The Guanine Nucleotide Exchange Factor p63RhoGEF, a Specific Link between Gq/11-coupled Receptor Signaling and RhoA
2005; Elsevier BV; Volume: 280; Issue: 12 Linguagem: Inglês
10.1074/jbc.m411322200
ISSN1083-351X
AutoresSusanne Lutz, Andrea Freichel-Blomquist, Yang Yang, Ulrich Rümenapp, Karl H. Jakobs, Martina Schmidt, Thomas Wieland,
Tópico(s)Cell Adhesion Molecules Research
ResumoThe monomeric GTPase RhoA, which is a key regulator of numerous cellular processes, is activated by a variety of G protein-coupled receptors, through either G12 or Gq family proteins. Here we report that p63RhoGEF, a recently identified RhoA-specific guanine nucleotide exchange factor, enhances the Rho-dependent gene transcription induced by agonist-stimulated Gq/11-coupled receptors (M3-cholinoceptor, histamine H1 receptor) or GTPase-deficient mutants of Gαq and Gα11. We further demonstrate that active Gαq or Gα11, but not Gα12 or Gα13, strongly enhances p63RhoGEF-induced RhoA activation by direct protein-protein interaction with p63RhoGEF at its C-terminal half. Moreover, the activation of p63RhoGEF by Gαq/11 occurs independently of and in competition to the activation of the canonical Gαq/11 effector phospholipase C β. Therefore, our results elucidate a new signaling pathway by which Gαq/11-coupled receptors specifically induce Rho signaling through a direct interaction of activated Gαq/11 subunits with p63RhoGEF. The monomeric GTPase RhoA, which is a key regulator of numerous cellular processes, is activated by a variety of G protein-coupled receptors, through either G12 or Gq family proteins. Here we report that p63RhoGEF, a recently identified RhoA-specific guanine nucleotide exchange factor, enhances the Rho-dependent gene transcription induced by agonist-stimulated Gq/11-coupled receptors (M3-cholinoceptor, histamine H1 receptor) or GTPase-deficient mutants of Gαq and Gα11. We further demonstrate that active Gαq or Gα11, but not Gα12 or Gα13, strongly enhances p63RhoGEF-induced RhoA activation by direct protein-protein interaction with p63RhoGEF at its C-terminal half. Moreover, the activation of p63RhoGEF by Gαq/11 occurs independently of and in competition to the activation of the canonical Gαq/11 effector phospholipase C β. Therefore, our results elucidate a new signaling pathway by which Gαq/11-coupled receptors specifically induce Rho signaling through a direct interaction of activated Gαq/11 subunits with p63RhoGEF. The Rho GTPase family belongs to the Ras superfamily and comprises more than 20 distinct proteins. The best characterized members (RhoA, Rac1, and Cdc42) were first identified in the early 1990s as regulators of actin cytoskeleton rearrangements. RhoA, Rac1, and Cdc42 induce stress fiber, lamellipodia, and filopodia formation, respectively (1Ridley A. Prog. Mol. Subcell. Biol. 1999; 22: 1-22Crossref PubMed Scopus (60) Google Scholar). Meanwhile, it became evident that Rho family proteins play a pivotal role in a variety of cellular processes, including secretion, smooth muscle contraction, migration, neurite retraction, and gene transcription (2Etienne-Manneville S. Hall A. Nature. 2002; 420: 629-635Crossref PubMed Scopus (3875) Google Scholar, 3Jaffe A. Hall A. Adv. Cancer Res. 2002; 84: 57-80Crossref PubMed Scopus (255) Google Scholar). As monomeric GTPases, Rho proteins cycle between an inactive GDP-bound and an active GTP-bound state. The activation step, i.e. the exchange of GDP by GTP, is catalyzed by a group of accessory proteins: the guanine nucleotide exchange factors (GEFs). 1The abbreviations used are: GEF, guanine nucleotide exchange factor; DH, Dbl homology; PH, pleckstrin homology; GST, glutathione S-transferase; GST·RBD, GST fusion protein containing the Rho binding domain of rhotekin; Lsc-RGS, RGS homology domain of Lsc; SRF, serum-response factor; GPCR, G protein-coupled receptor; LARG, leukemia-associated RhoGEF; PLC, phospholipase C; PKC, protein kinase C; GAP, GTPase-activating protein; M3R, M3-cholinoceptor; H1R, histamine H1 receptor; RGS, regulator of G protein signaling. About 60 different GEFs for Rho family members (RhoGEFs) are described so far. Most of them belong to the Dbl protein family, which share the typical tandem motif consisting of a Dbl homology (DH) and a pleckstrin homology (PH) domain (4Schmidt A. Hall A. Genes Dev. 2002; 16: 1587-1609Crossref PubMed Scopus (987) Google Scholar, 5Zheng Y. Trends Biochem. Sci. 2001; 26: 724-732Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). Besides this tandem motif, RhoGEFs often contain one or more additional signal transduction domains, such as SH2, SH3, PDZ, and additional PH domains. Therefore, they often function as molecular bridges between different signal transduction pathways (4Schmidt A. Hall A. Genes Dev. 2002; 16: 1587-1609Crossref PubMed Scopus (987) Google Scholar, 5Zheng Y. Trends Biochem. Sci. 2001; 26: 724-732Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). It is well established that apart from receptor tyrosine kinases, a large variety of G protein-coupled receptors (GPCRs), particularly those coupling to the G12/13 type of heterotrimeric G proteins, are upstream regulators of Rho proteins (6Sah V. Seasholtz T. Sagi S. Brown J. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 459-489Crossref PubMed Scopus (298) Google Scholar, 7Seasholtz T. Majumdar M. Brown J. Mol. Pharmacol. 1999; 55: 949-956Crossref PubMed Scopus (206) Google Scholar). A family of RhoA-specific GEFs, consisting of p115RhoGEF, PDZ-RhoGEF, and leukemia-associated RhoGEF (LARG), which mediates this activation process, has been identified (8Fukuhara S. Murga C. Zohar M. Igishi T. Gutkind J. J. Biol. Chem. 1999; 274: 5868-5879Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 9Fukuhara S. Chikumi H. Gutkind J. FEBS Lett. 2000; 485: 183-188Crossref PubMed Scopus (212) Google Scholar, 10Mao J. Yuan H. Xie W. Wu D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12973-12976Crossref PubMed Scopus (114) Google Scholar). All these proteins contain, in addition to the DH/PH tandem motif, a regulator of G protein signaling (RGS) homology domain for direct interaction with and activation by G12 type G proteins. Recently, clear evidence has been provided that Gαq and Gα11 as well as Gq/11-coupled receptors can induce potent RhoA activation (11Chikumi H. Vazquez-Prado J. Servitja J. Miyazaki H. Gutkind J. J. Biol. Chem. 2002; 277: 27130-27134Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 12Hadano S. Hand C.K. Osuga H. Yanagisawa Y. Otomo A. Devon R.S. Miyamoto N. Showguchi-Miyata J. Okada Y. Singaraja R. Figlewicz D. Kwiatkowski T. Hosler B.A. Sagie T. Skaug J. Nasir J. Brown Jr., R.H. Scherer S.W. Rouleau G.A. Hayden M.R. Ikeda J.E. Nat. Genet. 2001; 29: 166-173Crossref PubMed Scopus (594) Google Scholar, 13Vogt S. Grosse R. Schultz G. Offermanns S. J. Biol. Chem. 2003; 278: 28743-28749Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). This process is apparently independent of phospholipase C β (PLCβ) isozymes, the canonical Gαq/11 effectors, and their downstream signaling, i.e. Ca2+ mobilization and protein kinase C (PKC) activation (11Chikumi H. Vazquez-Prado J. Servitja J. Miyazaki H. Gutkind J. J. Biol. Chem. 2002; 277: 27130-27134Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 13Vogt S. Grosse R. Schultz G. Offermanns S. J. Biol. Chem. 2003; 278: 28743-28749Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Although the G12/13-activated LARG has been reported to additionally mediate Gαq-induced RhoA activation (13Vogt S. Grosse R. Schultz G. Offermanns S. J. Biol. Chem. 2003; 278: 28743-28749Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 14Booden M. Siderovski D. Der C. Mol. Cell. Biol. 2002; 22: 4053-4061Crossref PubMed Scopus (149) Google Scholar), a GEF specifically and selectively linking Gαq/11 proteins and Gq/11-coupled receptors to RhoA activation has not been identified so far. We and others (15Lutz S. Freichel-Blomquist A. Rümenapp U. Schmidt M. Jakobs K.H. Wieland T. Naunyn-Schmiedeberg's Arch. Pharmacol. 2004; 369: 540-546Crossref PubMed Scopus (42) Google Scholar, 16Souchet M. Portales-Casamar E. Mazurais D. Schmidt S. Leger I. Javre J. Robert P. Berrebi-Bertrand I. Bril I. Gout B. Debant A. Calmels T. J. Cell Sci. 2002; 115: 629-640Crossref PubMed Google Scholar) have recently identified a new RhoGEF of 580 amino acids in length and an apparent molecular mass of 63 kDa. It was therefore termed p63RhoGEF. The expression of p63RhoGEF in human heart and brain tissue was confirmed by both groups of investigators using Northern blot analysis and immunohistochemistry, and it was clearly demonstrated that p63RhoGEF activates specifically RhoA but not Rac1 or Cdc42. Sequence analysis revealed that this protein does not contain other distinct functional domains besides the typical DH/PH tandem motif. As we tried to identify regulatory mechanisms inducing a p63RhoGEF-mediated RhoA activation, we studied the influence of the stimulation of a variety of GPCRs on p63RhoGEF activity. The data presented herein will provide evidence that p63RhoGEF links specifically Gq/11-coupled receptors to RhoA by a direct interaction with GTP-liganded Gαq/11 proteins. This RhoGEF therefore represents a so far unknown Gαq effector molecule. Plasmids—The construction of plasmids encoding Myc-tagged p63RhoGEF and its deletion mutants in the pCMV-Tag3B vector was reported before (15Lutz S. Freichel-Blomquist A. Rümenapp U. Schmidt M. Jakobs K.H. Wieland T. Naunyn-Schmiedeberg's Arch. Pharmacol. 2004; 369: 540-546Crossref PubMed Scopus (42) Google Scholar). The coding sequences of the M3-cholinoceptor (M3R) and the histamine H1 receptor (H1R) was subcloned into the eukaryotic expression vector pcDNA3 (Invitrogen). pCDNA3-EE-GqQL was from UMR cDNA Resource Center. pCis-Gαq, pCis-GαqRC, pCis-Gα11, pCis-Gα11QL, pCis-Gα12, pCis-Gα12QL, pCis-Gα13, pCis-Gα13QL, and pCis-PLCβ2 were kindly provided by Dr M. I. Simon, Pasadena, CA. Cell Culture and Transfection—Culture of HEK-293 cells and COS-7 cells and transfection of the cells (250 ng of total DNA/well on a 48-well plate for SRF activation and up to 2 μg of DNA/6-well plate for RhoA pull-down assays) were performed as described before (15Lutz S. Freichel-Blomquist A. Rümenapp U. Schmidt M. Jakobs K.H. Wieland T. Naunyn-Schmiedeberg's Arch. Pharmacol. 2004; 369: 540-546Crossref PubMed Scopus (42) Google Scholar). Assays were performed 48 h after transfection in serum-starved cells. Preparation of RNA and Reverse Transcription-PCR—Total RNA from HEK-293 and COS-7 cells was prepared with RNeasy Minikit (Qiagen, Hilden, Germany). The RNA was transcribed into cDNA using oligo(dT) primers and a first strand synthesis kit (Roche Applied Science). PCR conditions for the amplification of p63RhoGEF cDNA fragments were as follows (50 μl): primer 0.4 μm each, dNTP 0.2 mm, 1× PCR buffer, Taq polymerase plus Q-Solution (Qiagen), and 2 μl of cDNA. 35 cycles (denaturation 95 °C, 30 s, annealing 56 °C, 30 s, elongation 72 °C, 90 s) followed by a final acquisition of 5 min at 72 °C were performed. Primer sequences were as follows: p63RhoGEF forward, 5′-GATGGTTGGATCATTCCAAACA-3′; p63RhoGEF reverse, 5′-GTTACAGCTCATCTTCATCCA-3′. The sequences of these primers are conserved in rat, mouse, and man. Assay of SRF Activation—HEK-293 cells or COS-7 cells seeded on 48-well plates were co-transfected with the indicated expression plasmids together with the pSRE.L-luciferase reporter plasmid and the pRL-TK control reporter vector. 48 h after transfection, cells were washed once with phosphate-buffered saline and lysed with passive lysis buffer (Promega). Luciferase activities were determined with the Dual-Luciferase reporter assay system (Promega) as described (15Lutz S. Freichel-Blomquist A. Rümenapp U. Schmidt M. Jakobs K.H. Wieland T. Naunyn-Schmiedeberg's Arch. Pharmacol. 2004; 369: 540-546Crossref PubMed Scopus (42) Google Scholar, 17Mao J. Yuan H. Xie W. Simon M. Wu D. J. Biol. Chem. 1998; 273: 27118-27123Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). The activity of the experimental reporter was normalized against the activity of the control vector. Pull-down Assay of Activated RhoA—The cellular level of GTP-loaded RhoA was determined using a GST fusion protein containing the Rho binding domain of rhotekin (GST·RBD) (15Lutz S. Freichel-Blomquist A. Rümenapp U. Schmidt M. Jakobs K.H. Wieland T. Naunyn-Schmiedeberg's Arch. Pharmacol. 2004; 369: 540-546Crossref PubMed Scopus (42) Google Scholar, 18Ren X. Schwartz M. Methods Enzymol. 2000; 325: 264-272Crossref PubMed Google Scholar). In brief, subconfluent monolayers of HEK-293 cells were transfected with the indicated amounts of plasmid DNA or the corresponding empty vectors using Polyfect (Qiagen) and cultured for 48 h. Thereafter, the cells were lysed in a buffer containing 1% Nonidet P-40, and the particular fraction was pelleted by centrifugation. 1 mg of the GTPase-containing supernatant was then incubated for 1 h at 4 °C with 40 μg of GST·RBD (expressed in and purified from Escherichia coli) bound to glutathione-Sepharose beads. After three times washing of the beads, bound proteins were eluted with sample buffer and separated by SDS-PAGE. RhoA was then detected by immunoblotting with a specific monoclonal antibody (Santa Cruz Biotechnology). Coimmunoprecipitation of Gα-proteins with p63RhoGEF—HEK-293 cells were seeded in 6-well plates and transfected at a confluence of 80% with 2 μg of the indicated cDNA constructs. 48 h after transfection, the cells were solubilized in 600 μl of immunoprecipitation buffer (10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 μm Pefabloc SC, 0.1% Triton X-100). After incubation on ice for 20 min and centrifugation (26,000 × g for 10 min), 2 μg of anti-c-Myc antibody (clone 9E10, Sigma) or polyclonal anti-EE-antiserum (1:150 dilution, Covance) were added to the clear supernatant (300 μg of protein) and incubated for 1 h at 4 °C. After the addition of a 40-μl 1:1 (v/v) slurry of anti-mouse-IgG-agarose conjugate (Sigma) or protein-A-Sepharose (Amersham Biosciences), the mixture was gently shaken for 4 h at 4 °C. Beads were washed three times with Tris-buffered saline, 0.1% Triton X-100, and 1 μm phenylmethylsulfonyl fluoride, and bound proteins were eluted with 25 μl of sample buffer for 5 min at 95 °C. Precipitated proteins were loaded onto a 12% polyacrylamide gel. After SDS-PAGE and transfer to nitrocellulose membranes, immunoprecipitated proteins were detected by immunoblotting with anti-c-Myc (clone 9E10, Sigma) and anti-Gα antibodies (Gramsch Laboratories, Schwabhausen, Germany). Phospholipase C Assay—For measurement of PLC activity, transfected COS-7 cells seeded on 12-well plates were incubated with 1 of μCi/ml myo-[3H]inositol (Amersham Biosciences) 48 h prior to the assay. Thereafter, the cells were washed once with Hanks' balanced salt solution followed by the addition of fresh Hanks' balanced salt solution supplemented with 10 mm LiCl, and if indicated, 1 mm carbachol. After a 30-min incubation at 37 °C, the reactions were stopped, and the formed [3H]inositol phosphates were determined as described (19Schmidt M. Hüwe S. Fasselt B. Homann D. Rümenapp U. Sandmann J. Jakobs K.H. Eur. J. Biochem. 1994; 225: 667-675Crossref PubMed Scopus (89) Google Scholar). Statistics—Statistical analysis was performed by analysis of variance followed by Tukey's multiple comparison test. A p value <0.05 was considered significant. Concentration-response curves were analyzed using iterative nonlinear regression analysis (GraphPAD Prism). Synergistic Activation of Rho-mediated Gene Transcription by p63RhoGEF and Stimulation of Gq/11-coupled Receptors—To study whether p63RhoGEF is activated by Gq/11-coupled receptors, we coexpressed p63RhoGEF together with the M3Rorthe H1R in HEK-293 cells, in which the mRNA encoding p63RhoGEF could be detected by reverse transcription-PCR (Fig. 1A, inset). Both GPCRs preferentially couple to Gq/11 proteins but can additionally activate other G proteins (20Kühn B. Schmid A. Harteneck C. Gudermann T. Schultz G. Mol. Endocrinol. 1996; 10: 1697-1707Crossref PubMed Scopus (74) Google Scholar, 21Offermanns S. Wieland T. Homann D. Sandmann J. Bombien E. Spicher K. Schultz G. Jakobs K. Mol. Pharmacol. 1994; 45: 890-898PubMed Google Scholar, 22Rümenapp U. Asmus M. Schablowski H. Wozniki M. Han L. Jakobs K. Fahimi-Vahid M. Michalek C. Wieland T. Schmidt M. J. Biol. Chem. 2001; 276: 2474-2479Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). To monitor RhoA activation in intact cells, we measured transcription of a serum-response element (SRE)-controlled reporter gene (SRE.L-luciferase). Overexpression of p63RhoGEF caused a slight increase in luciferase expression, whereas agonist (carbachol, histamine) activation of the M3R or H1R increased transcriptional activity about 35-fold (Fig. 1A). The combined expression of p63RhoGEF and the respective GPCR led to a strong enhancement of agonist-induced luciferase expression (about 90-fold). As shown in Fig. 1B, the agonist-induced transcriptional activity of the M3R and the H1R was further enhanced by the additional expression of Gαq but not of Gα12. In addition, coexpression of Gαq but not Gα12 unmasked the known constitutive activity of the H1R (23Bakker R. Wieland K. Timmerman H. Leurs R. Eur. J. Pharmacol. 2000; 387: R5-R7Crossref PubMed Scopus (150) Google Scholar, 24Bakker R. Schoonus S. Smit M. Timmerman H. Leurs R. Mol. Pharmacol. 2001; 60: 1133-1142Crossref PubMed Scopus (232) Google Scholar). A similar effect was detected upon coexpression of the H1R with p63RhoGEF (Fig. 1A). Coexpression of RGS2, which acts as GTPase-activating protein (GAP) and therefore as an inhibitor of Gαq/11, but not Gα12/13 proteins (25Heximer S.P. Srinivasa P.S. Bernstein L.S. Bernard J.L. Linder M.E. Hepler J.R. Blumer K.J. J. Biol. Chem. 1999; 274: 34253-34259Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 26Wieland T. Chen C.K. Naunyn-Schmiedeberg's Arch. Pharmacol. 1999; 360: 14-26Crossref PubMed Scopus (63) Google Scholar), nearly eliminated the luciferase production induced by the activated M3R alone and largely suppressed the synergistic stimulation detected upon coexpression of p63RhoGEF (Fig. 1C). In contrast, the expression of the RGS domain of the mouse ortholog of p115RhoGEF Lsc (Lsc-RGS), which is a Gα12/13-specific GAP and thereby suppresses Gα12/13-mediated responses in HEK-293 cells (22Rümenapp U. Asmus M. Schablowski H. Wozniki M. Han L. Jakobs K. Fahimi-Vahid M. Michalek C. Wieland T. Schmidt M. J. Biol. Chem. 2001; 276: 2474-2479Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), only slightly reduced (about 10–20%) the M3R-induced luciferase production. The transcriptional activity induced by the agonist-stimulated M3R coexpressed with p63RhoGEF was not affected by Lsc-RGS expression. The expression of both RGS proteins was verified by immunoblotting (not shown). Synergistic Activation of Rho-mediated Gene Transcription by p63RhoGEF and Gαq/11 but Not Gα12/13 Proteins—As the data obtained so far argued for an activation of p63RhoGEF by Gq/11 proteins, we studied the influence of various Gα subunits on p63RhoGEF-induced luciferase expression. Overexpression of wild-type Gαq or Gα11 only marginally (2–3-fold) increased luciferase production. Upon coexpression of p63RhoGEF, the transcriptional activity was increased by 20–40-fold (Fig. 2, A and B). As reported before (11Chikumi H. Vazquez-Prado J. Servitja J. Miyazaki H. Gutkind J. J. Biol. Chem. 2002; 277: 27130-27134Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 17Mao J. Yuan H. Xie W. Simon M. Wu D. J. Biol. Chem. 1998; 273: 27118-27123Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), overexpression of wild-type Gα12 and Gα13 induced strong increases in luciferase production, by 25- and 75-fold, respectively (Fig. 2C). Coexpression of p63RhoGEF with either Gα12 or Gα13 did not lead to further increases in transcriptional activity. The ineffectiveness of p63RhoGEF to enhance gene transcription by Gα12 and Gα13 was not due to a ceiling effect in the experimental setting. Expression of the GTPase-deficient mutants of Gαq (GαqRC) and Gα11 (Gα11QL) induced transcriptional activity in a range similar to that observed with Gα12 or Gα13 (compare Fig. 2, A and C). Upon coexpression of p63RhoGEF, the luciferase production induced by GαqRC and Gα11QL was enhanced in a synergistic manner reaching levels of more than 100-fold. In contrast, the strong transcriptional activity (more than 100-fold) induced by the GTPase-deficient mutants of Gα12 (Gα12QL) and Gα13 (Gα13QL) was not enhanced but was even slightly reduced by coexpression of p63RhoGEF (Fig. 2C). As reported before (15Lutz S. Freichel-Blomquist A. Rümenapp U. Schmidt M. Jakobs K.H. Wieland T. Naunyn-Schmiedeberg's Arch. Pharmacol. 2004; 369: 540-546Crossref PubMed Scopus (42) Google Scholar), the p63RhoGEF-induced luciferase production, even when stimulated by GαqRC or Gα11QL, was largely suppressed by the RhoA–C-inactivating C3 ADP-ribosyl transferase (27Aktories K. Schmidt G. Just I. Biol. Chem. 2000; 381: 421-426Crossref PubMed Scopus (139) Google Scholar) (data not shown). Activated Gαq/11 Proteins Largely Stimulate p63RhoGEF-induced RhoA Activation—The measurements of Rho-dependent gene transcription indicated a stimulation of p63RhoGEF by activated Gαq/11 but not by activated Gα12/13 proteins. To substantiate this hypothesis, the amount of GTP-liganded RhoA was analyzed using the RhoA binding domain of rhotekin (GST·RBD) for pull-down of RhoA-GTP (18Ren X. Schwartz M. Methods Enzymol. 2000; 325: 264-272Crossref PubMed Google Scholar) in lysates of transfected HEK-293 cells (15Lutz S. Freichel-Blomquist A. Rümenapp U. Schmidt M. Jakobs K.H. Wieland T. Naunyn-Schmiedeberg's Arch. Pharmacol. 2004; 369: 540-546Crossref PubMed Scopus (42) Google Scholar). Under the conditions used (submaximally effective amounts of plasmid DNA encoding p63RhoGEF and Gα subunits), expression of either p63RhoGEF, GαqRC, or Gα11QL alone only slightly increased the amount of RhoA-GTP (Fig. 3). Combined expression of p63RhoGEF and GαqRC or Gα11QL induced a substantial increase in RhoA-GTP. In contrast, the combined overexpression of p63RhoGEF with either Gα12QL or Gα13QL did not induce a synergistic activation of RhoA. In accordance with the data observed in the SRF activation assay, coexpression of p63RhoGEF slightly inhibited the RhoA activation by the permanent active Gα12/13 mutants. Activated Gαq/11 Proteins Interact Directly with the C-terminal Half of p63RhoGEF—To investigate whether p63RhoGEF interacts with activated Gαq/11 proteins directly, we expressed various Gα-proteins and p63RhoGEF together in HEK-293 cells. Upon precipitation of the N-terminally Myc-tagged p63RhoGEF with anti-c-Myc antibodies, the immunoprecipitates were analyzed for coprecipitated Gα proteins. As shown in Fig. 4B, the GTPase-deficient mutants GαqRC or Gα11QL were coprecipitated by the anti-c-Myc antibody from cells transfected with the respective eukaryotic expression vectors. In contrast, Gαq was not detected in the p63RhoGEF immunoprecipitates from cells overexpressing wild-type Gαq. These data indicate that only activated Gαq and Gα11 apparently exhibit high affinity binding to p63RhoGEF and thus can be precipitated in a complex with p63RhoGEF. To verify this hypothesis, we overexpressed an EE-tagged version of GαqQL (EE-GαqQL) together with p63RhoGEF and precipitated EE-tagged proteins. As shown in Fig. 4C, p63RhoGEF was coprecipitated together with EE-GαqQL from lysates of cells coexpressing EE-GαqQL and p63RhoGEF. To identify the part of p63RhoGEF in which this interaction takes place, we performed similar experiments with two truncated mutants of p63RhoGEF, i.e. p63-DH (amino acids 138–379), which mainly consists of the DH domain, responsible for the guanine nucleotide exchange activity at RhoA (15Lutz S. Freichel-Blomquist A. Rümenapp U. Schmidt M. Jakobs K.H. Wieland T. Naunyn-Schmiedeberg's Arch. Pharmacol. 2004; 369: 540-546Crossref PubMed Scopus (42) Google Scholar, 16Souchet M. Portales-Casamar E. Mazurais D. Schmidt S. Leger I. Javre J. Robert P. Berrebi-Bertrand I. Bril I. Gout B. Debant A. Calmels T. J. Cell Sci. 2002; 115: 629-640Crossref PubMed Google Scholar) and p63-ΔN, consisting of the C-terminal half (amino acids 295–580) of p63RhoGEF with the PH domain but lacking the DH domain (Fig. 4A). The mutant p63-DH was precipitated to a similar extent as full-length p63RhoGEF (p63-FL) by the anti-c-Myc antibody but did not form a detectable complex with any of the Gαq/11 proteins. In contrast, similar to p63-FL, p63-ΔN coprecipitated GαqRC and Gα11QL but not wild-type Gαq (Fig. 4, B and D). No coprecipitation with any p63RhoGEF construct was observed with either wild-type Gα12 and Gα13 (not shown) or their GTPase-deficient mutants, Gα12QL and Gα13QL (Fig. 4D). The Recombinant Gαq/11 Binding Domain of p63RhoGEF Inhibits Gαq/11 and M3R-induced Rho-mediated Gene Transcription—As the N-terminally truncated mutant p63-ΔN specifically bound activated Gαq/11 proteins, we used the expression of this mutant to study the role of endogenous RhoGEFs in RhoA activation by Gα proteins and the M3R. In contrast to p63-FL, coexpression of p63-ΔN with GαqRC largely reduced the luciferase production induced by the GTPase-deficient Gαq mutant (Fig. 5A). On the other hand, p63-ΔN and p63-FL both weakly inhibited the transcriptional activity induced by Gα13. These data exclude a nonspecific inhibition of RhoA activation by p63-ΔN. Most important, expression of p63-ΔN, which by itself did not induce any transcriptional activity, potently suppressed (by up to 90%) the luciferase production induced by the carbachol-activated M3R (Fig. 5B). p63RhoGEF Competes with PLCβ for Activated Gαq/11 Proteins—The direct interaction of p63RhoGEF with Gαq/11 proteins finally prompted us to study whether p63RhoGEF and PLCβ isozymes, which also directly interact with Gαq/11 proteins (28Rhee S. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1227) Google Scholar), may compete with each other for activation. For this, the effect of p63RhoGEF on Gαq- and M3R-induced luciferase production and PLC activation was examined in COS-7 cells, which are better suited to measure agonist-stimulated PLC activity in response to transient M3R expression than HEK-293 cells. Similar to HEK-293 cells, COS-7 cells express p63RhoGEF endogenously (Fig. 1A, inset). Also, in these cells, coexpression of p63RhoGEF largely enhanced the GαqRC-induced transcriptional activity, by about 10-fold (Fig. 6A). On the other hand, p63RhoGEF, which had no effect on basal PLC activity, significantly reduced the PLC stimulation induced by GαqRC by about 40% (Fig. 6B). An even stronger inhibition, of about 70%, by p63RhoGEF was observed on the M3R-induced PLC stimulation (Fig. 6D). Vice versa, the effect of an overexpression of PLCβ2onM3R-induced PLC activity and luciferase production was studied in COS-7 cells. Overexpression of PLCβ2 significantly increased the carbachol-induced inositol phosphate production from 4- to 5.5-fold (data not shown). As shown in Fig. 6C, the carbachol-induced luciferase production was reduced by about 50% in cells coexpressing PLCβ2 and p63RhoGEF. To further exclude a contribution of the PLC/PKC pathway on the regulation of p63RhoGEF, we additionally studied the influence of the PLC inhibitor U-73122 and the broad range PKC inhibitor bisindolylmaleimide IX on GαqQL- and p63RhoGEF-induced transcriptional activity in COS-7 cells overexpressing PLCβ2. Neither U-73122 (2.5 μm) nor bisindolylmaleimide IX (0.5 μm) altered the transcriptional activity induced by p63RhoGEF, GαqQL, or their combination (data not shown). It is meanwhile well documented that activation of heterotrimeric G proteins of the Gq/11 subfamily induce Rho activation in a variety of cells and tissues (11Chikumi H. Vazquez-Prado J. Servitja J. Miyazaki H. Gutkind J. J. Biol. Chem. 2002; 277: 27130-27134Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 12Hadano S. Hand C.K. Osuga H. Yanagisawa Y. Otomo A. Devon R.S. Miyamoto N. Showguchi-Miyata J. Okada Y. Singaraja R. Figlewicz D. Kwiatkowski T. Hosler B.A. Sagie T. Skaug J. Nasir J. Brown Jr., R.H. Scherer S.W. Rouleau G.A. Hayden M.R. Ikeda J.E. Nat. Genet. 2001; 29: 166-173Crossref PubMed Scopus (594) Google Scholar, 13Vogt S. Grosse R. Schultz G. Offermanns S. J. Biol. Chem. 2003; 278: 28743-28749Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 29Sah V. Hoshijima M. Chien K. Brown J. J. Biol. Chem. 1996; 271: 31185-31190Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). There is increasing evidence that this activation of Rho occurs independently of the PLC/PKC pathway. Most likely, so far unidentified GEFs are involved (11Chikumi H. Vazquez-Prado J. Servitja J. Miyazaki H. Gutkind J. J. Biol. Chem. 2002; 277: 27130-27134Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 13Vogt S. Grosse R. Schultz G. Offermanns S. J. Biol. Chem. 2003; 278: 28743-28749Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). In this report, we presented several lines of evidence that p63RhoGEF, a novel member of the Dbl family of GEFs, might represent the specific GEF, or at least one of the GEFs, mediating this response. Firstly, p63RhoGEF largely enhanced the transcriptional activity of the primarily Gq/11-coupled M3R and H1R in a manner sensitive to RGS2, a negative regulator of Gq/11 activity (25Heximer S.P. Srinivasa P.S. Bernstein L.S. Bernard J.L. Linder M.E. Hepler J.R. Blumer K.J. J. Biol. Chem. 1999; 274: 34253-34259Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 26Wieland T. Chen C.K. Naunyn-Schmiedeberg's Arch. Pharmacol. 1999; 360: 14-26Crossref PubMed Scopus (63) Google Scholar), but insensitive to the G12/13-specific Lsc-RGS (22Rümenapp U. Asmus M. Schablowski H. Wozniki M. Han L. Jakobs K. Fahimi-Vahid M. Michalek C. Wieland T. Schmidt M. J. Biol. Chem. 2001; 276: 2474-2479Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Secondly, it largely enhanced the Rho-mediated gene transcription induced by the GTPase-deficient mutants of Gαq and Gα11 but not of Gα12 and Gα13. This increase in transcriptional activity was completely sensitive to the RhoA-inactivating C3 ADP-ribosyl transferase of Clostridium botulinum (27Aktories K. Schmidt G. Just I. Biol. Chem. 2000; 381: 421-426Crossref PubMed Scopus (139) Google Scholar). Thirdly, coexpression of p63RhoGEF and activated mutants of Gαq and Gα11 largely and synergistically increased the cellular amount of activated RhoA proteins. Finally, p63RhoGEF directly interacted with activated Gαq and Gα11 proteins. This interaction could be detected by means of coimmunoprecipitations as well as by functional inhibition of Gαq-induced gene transcription by the Gαq/11 binding domain of p63RhoGEF. Therefore, our data indicate that p63RhoGEF is the first known GEF that is specifically and so far exclusively regulated by activated Gα subunits of the Gq family. Furthermore, the GEF-deficient mutant p63-ΔN efficiently suppressed Rho-mediated gene transcription induced by the activated M3R and GαqRC, but not Gα13, indicating that p63RhoGEF is in fact specifically involved in RhoA activation by Gq type G proteins and Gq/11-coupled GPCRs, e.g. the M3R and H1R. Thus, p63-ΔN apparently binds to activated Gαq/11 proteins and thereby prevents binding and activation of endogenous p63RhoGEF (or another functionally related RhoGEF) required for RhoA activation by these Gα proteins and the Gq/11-coupled M3R. This hypothesis is on the one hand corroborated by the finding that agonist-induced gene transcription by these receptors was enhanced by coexpression of Gαq but not by Gα12. On the other hand, the stimulatory effect of the M3R was strongly, but not fully, blunted by either p63-ΔN or RGS2, which inactivates and traps activated Gαq/11 proteins (25Heximer S.P. Srinivasa P.S. Bernstein L.S. Bernard J.L. Linder M.E. Hepler J.R. Blumer K.J. J. Biol. Chem. 1999; 274: 34253-34259Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 30Anger T. Zhang W. Mende U. J. Biol. Chem. 2004; 279: 3906-3915Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The rather small remaining part of the M3R action might therefore represent coupling to G12 type G proteins, which is in line with the known G protein coupling specificity of this GPCR (21Offermanns S. Wieland T. Homann D. Sandmann J. Bombien E. Spicher K. Schultz G. Jakobs K. Mol. Pharmacol. 1994; 45: 890-898PubMed Google Scholar, 22Rümenapp U. Asmus M. Schablowski H. Wozniki M. Han L. Jakobs K. Fahimi-Vahid M. Michalek C. Wieland T. Schmidt M. J. Biol. Chem. 2001; 276: 2474-2479Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). This interpretation is further corroborated by the small inhibitory effect (about 15%, Fig. 1C) of the Gα12/13-specific GAP Lsc-RGS (22Rümenapp U. Asmus M. Schablowski H. Wozniki M. Han L. Jakobs K. Fahimi-Vahid M. Michalek C. Wieland T. Schmidt M. J. Biol. Chem. 2001; 276: 2474-2479Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Screening data bases of the human genome project and the mouse genome project revealed that p63RhoGEF, in contrast to other known RhoA-specific GEFs, has no close homolog. Besides the existence of a DH/PH tandem motif, p63RhoGEF is especially not related to the members of the p115RhoGEF family, which mediate the activation of RhoA by G12/13 proteins(8Fukuhara S. Murga C. Zohar M. Igishi T. Gutkind J. J. Biol. Chem. 1999; 274: 5868-5879Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 9Fukuhara S. Chikumi H. Gutkind J. FEBS Lett. 2000; 485: 183-188Crossref PubMed Scopus (212) Google Scholar, 10Mao J. Yuan H. Xie W. Wu D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12973-12976Crossref PubMed Scopus (114) Google Scholar,31Hart M. Jiang X. Kozasa T. Roscoe W. Singer W. Gilman A. Sternweis P. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (677) Google Scholar). Accordingly, we could not detect any interaction of p63RhoGEF with or activation by Gα12 family members. Moreover, p63RhoGEF does not contain the RGS homology domain, which is involved in the interaction of the p115 RhoGEF family members with activated Gα subunits. The activation of p63RhoGEF is apparently induced by a direct interaction of activated Gαq/11 proteins with the C-terminal half of the p63RhoGEF molecule, containing the PH domain. This interaction likely results in the relief of the described autoinhibition of the GEF activity (15Lutz S. Freichel-Blomquist A. Rümenapp U. Schmidt M. Jakobs K.H. Wieland T. Naunyn-Schmiedeberg's Arch. Pharmacol. 2004; 369: 540-546Crossref PubMed Scopus (42) Google Scholar, 32Rümenapp U. Freichel-Blomquist A. Wittinghofer B. Jakobs K. Wieland T. Biochem. J. 2002; 366: 721-728Crossref PubMed Google Scholar). Therefore, our data indicate that the C-terminal Gαq/11 binding site of p63RhoGEF has to be quite distinct from the interaction site by which p115 family members interact with activated Gα subunits. With regard to this interaction site, it is noteworthy, however, that the p115RhoGEF family member, LARG, has been shown to interact with and be activated by Gαq (13Vogt S. Grosse R. Schultz G. Offermanns S. J. Biol. Chem. 2003; 278: 28743-28749Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 14Booden M. Siderovski D. Der C. Mol. Cell. Biol. 2002; 22: 4053-4061Crossref PubMed Scopus (149) Google Scholar). Whether this interaction is mediated by the RGS homology domain of LARG is still a matter of debate. One report indicated a binding of activated Gαq to the RGS domain of LARG and a productive coupling of activated Gαq to RhoA via LARG (14Booden M. Siderovski D. Der C. Mol. Cell. Biol. 2002; 22: 4053-4061Crossref PubMed Scopus (149) Google Scholar). Another report showed that a LARG mutant lacking the DH domain but not the RGS homology domain interfered with Gαq-induced RhoA activation (13Vogt S. Grosse R. Schultz G. Offermanns S. J. Biol. Chem. 2003; 278: 28743-28749Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). However, a third publication did not detect an interaction of activated Gαq with the RGS homology domain of LARG, which also failed to inhibit GαqQL-induced luciferase production (11Chikumi H. Vazquez-Prado J. Servitja J. Miyazaki H. Gutkind J. J. Biol. Chem. 2002; 277: 27130-27134Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). In contrast to the data on the interaction of Gαq with the LARG-RGS homology domain, the interaction of Gα12 family members with this domain and a productive coupling to RhoA were found by both groups (11Chikumi H. Vazquez-Prado J. Servitja J. Miyazaki H. Gutkind J. J. Biol. Chem. 2002; 277: 27130-27134Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 14Booden M. Siderovski D. Der C. Mol. Cell. Biol. 2002; 22: 4053-4061Crossref PubMed Scopus (149) Google Scholar). Another line of evidence for p63RhoGEF to be a so far unknown effector molecule for Gq proteins resulted from the experiments analyzing the influence of the canonical Gαq effector molecule PLCβ (28Rhee S. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1227) Google Scholar) on the activation of p63RhoGEF and vice versa. The overexpression of p63RhoGEF inhibited Gαq-stimulated PLC activity as well as the overexpression of PLCβ2 inhibited the Gαq-induced gene transcription. These data argue for a direct competition of p63RhoGEF and PLCβ isoforms for activated Gαq/11 proteins. In line with the inhibitory effect of PLCβ2 and in accordance with previous observations, which indicated the existence of a Gq/11-activated RhoGEF not regulated by PLC and the subsequent Ca2+ mobilization and PKC activation (11Chikumi H. Vazquez-Prado J. Servitja J. Miyazaki H. Gutkind J. J. Biol. Chem. 2002; 277: 27130-27134Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), the M3R- and GαqQL-induced gene transcription was not influenced by the inhibition of PLC and PKC activity, by U73122 (33Bleasdale J. Bundy G. Bunting S. Fitzpatrick F. Huff R. Sun F. Pike J. Adv. Prostaglandin Thromboxane Leukotriene Res. 1989; 19: 590-593PubMed Google Scholar) and bisindolylmaleimide IX (34Davis P. Hill C. Lawton G. Nixon J. Wilkinson S. Hurst S. Keech E. Turner S. J. Med. Chem. 1992; 35: 177-184Crossref PubMed Scopus (180) Google Scholar), respectively. In summary, the data presented herein define a new signaling pathway for GPCRs, with p63RhoGEF serving as a direct Gαq/11 effector molecule. It directly links these GPCRs to RhoA- and RhoA-dependent cellular processes, apparently in competition with the canonical PLCβ/PKC pathway. We thank S. Alexa and A. Hahn for expert technical assistance. The gifts of various plasmids and antisera by J. H. Kehrl, A. Hall, B. Moepps, M. I. Simon, T. Walther, J. Mao, M. A. Schwartz, and D. Wu are greatly appreciated. Download .pdf (.04 MB) Help with pdf files
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