Specific Involvement of G Proteins in Regulation of Serum Response Factor-mediated Gene Transcription by Different Receptors

Regulation of serum response factor (SRF)-mediated gene transcription by G protein subunits and G protein-coupled receptors was investigated in transfected NIH3T3 cells and in a cell line that was derived from mice lacking Gαq and Gα11. We found that the constitutively active forms of the α subunits of the Gqand G12 class of G proteins, including Gαq, Gα11, Gα14, Gα16, Gα12, and Gα13, can activate SRF in NIH3T3 cells. We also found that the type 1 muscarinic receptor (m1R) and α1-adrenergic receptor (AR)-mediated SRF activation is exclusively dependent on Gαq/11, while the receptors for thrombin, lysophosphatidic acid (LPA), thromboxane A2, and endothelin can activate SRF in the absence of Gαq/11. Moreover, RGS12 but not RGS2, RGS4, or Axin was able to inhibit Gα12 and Gα13-mediated SRF activation. And RGS12, but not other RGS proteins, blocked thrombin- and LPA-mediated SRF activation in the Gαq/11-deficient cells. Therefore, the thrombin, LPA, thromboxane A2, and endothelin receptors may be able to couple to Gα12/13. On the contrary, receptors including β2- and α2-ARs, m2R, the dopamine receptors type 1 and 2, angiotensin receptors types 1 and 2, and interleukin-8 receptor could not activate SRF in the presence or absence of Gαq/11, suggesting that these receptors cannot couple to endogenous G proteins of the G12 or Gqclasses. Regulation of serum response factor (SRF)-mediated gene transcription by G protein subunits and G protein-coupled receptors was investigated in transfected NIH3T3 cells and in a cell line that was derived from mice lacking Gαq and Gα11. We found that the constitutively active forms of the α subunits of the Gqand G12 class of G proteins, including Gαq, Gα11, Gα14, Gα16, Gα12, and Gα13, can activate SRF in NIH3T3 cells. We also found that the type 1 muscarinic receptor (m1R) and α1-adrenergic receptor (AR)-mediated SRF activation is exclusively dependent on Gαq/11, while the receptors for thrombin, lysophosphatidic acid (LPA), thromboxane A2, and endothelin can activate SRF in the absence of Gαq/11. Moreover, RGS12 but not RGS2, RGS4, or Axin was able to inhibit Gα12 and Gα13-mediated SRF activation. And RGS12, but not other RGS proteins, blocked thrombin- and LPA-mediated SRF activation in the Gαq/11-deficient cells. Therefore, the thrombin, LPA, thromboxane A2, and endothelin receptors may be able to couple to Gα12/13. On the contrary, receptors including β2- and α2-ARs, m2R, the dopamine receptors type 1 and 2, angiotensin receptors types 1 and 2, and interleukin-8 receptor could not activate SRF in the presence or absence of Gαq/11, suggesting that these receptors cannot couple to endogenous G proteins of the G12 or Gqclasses. lysophosphatidic acid adrenergic receptor angiotensin C. butulinum C3 transferase cAMP response element β-galactosidase G protein-coupled receptors interleukin-8 muscarinic cholinergic receptor type 1 and type 2, respectively pertussis toxin regulator of G protein signaling serum response element serum response factor green fluorescence protein. Hormones, neurotransmitters, and many other biologically active molecules, such as lysophosphatidic acid (LPA),1 thrombin, catecholamines, endothelin, etc., transduce their signals through heterotrimeric G proteins (1Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4700) Google Scholar, 2Birnbaumer L. Abramowitz J. Brown A.M. Biochim. Biophys. Acta. 1990; 90: 163-224Crossref Scopus (961) Google Scholar). Molecular cloning has revealed at least four classes of G protein α subunits: Gαs, Gαi, Gαq, and Gα12 (3Simon M.I. Strathman M.P. Gautum M. Science. 1991; 252: 802-808Crossref PubMed Scopus (1580) Google Scholar). The Gαs subunits and Gαi subunits regulate adenylyl cyclase activities, while the Gαq subunits regulate phospholipase C activities. However, the function of the Gα12 class of G proteins, which includes Gα12 and Gα13, remains to be elucidated. Activated forms of Gα12 and Gα13, when transfected into fibroblast cells, were shown to induce transformation phenotypes (4Jiang H. Wu D. Simon M.I. FESB Lett. 1993; 330: 319-322Crossref PubMed Scopus (101) Google Scholar, 5Hermouet S. Merendino J.J. Gutkind J.S. Spiegel A.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 455-459Crossref Scopus (104) Google Scholar, 6Xu N. Voyno-Yasenetskaya T. Gutkind J.S. Biochem. Biophys. Res. Commun. 1994; 201: 603-609Crossref PubMed Scopus (87) Google Scholar), suggesting that this class of G proteins may be involved in cell growth regulation. Moreover, Gα12 and Gα13 were shown to induce formation of stress fibers in fibroblast cells through small G protein RhoA (7Buhl 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). This observation was supported by the report that Gα12 activated serum response factor (SRF) through RhoA (8Fromm C. Coso O.A. Montaner S. Xu N. Gutkind J.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10098-10103Crossref PubMed Scopus (196) Google Scholar). The in vivo function of Gα13 was also investigated using the gene-targeting technique in mice. Mice lacking Gα13 are embryonic lethal apparently due to the failure to develop vasculature structures, indicating that Gα13 may be involved in the function of endothelial cells (9Offermanns S. Mancino V. Revel J.P. Simon M.I. Science. 1997; 275: 533-536Crossref PubMed Scopus (287) Google Scholar). In the same study, thrombin-mediated chemotaxis of fibroblasts lacking Gα13 was blocked, indicating that the thrombin receptor couples to Gα13. This is consistent with the observation that thrombin as well as a thromboxane A2 receptor agonist could stimulate the binding of a photo-affinity GTP analog to Gα13 (10Offermanns S. Laugwitz K.L. Spicher K. Schultz G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 504-508Crossref PubMed Scopus (392) Google Scholar). However, there were contradictory reports with regard to the involvement of the Gq class of G proteins in RhoA and SRF activation (7Buhl 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, 8Fromm C. Coso O.A. Montaner S. Xu N. Gutkind J.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10098-10103Crossref PubMed Scopus (196) Google Scholar, 16Heximer S.P. Watson N. Linder M.E. Blumer K.J. Hepler J.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14389-14393Crossref PubMed Scopus (312) Google Scholar). RGS (regulator of G protein signaling) proteins belong to a growing family of proteins that contains homologous RGS domains (11Koelle M.R. Curr. Opin. Cell Biol. 1997; 9: 143-147Crossref PubMed Scopus (174) Google Scholar, 12Dohlman H.G. Thorner J. J. Biol. Chem. 1997; 272: 3871-3874Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar). Some of these proteins such as GIAP and RGS4 were shown to inhibit G protein-mediated signaling by interacting with the Gαiand Gαo subunits and stimulating their GTPase activities (13Berman D.M. Wilkie T.M. Gilman A.G. Cell. 1996; 86: 445-452Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar), which are also referred to as GTPase-activating protein activities. RGS2 and RGS4 were found to inhibit Gαq-mediated activation of phospholipase Cβ (14Hepler J.R. Berman D.M. Gilman A.G. Kozasa T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 428-432Crossref PubMed Scopus (335) Google Scholar, 15Yan Y. Chi P.P. Bourne H.R. J. Biol. Chem. 1997; 272: 11924-11927Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 16Heximer S.P. Watson N. Linder M.E. Blumer K.J. Hepler J.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14389-14393Crossref PubMed Scopus (312) Google Scholar), implying that they may function as GTPase-activating protein for Gαq. Interestingly, most of these RGS proteins were unable to inhibit Gs-mediated signaling, and they could not stimulate the GTPase activity of the Gαs proteins (13Berman D.M. Wilkie T.M. Gilman A.G. Cell. 1996; 86: 445-452Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar,14Hepler J.R. Berman D.M. Gilman A.G. Kozasa T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 428-432Crossref PubMed Scopus (335) Google Scholar). Regulation of the G12/13 proteins by RGS proteins has not been investigated. In this report, we characterized the abilities of the G protein subunits and of a number of GPCRs to stimulate SRF-mediated gene transcription using a cotransfection system. We found that the α subunits of the Gq and G12 classes of G proteins can induce SRF activation in a C3-dependent manner. We also found that the activation of SRF by some of GPCRs depends exclusively on Gαq/11, while others do not. Those that activate SRF independently of Gαq/11 may act through Gα12/13. In addition, we, for the first time, identified a RGS protein, RGS12, that can inhibit the Gα12/13function. -NIH3T3 and the Gαq/11-deficient cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum at 37 °C under 5% CO2. The Gαq/11-deficient cell line was established from mice lacking both Gαq and Gα11 (17Offermanns S. Zhao L.P. Gohla A. Sarosi I. Simon M.I. Wilkie T.M. EMBO J. 1998; 17: 4304-4312Crossref PubMed Scopus (203) Google Scholar). For transfection, cells (5 × 104 cells/well) were seeded into 24-well plates the day before transfection. Cells were transfected with 0.5 μg of DNA/well using LipofectAMINE Plus (Life Technologies, Inc.), as suggested by the manufacturer. The transfection was stopped after 3 h by switching to culture medium containing 0.5% fetal bovine serum. Cell extracts were collected 24 h later for luciferase assays. Luciferase assays were performed using Boehringer Mannheim Constant Light Luciferase Assay Kit as instructed. Cell lysates were first taken for determining in a Wallac multicounter the fluorescence intensity emitted by GFP proteins, which are cotransfected with the luciferase reporter gene plasmid and used to normalize the transfection efficiency. The Wallac counter (Wallac AG&G, Finland) is capable of counting both fluorescence and luminescence. Then, the luciferase substrate was added to the cell lysates, and luciferase activities were determined by measuring luminescence intensity using the same counter. Luminescence intensities were normalized against fluorescence intensities. DNA concentrations were adjusted if transfection of any of the cDNAs resulted in significant differences between normalized and non-normalized data. All of the G protein subunits and GPCRs were in pCMV expression vectors as described previously (18Wu D. Lee C.-H. Rhee S.G. Simon M.I. J. Biol. Chem. 1992; 267: 1811-1817Abstract Full Text PDF PubMed Google Scholar, 19Wu D. Katz A. Lee C.-H. Jiang H. Simon M.I. J. Biol. Chem. 1992; 267: 25798-25802Abstract Full Text PDF PubMed Google Scholar, 20Wu D. LaRosa G.J. Simon M.I. Science. 1993; 261: 101-103Crossref PubMed Scopus (332) Google Scholar). The SRE.L-luciferase reporter plasmid was constructed as described in Ref. 7Buhl 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, except the luciferase gene was used as the reporter instead of the chloramphenicol acetyltransferase gene. The CRE-luciferase reporter gene plasmid was purchased from Strategene, La Jolla, CA. RGS2, RGS4, and RGS12 (kindly provided by Sheng-Cai Lin) were also in the pCMV vector. Axin (kindly provided by F. Costantini) was in the pcDNA3 vector (Invitrogen). C3 was kindly provided by Alan Hall. A cotransfection system was used to characterize signal transduction pathways mediated by G proteins that lead to the regulation of SRF-dependent gene transcription. SRF-mediated gene transcription was evaluated by determining the activity of luciferase, the production of which is regulated by a transcription regulatory sequence element, called SRE.L. SRE.L is a derivative of c-Fos serum response element (SRE), to which SRF but not tertiary complex factor binds (21Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1202) Google Scholar). Thus, SRE.L-mediated production of luciferase mainly depends on the activity of SRF. The abilities of various G protein subunits to regulate SRE.L-mediated gene transcription were determined by cotransfecting NIH3T3 cells with the reporter gene plasmid and cDNA encoding one of constitutively active Gα subunits. We found that cells expressing activated α subunit of Gq, G11, G14, G16, G12, or G13, produced markedly higher levels of luciferase than those expressing the control β-galactosidase (LacZ), whereas expression of activated Gαi or Gαo did not (Fig. 1). This indicates that the α subunits of the Gq and G12 classes of G proteins can lead to SRF activation. The finding that C3 blocked SRF activation by the G protein α subunits suggests that the small GTP-binding protein RhoA (Fig. 1) may mediate the SRF activation. C3 (Clostridium butulinum C3 transferase) is a specific RhoA inactivator, which ADP-ribosylates RhoA (21Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1202) Google Scholar). In our transfection system, C3 inhibited only RhoA-induced but not Cdc42- (Fig. 1) or Rac1- (data not shown) induced SRF activation, indicating that C3 acted specifically. Activation of SRF by the Gq and G12 family of G proteins allows us to determine which receptors can couple to these G proteins to activate SRF. Many cells, including fibroblasts, contain endogenous receptors for thrombin and LPA, which belong to a superfamily of GPCR. In addition, the thrombin receptor was shown previously to couple to the G12/13 proteins (10Offermanns S. Laugwitz K.L. Spicher K. Schultz G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 504-508Crossref PubMed Scopus (392) Google Scholar) so that it may function as a positive control. We found that both thrombin and LPA were able to stimulate SRE.L-mediated gene transcription in NIH3T3 cells transfected with the reporter gene plasmids (Fig. 2 A). This result suggests that NIH3T3 cells contain endogenous receptors for thrombin and LPA. A number of other GPCRs were also tested for their abilities to stimulate SRE.L-mediated gene transcription in transfected 3T3 cells. Cells expressing m1R and α1-AR showed marked increases in luciferase activities in response to carbachol and norepinephrine, respectively (Fig. 1 B). Neither carbachol nor norepinephrine elicited any change in the luciferase activity in cells transfected with the reporter gene plasmid alone (Fig. 1 A), indicating that there are no endogenous receptors for carbachol and norepinephrine in NIH3T3 cells. A mutant of α1-AR, α1-ARΔ2, was also tested in the same cotransfection system. α1-ARΔ2, which was unable to couple to Gαq/11to activate phospholipase C (22Wu D. Jiang H. Simon M.I. J. Biol. Chem. 1995; 270: 9828-9832Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), lost the ability to activate SRF in the presence of ligand norepinephrine. This suggests that α1-AR-mediated SRF activation appears to depend on Gαq/11 in fibroblasts. The fact that the expression of C3 was able to abolish thrombin-, LPA-, norepinephrine-, and carbachol-induced SRF activation suggests that the SRF activation by these ligands be mediated by RhoA (Fig. 2, A and B). Furthermore, we tested Gi-coupling m2R and IL-8 receptor and Gs-coupling β2-AR. None of these receptors was able to stimulate SRF activity in response to their ligands (Fig. 2 B). These results are consistent with the observation that the Gi and Go are not involved in regulation of SRF. It has been demonstrated by various approaches that m1R and α1-AR couple to the Gq proteins (18Wu D. Lee C.-H. Rhee S.G. Simon M.I. J. Biol. Chem. 1992; 267: 1811-1817Abstract Full Text PDF PubMed Google Scholar, 23Berstein G. Blank J.L. Smrcka A.V. Higashijima T. Sternweis P.C. Exton J.H. Ross E.M. J. Biol. Chem. 1992; 267: 8081-8088Abstract Full Text PDF PubMed Google Scholar). Although the receptors for thrombin and LPA have not been rigorously tested for their abilities to couple to the G proteins of the Gq family, they were shown previously to stimulate inositol phosphate accumulation (24Noh D.Y. Shin S.H. Rhee S.G. Biochim. Biophys. Acta. 1995; 1242: 99-113Crossref PubMed Scopus (254) Google Scholar), suggesting that they may couple to the Gq proteins. To test the roles of Gαq/11 in SRF activation by these receptors, a fibroblast cell line derived from mice lacking Gq/11 was used. Thrombin, LPA, carbachol, and norepinephrine were added to the Gαq/11-deficient cells that were transfected with the SRE.L-luciferase reporter gene plasmid. Both thrombin and LPA were able to stimulate SRE.L-mediated gene transcription, which can be blocked by C3 (Fig. 3 A). Thrombin and LPA showed EC50 values of about 5 units and 4 μm in stimulation of luciferase activities, respectively (Fig. 4, A and B). Thus, this cell line, like NIH3T3 cells, also contains endogenous thrombin and LPA receptors, and the receptors for thrombin and LPA are able to activate SRF independent of Gαq/11. The Gαq/11-deficient cells were also cotransfected with the cDNA encoding the thrombin receptor and the reporter gene plasmid, and cells expressing the recombinant thrombin receptors showed higher thrombin-induced production of luciferase than those transfected with the control plasmid (Fig. 3 A). Therefore, both endogenous and recombinant thrombin receptors are able to activate SRF in a Gαq/11-independent way.Figure 4Dose-dependent stimulation of SRF. The Gq/11-deficient cells were cotransfected with 0.15 μg of SRE.L-luciferase reporter plasmid, 0.15 μg of GFP expression construct, and 0.2 μg of LacZ (A, B), the endothelin receptor type 1a (C), and thromboxane A2 receptor (D). The next day, cells were lysed 6 h after the addition of thrombin (A), LPA (B), endothelin (C), and U46619 (D) with concentrations indicated in the figure. Data are processed and presented as described in the legend to Fig. 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The inability of norepinephrine and carbachol to elicit reporter gene transcription (data not shown) may be due to the lack of endogenous receptors. Therefore, these two ligands were tested in the Gαq/11-deficient cells transiently expressing α1-AR and m1R, respectively. The ligands were still unable to elicit responses, even in the presence of the recombinant receptors (Fig. 3 B). However, the responses of cells to norepinephrine were restored when Gαq was reintroduced back into the Gαq/11-deficient cells by cotransfection with α1-AR and the reporter gene (Fig. 3 B). The same result was also observed for carbachol when m1R and Gαq were coexpressed in the Gαq/11-deficient cells (Fig. 3 B). Therefore, we conclude that α1-AR and m1R are dependent exclusively on Gαq/11 in SRF activation. This conclusion further strengthens the idea that the Gq proteins are capable of activating RhoA and SRF. Since α1-AR is able to couple to all the members of the Gq class of G proteins, including Gα14 and Gα16 (19Wu D. Katz A. Lee C.-H. Jiang H. Simon M.I. J. Biol. Chem. 1992; 267: 25798-25802Abstract Full Text PDF PubMed Google Scholar), the inability of α1-AR to activate SRF in the Gαq/11-deficient cell line suggests that there are not sufficient levels of endogenous Gα14 and Gα16 in this cell line. Thus, if any receptor can induce SRF activation in this cell line, it would suggest that this receptor is able to couple to G proteins other than the Gq class, which would be the G12/13 proteins. Thus, the Gαq/11-deficient cell line may be used for testing the coupling of receptors to the G proteins of the G12 class. We tested a number of receptors in this Gαq/11-deficient cell line, including the endothelin receptors 1a and 1b, thromboxane A2 receptor, angiotensin (AT) receptors type 1a and type 2, α2- and β2-AR, and dopamine receptors type 1 and 2. Endothelin was able to activate the SRF.L-mediated gene transcription in cells expressing endothelin receptor 1a (Fig. 3 C) or 1b (data not shown) with EC50 values of about 0.25 nm (Fig. 4 C and data not shown), while the thromboxane A2 receptor agonist U46619 gave an EC50 value of 1 nm (Figs. 3 C and 4 D). Angiotensin, norepinephrine, isoprenaline, and dopamine failed to elicit any SRF activation in cells expressing AT receptors, α1-AR, β2-AR, and dopamine receptors, respectively (Fig. 3 C). These results suggest that endothelin receptors and the thromboxane A2 receptor may be able to couple to G12/13. The effects of Gβγ subunits on SRF activation were also tested in the same cotransfection system. Cells coexpressing β1 and γ1 or β1 and γ5 showed approximately 1-fold more luciferase activity than those expressing the control LacZ (Fig. 5 A). This result suggests that Gβγ subunits may also be involved in regulation of SRF, but with less potency than the Gαq and Gα12 subunits. To further investigate the role of Gβγ in SRF activation, we tested if Gβγ has any effect on Gα-mediated SRF activation by coexpressing activated Gα13 or Gαq with Gβγ. Interestingly, cells coexpressing Gα13QL and Gβ1γ1 or Gβ1γ5 showed higher luciferase activities than the sum of the activities shown by cells expressed Gβγ and Gα13 alone. This suggests that Gα13 may work synergistically with Gβγ in activation of SRF. Similar synergistic effects were also observed with Gαq and Gβγ. To confirm the above observation, the Gi-coupled m2-muscarinic receptor (m2R) was expressed in NIH3T3 cells, and carbachol-induced SRF-mediated transcription was determined. As expected, cells expressing m2R showed marginal carbachol-mediated increases in luciferase activities (Fig. 5 A), which may be attributed to Gβγ released from the Gi proteins, since Gαi does not activate SRF (Fig. 1). However, when m2R was coexpressed with m1R in NIH3T3 cells, there was a synergistic response to the muscarinic ligand carbachol (Fig. 5 B),i.e. carbachol induced more luciferase activities in cells coexpressing m1R and m2R than those expressing m1R or m2R alone. Furthermore, Pertussis toxin (Ptx), which did not inhibit the response to carbachol in cells expressing m1R, inhibited the response to carbachol in cells coexpressing m1R and m2R (Fig. 5 B). It appears that Ptx blocked only the part contributed by m2R. Ptx is a bacteria toxin that ADP-ribosylates the α subunits of the Gi, Go, and Gt proteins and blocks the activation of these G proteins by receptors. Therefore, it is reasonable to conclude that Gβγ works synergistically with Gαq and Gα13 in regulation of SRF. The wild-type Gα12 and Gα13, when expressed in NIH3T3 cells, also showed significant stimulation of SRF-mediated transcription (Fig. 6 A), although the activity is usually one-fifth of that of the QL mutant when the same amount of DNA is used in transfection (data not shown). The relative high activities of the wild-type Gα12 and Gα13 may be due to the slow intrinsic GTPase activities of these Gα subunits (10Offermanns S. Laugwitz K.L. Spicher K. Schultz G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 504-508Crossref PubMed Scopus (392) Google Scholar, 25Macrez-Lepretre N. Kalkbrenner F. Morel J.L. Schultz G. Mironneau J. J. Biol. Chem. 1997; 272: 10095-10102Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 26Singer W.D. Miller R.T. Sternweis P.C. J. Biol. Chem. 1994; 269: 19796-19802Abstract Full Text PDF PubMed Google Scholar). The SRF activation by the wild-type Gα12 and Gα13 allowed us to test if the RGS proteins could inhibit Gα12/13-mediated effects. We tested RGS2, RGS4, RGS12, and Axin. RGS2 and RGS4 are among the well characterized RGS proteins, which show GTPase-activating protein activities for the Gi and/or Gqfamilies of α subunits (13Berman D.M. Wilkie T.M. Gilman A.G. Cell. 1996; 86: 445-452Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar, 14Hepler J.R. Berman D.M. Gilman A.G. Kozasa T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 428-432Crossref PubMed Scopus (335) Google Scholar, 27Huang C. Hepler J.R. Gilman A.G. Mumby S.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6159-6163Crossref PubMed Scopus (150) Google Scholar, 28Tesmer J.J. Berman D.M. Gilman A.G. Sprang S.R. Cell. 1997; 89: 251-261Abstract Full Text Full Text PDF PubMed Scopus (682) Google Scholar). RGS12 (29Snow B.E. Antonio L. Suggs S. Gutstein H.B. Siderovski D.P. Biochem. Biophys. Res. Commun. 1997; 233: 770-777Crossref PubMed Scopus (99) Google Scholar) and Axin (30Zeng L. Fagotto F. Zhang T. Hsu W. Vasicek T.J. Perry W.L.R. Lee J.J. Tilghman S.M. Gumbiner B.M. Costantini F. Cell. 1997; 90: 181-192Abstract Full Text Full Text PDF PubMed Scopus (784) Google Scholar) are two recently cloned proteins that contain the RGS domains but have not been tested for their abilities to regulate G protein-mediated signaling. Coexpression of RGS12 significantly inhibited Gα12- and Gα13-induced SRF activation (Fig. 6 A), whereas RGS2, Axin (Fig. 6 A), and RGS4 (data not shown) showed little effects. Moreover, expression of RGS12 did not inhibit activated RhoA-mediated SRF activation (data not shown), indicating that inhibition of Gα12/13-mediated effects by RGS12 is not due to nonspecific inhibition of downstream proteins. The inhibition by RGS12 is also unlikely to be the result of the changes in the expression levels of cotransfected G proteins, since coexpression of RGS12, Axin, or RGS2 did not alter the expression levels of Gα12 or Gα13 (Fig. 6 E). Therefore, RGS12 is likely to affect the function of Gα12/13 directly. The effects of the RGS proteins on LPA and thrombin-mediated SRF activation were also tested in the Gαq/11-deficient fibroblast cell line, where LPA and thrombin-mediated SRF activation is presumably mediated by the G12 family of G proteins. The Gαq/11-deficient cells were cotransfected with SRE.L reporter plasmids and cDNA encoding RGS2, RGS4, RGS12, or Axin, and LPA-induced production of luciferase was determined. Cells expressing RGS12 showed consistently lower luciferase activities than those expressing the control LacZ, RGS2, RGS4, or Axin (Fig. 6 B). RGS12 was also able to inhibit thrombin-mediated SRF activation in the Gαq/11-deficient cells (data not shown). Since RGS12 was able to inhibit Gα12/13 (Fig. 6 A), RGS12-mediated attenuation of LPA and thrombin-induced SRF activation may result from the inhibition of endogenous Gα12/13proteins by RGS12 in the Gαq/11-deficient cells. The effects of RGS12 on other G protein-mediated signaling pathways were also investigated. RGS12 as well as RGS2, RGS4, or Axin did not affect β-adrenergic agonist isoprenaline-induced CRE-mediated gene transcription (Fig. 6 C), suggesting that none of the RGS proteins could inhibit the Gs function. However, all the RGS proteins except Axin were also able to inhibit norepinephrine-induced SRF activation in 3T3 cells expressing α1-AR (Fig. 6 D). As demonstrated earlier, α1-AR-mediated SRF activation is dependent on Gαq/11 proteins. Thus, inhibition of α1-AR-mediated SRF by the RGS proteins suggests that these RGS proteins may inhibit Gαq/11. Both RGS2 and RGS4 were also able to inhibit norepinephrine-induced phospholipase C activation in COS-7 cells coexpressing α1-AR. 2D. Wu, unpublished data. Thus, the action of RGS12 may not be specific to the G12 class of G proteins. In this report, we have characterized the involvement of G protein subunits in activation of SRF by a number of GPCRs. Our findings that Gα12 and Gα13 can activate SRF are consistent with previous reports that Gα12 activates SRF through RhoA (8Fromm C. Coso O.A. Montaner S. Xu N. Gutkind J.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10098-10103Crossref PubMed Scopus (196) Google Scholar) and that Gα12/13 induces formation of stress fibers via RhoA (7Buhl 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). The literature, however, appears to be inconsistent with regard to the involvement of Gαq in SRF activation (8Fromm C. Coso O.A. Montaner S. Xu N. Gutkind J.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10098-10103Crossref PubMed Scopus (196) Google Scholar, 31Sah V.P. Hoshijima M. Chien K.R. Brown J.H. J. Biol. Chem. 1996; 271: 31185-31190Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). Our findings described in this report demonstrate that not only Gαq, but also all other members of this class of G proteins can activate SRF probably through RhoA. The fact that m1R and α1-AR use Gαq/11 exclusively in activation of SRF strongly supports the involvement of Gq in SRF activation. Not all Gq-coupled receptors, however, activate SRF in Gαq/11-dependent pathways. Receptors, including the endothelin receptors and thromboxane receptor A2, both of which are known to couple to Gq to activate phospholipase C, can activate SRF in a Gαq/11-independent way (32Wange R.L. Smrcka A. Sternweis P. Exton J. J. Biol. Chem. 1991; 266: 11409-11412Abstract Full Text PDF PubMed Google Scholar). Moreover, receptors for thrombin and LPA can also lead to RhoA and SRF activation independently of Gαq/11, although these two receptors may also be able to couple to the Gi proteins in addition to the Gq proteins. Interestingly, among those we tested none of the receptors that are previously known to predominantly couple to Gi or Gs can induce activation of SRF in the presence or absence of Gαq/11. Unlike the Gα subunits of the Gq and G12classes, the expression of activated Gαo or Gαi does not activate SRF. In addition, Gsdoes not appear to activate SRF because the β-AR agonist isoprenaline did not stimulate SRF-mediated transcription in cells expressing β2-AR, while isoprenaline was able to stimulate CRE-mediated transcription. The inability of β-AR to activate SRF also suggests that β2-AR cannot couple to endogenous G12/13 in the fibroblasts. It is also apparent that m1R and α1-AR as well as α2-AR, β1-AR, the IL-8 receptor, and D1 and D2 receptors are unable to couple to Gα12 or Gα13, because these receptors could not activate SRF in the Gαq/11-deficient cells. The thromboxane A2 and thrombin receptors were shown previously to couple to Gα12 and Gα13 using different methods (9Offermanns S. Mancino V. Revel J.P. Simon M.I. Science. 1997; 275: 533-536Crossref PubMed Scopus (287) Google Scholar, 10Offermanns S. Laugwitz K.L. Spicher K. Schultz G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 504-508Crossref PubMed Scopus (392) Google Scholar, 33Post G.R. Collins L.R. Kennedy E.D. Moskowitz S.A. Aragay A.M. Goldstein D. Brown J.H. Mol. Biol. Cell. 1996; 7: 1679-1690Crossref PubMed Scopus (66) Google Scholar). Thus, SRF activation by these receptors should at least in part be mediated by Gα12/13. The LPA receptor and endothelin receptors may also be able to couple to Gα12/13 unless there exist yet-to-be identified G protein α subunits that can also activate SRF in the Gαq/11-deficient mouse fibroblast cells. Under the same premise, the inhibition of LPA and thrombin-induced SRF activation by RGS12 may be attributed to the inhibition of Gα12/13. In fact, RGS12 can inhibit recombinant Gα12 and Gα13-mediated SRF activation (Fig. 6 A). The inability of RGS2 and RGS4 to inhibit Gα12/13-, LPA-, or thrombin-mediated SRF activation indicates that these two RGS proteins do not act on Gα12/13. Therefore, there is apparent specificity in interactions between RGS proteins and G proteins. Although Axin contains a RGS domain, it is unable to regulate any of the known G proteins. Gβγ subunits are also involved in regulation of SRF, especially together with Gαq and Gα13. Since SRF is downstream of the signal cascade, we do not know where the Gα-mediated pathways interact with the Gβγ-mediated ones. It appears that the interactions are upstream of RhoA because C3 was able to completely abolish carbachol-mediated effects in cells coexpressing m1R and m2R. It is also not clear how Gαq and Gα13 regulate RhoA and SRF. Recent studies suggested that the Tec family of nonreceptor tyrosine kinases may be regulated by Gαq (34Bence K. Ma W. Kozasa T. Huang X.Y. Nature. 1997; 389: 296-299Crossref PubMed Scopus (167) Google Scholar) and Gα12/13 and that Tec kinases regulate RhoA in a C3-dependent manner. 3J. Mao and D. Wu, unpublished data. In addition, Gβγ was shown to regulate Btk, a member of the Tec family, via phosphatidylinositide 3-kinase (35Li Z.M. Wahl M.I. Eguinoa A. Stephens L.R. Hawkins P.T. Witte O.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13820-13825Crossref PubMed Scopus (186) Google Scholar). Thus, the synergistic activation of SRF by Gα and Gβγ may lie in the kinases. Further studies are needed to better understand these questions. We thank Huiping Jiang and Mark Betz for reading the manuscript. We also thank Alan Hall, Solvio Gutkind, Sheng-Cai Lin, and F. Costantini for providing cDNAs.