BAPTA/AM, and Fura-2/AM were purchased from Calbiochem (Schwalbach, Germany). Plasmids—HA-ΔDH/PH-LARG lacking the DH and the PH domain was generated by EcoNI and EcoRI digestion of full-length LARG and in-frame re-ligation using MungBean-nuclease and T4-DNA polymerase. Deletion was confirmed by sequencing (Li-cor 4200, Li-cor, Inc.). The modifying enzymes were purchased from New England Biolabs (Frankfurt, Germany), the ligase was obtained from Takara (Verviers, Belgium). Cell Lines and Transfection—MEF cell lines were generated as described (25Zywietz A. Gohla A. Schmelz M. Schultz G. Offermanns S. J. Biol. Chem. 2001; 276: 3840-3845Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells were transiently transfected using LipofectAMINE™ (Invitrogen, Karlsruhe, Germany) according to the manufacturer's protocol. Serum starvation and pertussis toxin treatment were done for 24 h. Western Blot Analysis—Lysates of MEFs were analyzed by Western blotting after SDS-polyacrylamide gel electrophoresis and visualized by chemiluminescence detection using sheep anti-mouse (Amersham Biosciences) or goat anti-rabbit antibodies (Cell Signaling, Frankfurt, Germany) coupled to horseradish peroxidase and were visualized using ECL reagent (Amersham Biosciences). Monoclonal antibodies against RhoA (26C4) or c-Myc (9E10) and rabbit polyclonal antibodies against Gα13 (A-20) and Gαq/11 (C-19) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, USA). Anti-Gα12 rabbit polyclonal antibody was described J.M. A. A. Offermanns S. J. 2002; PubMed Scopus Google Scholar). Monoclonal antibody was obtained from in rabbit against PDZ-RhoGEF, and LARG were described J.M. A. A. Offermanns S. J. 2002; PubMed Scopus Google Scholar). from MEFs was using the according to the manufacturer's protocol. was using and the from was with to of of various G-protein-coupled which possible were by different using The were and and and and and and and and and and and and and and of of cells were on MEFs were for with Fura-2/AM in and bovine serum were analyzed on a Germany) Germany). The was with a and was and and through a and the of the was as described T. A. and G. J. Biol. Scholar). were in and as described T. M. 2003; 22: PubMed Scopus Google Scholar). anti-mouse were generated using a and a Rho of Rho was by a modified described by and M.A. J. 1999; PubMed Scopus Google Scholar). cells were on and were transfected with RhoA and or for of endogenous serum starvation and pertussis toxin treatment for 24 cells were with different agonists and in and and were for with (Amersham Biosciences) coupled with proteins to the Rho-binding domain of M.A. J. 1999; PubMed Scopus Google Scholar). were with of and and and were by the addition of and analyzed using RhoA and transfected was using a mouse antibody (26C4) and antibody (Santa Cruz embryonic cell lines were generated from as well as from deficient for and Gα12/Gα13 S. Gohla A. Simon M.I. T.M. J. 1998; PubMed Scopus Google Scholar, S. Offermanns S. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). Western of lysates from cell lines confirmed the of and Gα12/Gα13 in cells We whether the different cell lines receptors for various which have been shown to induce the activation of Gq/G11 as well as G12/G13, like the lysophosphatidic acid and sphingosine or like thrombin using we that as well as Gαq/Gα11- and Gα12/Gα13-deficient MEFs receptors for sphingosine and LPA and bradykinin as well as for thrombin expression was for or receptors as well as or receptors. and receptors have been shown to couple to G12/G13, as well as to G-proteins M.J. T. S. A.J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), and is that and receptors have a G-protein coupling N. J. Annu. Rev. Pharmacol. Toxicol. 2001; PubMed Scopus Google Scholar). the thrombin receptor has also been shown to couple to G-protein (11Klages B. Brandt U. Simon M.I. Schultz G. Offermanns S. J. Cell Biol. 1999; 144: 745-754Crossref PubMed Scopus (305) Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, S. K. Schultz G. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar, A.J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). the of receptors not between cell lines that are well for a of In we used LPA, bradykinin and thrombin to stimulate cells through their endogenous receptors. To study the role of Gq/G11 and G12/G13 in Rho we restricted coupling of receptors to G-protein by cells with pertussis toxin which receptors from that treatment α-subunits of in Gα12/Gα13-deficient cells under the used of the was able to induce in Gαq/Gα11-deficient cells were induced in and cells and data not In pertussis toxin-treated Gαq/Gα11-deficient cells, in which coupling of receptors is restricted to G12/G13, and LPA in an activation of RhoA This that G12/G13 are able to RhoA activation independently of signaling via which induced Rho activation in cells on Rho activity in Gαq/Gα11-deficient cells, that the bradykinin receptor is not coupled to G12/G13, but induce Rho activation through Gq/G11 To the potential ability of Gq/G11 to Rho activation more we the of LPA, and bradykinin on RhoA activation in Gα12/Gα13-deficient cells, in which coupling of receptors is restricted to receptor agonists were able to activate RhoA through Gq/G11 we whether Gq/G11-mediated RhoA activation a phospholipase C-β or in Rho activation in Gα12/Gα13-deficient cells was not by the phospholipase C-β U73122 or the Ca2+-chelator BAPTA, blocked induction of in the same cells and These data that GPCRs can RhoA activation via Gq/G11 in a phospholipase C-β-independent To and Gq/G11-mediated RhoA activation through we for RhoA activation in and Gα12/Gα13-deficient cells and RhoA activation in cells and in Gαq/Gα11-deficient cells with and of and However, G-protein coupling was restricted to Gq/G11 in Gα12/Gα13-deficient cells, thrombin was and RhoA activation was thrombin of While G12/G13 and Gq/G11 can RhoA activation through Gq/G11 of than activation through G12/G13. The that RhoA activation via Gq/G11 independently of phospholipase C-β to the of RGS RhoGEF proteins, which have been to directly G12/G13 to Rho activation. the members of of RhoGEF proteins, p115 RhoGEF, PDZ-RhoGEF, and LARG, we LARG to be in MEFs This is with the expression of LARG with and PDZ-RhoGEF, which are in and neuronal cells, J.M. A. A. Offermanns S. J. 2002; PubMed Scopus Google Scholar, Oncogene. 1997; PubMed Scopus Google Scholar, K. A. K. T. K. 2001; PubMed Scopus Google Scholar). To the potential role of LARG in Gq/G11-mediated Rho we a mutant of LARG lacking the DH and PH for its RhoGEF activity of in Gα12/Gα13-deficient cells blocked rapid actin stress fiber formation induced by LPA expression of also the cellular in the of 10% fetal serum was to activation of RhoA in response to thrombin and LPA in Gα12/Gα13-deficient cells These data that LARG is involved in the phospholipase C-β-independent RhoA activation via GPCRs have been shown to be able to the activation of the small GTPase RhoA (6Sah V.P. Seasholtz T.M. Sagi S.A. Brown J.H. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 459-489Crossref PubMed Scopus (298) Google Scholar, 15Fukuhara S. Chikum i H. Gutkind J.S. Oncogene. 2001; 20: 1661-1668Crossref PubMed Scopus (193) Google Scholar). Activation of RhoA via GPCRs is involved in a variety of functions like regulation of cell cell and cell that stimulate RhoA activity couple to G12/G13, and The that Rho activation through GPCRs is not affected by and receptors coupling to G-proteins are to stimulate Rho activity that is to be directly involved in RhoA activation via GPCRs T.M. Majumdar M. Brown J.H. Mol. Pharmacol. 1999; 55: 949-956Crossref PubMed Scopus (204) Google Scholar, 6Sah V.P. Seasholtz T.M. Sagi S.A. Brown J.H. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 459-489Crossref PubMed Scopus (298) Google Scholar). It has been to the of G12/G13 and Gq/G11 in Rho activation are of G-protein has that G12 and can the activation of and the described RhoGEF proteins, LARG, and have been shown to G12/G13 to RhoA activation (13Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (672) Google Scholar, 14Kozasa T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. Gilman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Crossref PubMed Scopus (736) Google Scholar, 15Fukuhara S. Chikum i H. Gutkind J.S. Oncogene. 2001; 20: 1661-1668Crossref PubMed Scopus (193) Google Scholar, 16Suzuki N. Nakamura S. Mano H. Kozasa T. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 733-738Crossref PubMed Scopus (173) Google Scholar). However, is not whether Gq/G11 are directly involved in the rapid RhoA activation by extracellular in lacking demonstrated that RhoA activation does not on activation of Gq/G11 B. B. Offermanns S. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). Sagi (24Sagi S.A. Seasholtz T.M. Kobiashvili M. Wilson B.A. Toksoz D. Brown J.H. J. Biol. Chem. 2001; 276: 15445-15452Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) that a constitutively active mutant of Gαq was to induce RhoA activation. In contrast, recent reports clearly show that of constitutively active mutants of Gαq in NIH3T3 cells or in HEK293T cells results in of active RhoA (21Booden M.A. Siderovski D.P. Der C.J. Mol. Cell. Biol. 2002; 22: 4053-4061Crossref PubMed Scopus (148) Google Scholar, 22Dutt P. Kjolle L. Giel M. Hall A. Toksoz D. FEBS Lett. 2002; 531: 565-569Crossref PubMed Scopus (40) Google Scholar, 23Chikumi H. Vazquez Prado J. Servitja J.M. Miyazaki H. Gutkind J.S. J. Biol. Chem. 2002; 277: 27130-27134Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). studies on the effects of transfected constitutively active mutants of Gαq not a of Gq proteins in RhoA activation by extracellular signals via receptors. We a genetic approach to study the activation of RhoA through endogenous receptors in MEFs lacking or In cells, receptor coupling is restricted to G12/G13 and Our data that RhoA activation is induced through G12/G13. genetic that Gq/G11 proteins can also the rapid RhoA activation via GPCRs. we a in the by which thrombin induced RhoA activation via G12/G13 and Rho activation through Gq/G11 of than Rho activation via G12/G13. The potency for Rho activation could be to different by which the activated receptor to different G-protein For the which to G12/G13 and Gq/G11 has been shown to activate G12/G13 in S. K. Schultz G. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar). However, is not whether is a of and receptors. is that RhoGEF proteins are to regulation via than through While is well established that RhoA activation RhoGEF proteins like PDZ-RhoGEF, LARG, and the of Gq/G11-mediated Rho activation is We could not effects of or of the phospholipase C-β U73122 on Gq/G11-mediated Rho activation that Gq/G11 Rho activation in a manner of of This is with data that activation of protein kinase or of is not able to RhoA activation and that of does not with Rho activation by an active mutant of Gαq H. Vazquez Prado J. Servitja J.M. Miyazaki H. Gutkind J.S. J. Biol. Chem. 2002; 277: 27130-27134Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). is not involved in Gq/G11-mediated RhoA is to that Gq/G11 activate RhoA by a to G12/G13. recent data that the RGS domain of LARG but not of p115 RhoGEF can interact with Gαq with to the state of (21Booden M.A. Siderovski D.P. Der C.J. Mol. Cell. Biol. 2002; 22: 4053-4061Crossref PubMed Scopus (148) Google Scholar). In contrast, the constitutively active mutant of Gαq not show a with the RGS or the full-length of LARG, PDZ-RhoGEF, or H. Vazquez Prado J. Servitja J.M. Miyazaki H. Gutkind J.S. J. Biol. Chem. 2002; 277: 27130-27134Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, S. C. M. T. Gutkind J.S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, S. H. Gutkind J.S. FEBS Lett. 2000; PubMed Scopus Google Scholar). However, mutants of and which the including the RGS domain were able to interact with H. Vazquez Prado J. Servitja J.M. Miyazaki H. Gutkind J.S. J. Biol. Chem. 2002; 277: 27130-27134Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). In was shown that expression of full-length LARG but not of a LARG which the and RGS RhoA activation by constitutively active Gαq (21Booden M.A. Siderovski D.P. Der C.J. Mol. Cell. Biol. 2002; 22: 4053-4061Crossref PubMed Scopus (148) Google Scholar), that an of the RGS domain with Gαq is involved in RhoA activation. However, expression of the RGS of LARG and not with serum response an which is by Rho H. Vazquez Prado J. Servitja J.M. Miyazaki H. Gutkind J.S. J. Biol. Chem. 2002; 277: 27130-27134Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, S. H. Gutkind J.S. FEBS Lett. 2000; PubMed Scopus Google Scholar). is that active Gαq can interact with LARG and other RGS domain RhoGEF proteins, the of In we used a LARG mutant that the RhoGEF domain to study the potential of LARG in RhoA activation. of blocked RhoA activation as well as actin stress fiber formation in Gα12/Gα13-deficient cells, that LARG is involved in Gq/G11-mediated RhoA activation. the of Rho activation by Gq/G11 is to that by G12/G13 to be In on a genetic data clearly that G12/G13 and can the activation of RhoA via G-protein-coupled receptors. The Gq/G11-mediated Rho activation occurs independently of of phospholipase and the RhoGEF protein LARG. We M. A. for the LARG