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• W2041361220 abstract "Transmembrane signaling through Gαq-coupled receptors is linked to physiological processes such as cardiovascular development and smooth muscle function. Recent crystallographic studies have shown how Gαq interacts with two activation-dependent targets, p63RhoGEF and G protein-coupled receptor kinase 2 (GRK2). These proteins bind to the effector-binding site of Gαq in a manner that does not appear to physically overlap with the site on Gαq bound by regulator of G-protein signaling (RGS) proteins, which function as GTPase-activating proteins (GAPs). Herein we confirm the formation of RGS-Gαq-GRK2/p63RhoGEF ternary complexes using flow cytometry protein interaction and GAP assays. RGS2 and, to a lesser extent, RGS4 are negative allosteric modulators of Gαq binding to either p63RhoGEF or GRK2. Conversely, GRK2 enhances the GAP activity of RGS4 but has little effect on that of RGS2. Similar but smaller magnitude responses are induced by p63RhoGEF. The fact that GRK2 and p63RhoGEF respond similarly to these RGS proteins supports the hypothesis that GRK2 is a bona fide Gαq effector. The results also suggest that signal transduction pathways initiated by GRK2, such as the phosphorylation of G protein-coupled receptors, and by p63RhoGEF, such as the activation of gene transcription, can be regulated by RGS proteins via both allosteric and GAP mechanisms. Transmembrane signaling through Gαq-coupled receptors is linked to physiological processes such as cardiovascular development and smooth muscle function. Recent crystallographic studies have shown how Gαq interacts with two activation-dependent targets, p63RhoGEF and G protein-coupled receptor kinase 2 (GRK2). These proteins bind to the effector-binding site of Gαq in a manner that does not appear to physically overlap with the site on Gαq bound by regulator of G-protein signaling (RGS) proteins, which function as GTPase-activating proteins (GAPs). Herein we confirm the formation of RGS-Gαq-GRK2/p63RhoGEF ternary complexes using flow cytometry protein interaction and GAP assays. RGS2 and, to a lesser extent, RGS4 are negative allosteric modulators of Gαq binding to either p63RhoGEF or GRK2. Conversely, GRK2 enhances the GAP activity of RGS4 but has little effect on that of RGS2. Similar but smaller magnitude responses are induced by p63RhoGEF. The fact that GRK2 and p63RhoGEF respond similarly to these RGS proteins supports the hypothesis that GRK2 is a bona fide Gαq effector. The results also suggest that signal transduction pathways initiated by GRK2, such as the phosphorylation of G protein-coupled receptors, and by p63RhoGEF, such as the activation of gene transcription, can be regulated by RGS proteins via both allosteric and GAP mechanisms. Heterotrimeric GTP-binding (G) proteins (Gαβγ) relay the extracellular signals received by G protein-coupled receptors (GPCRs) 3The abbreviations used are: GPCR, G protein-coupled receptor; RGS, regulator of G-protein signaling; PDE, phosphodiesterase; FCPIA, flow cytometry protein interaction assay; AF, Alexa Fluor; MFI, median fluorescence intensity; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; GRK2, G protein-coupled receptor kinase 2; DTT, dithiothreitol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GTPγS, guanosine 5′-3-O-(thio)triphosphate.3The abbreviations used are: GPCR, G protein-coupled receptor; RGS, regulator of G-protein signaling; PDE, phosphodiesterase; FCPIA, flow cytometry protein interaction assay; AF, Alexa Fluor; MFI, median fluorescence intensity; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; GRK2, G protein-coupled receptor kinase 2; DTT, dithiothreitol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GTPγS, guanosine 5′-3-O-(thio)triphosphate. to effector enzymes and channels in the cell (1.Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Google Scholar). Activated GPCRs catalyze nucleotide exchange on Gα subunits, thereby converting the inactive (GDP-bound) Gαβγ heterotrimer into activated Gα·GTP and Gβγ subunits. These subunits then interact with downstream effectors to elicit intracellular responses (2.Neves S.R. Ram P.T. Iyengar R. Science. 2002; 296: 1636-1639Crossref PubMed Scopus (949) Google Scholar). Duration of signaling is limited by the rate of GTP hydrolysis on the Gα subunit. After returning to the GDP-bound state, Gα reforms the inactive Gαβγ heterotrimer, which can then undergo additional rounds of receptor-mediated activation. For some families of Gα subunits, the rate of GTP hydrolysis can be accelerated by direct interactions with effectors (3.Berstein G. Blank J.L. Jhon D.Y. Exton J.H. Rhee S.G. Ross E.M. Cell. 1992; 70: 411-418Abstract Full Text PDF PubMed Google Scholar, 4.Kozasa 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 (735) Google Scholar) or regulator of G protein signaling (RGS) proteins (5.Berman D.M. Wilkie T.M. Gilman A.G. Cell. 1996; 86: 445-452Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar, 6.Watson N. Linder M.E. Druey K.M. Kehrl J.H. Blumer K.J. Nature. 1996; 383: 172-175Crossref PubMed Scopus (472) Google Scholar). Although RGS proteins are generally thought of as inhibitors of heterotrimeric G protein signaling mediated by the Gαi and Gαq/11 families, they may also serve to spatially focus the signals being propagated (7.Zhong H. Wade S.M. Woolf P.J. Linderman J.J. Traynor J.R. Neubig R.R. J. Biol. Chem. 2003; 278: 7278-7284Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) or to regulate the steady-state flux through a specific signaling cascade (8.Mukhopadhyay S. Ross E.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9539-9544Crossref PubMed Scopus (152) Google Scholar). Comparison of the crystal structures of the Gαi-RGS4 (9.Tesmer J.J.G. Berman D.M. Gilman A.G. Sprang S.R. Cell. 1997; 89: 251-261Abstract Full Text Full Text PDF PubMed Google Scholar) and Gαs-adenylyl cyclase (10.Tesmer J. Sunahara R. Gilman A. Sprang S. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (666) Google Scholar) complexes revealed that RGS proteins and effectors interact with discrete footprints on the surface of Gα and have the potential to bind simultaneously (11.Sunahara R.K. Tesmer J.J.G. Gilman A.G. Sprang S.R. Science. 1997; 278: 1943-1947Crossref PubMed Scopus (262) Google Scholar, 12.Sprang S.R. Chen Z. Du X. Adv. Protein Chem. 2007; 74: 1-65Crossref PubMed Scopus (79) Google Scholar). Direct experimental support for an RGS-Gα-effector ternary complex came from analysis of the interactions of transducin (Gαt) with RGS proteins and the γ subunit of cGMP phosphodiesterase (PDEγ). Both PDEγ (13.Arshavsky V. Bownds M.D. Nature. 1992; 357: 416-417Crossref PubMed Scopus (218) Google Scholar) and RGS9 (14.He W. Cowan C.W. Wensel T.G. Neuron. 1998; 20: 95-102Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar) are required for physiological rates of GTP hydrolysis on Gαt. Although PDEγ has no GAP activity on its own, it can stimulate RGS9-mediated GAP activity by up to ∼3-fold (14.He W. Cowan C.W. Wensel T.G. Neuron. 1998; 20: 95-102Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). Mutagenesis studies (15.McEntaffer R.L. Natochin M. Artemyev N.O. Biochemistry. 1999; 38: 4931-4937Crossref PubMed Scopus (25) Google Scholar), biophysical measurements (16.Skiba N.P. Yang C.S. Huang T. Bae H. Hamm H.E. J. Biol. Chem. 1999; 274: 8770-8778Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), and ultimately the crystal structure of the RGS9-Gαt/i1-PDEγ ternary complex (17.Slep K.C. Kercher M.A. He W. Cowan C.W. Wensel T.G. Sigler P.B. Nature. 2001; 409: 1071-1077Crossref PubMed Scopus (221) Google Scholar) were all consistent with a model of allosteric modulation between the effector and RGS binding sites of Gαt, with little or no direct functional interaction between PDEγ and RGS9. It has been proposed that this PDEγ-regulated GAP activity prevents a “short circuit” of the phototransduction cascade via premature hydrolysis of Gαt·GTP before effectors can functionally interact with the G protein (18.Nekrasova E. Berman D. Rustandi R. Hamm H. Gilman A. Arshavsky V. Biochemistry. 1997; 36: 7638-7643Crossref PubMed Scopus (52) Google Scholar). Conversely, PDEγ inhibits the GAP activity of other RGS proteins (RGS4, GAIP, and RGS16/RGSr) most likely through a negative allosteric mechanism (18.Nekrasova E. Berman D. Rustandi R. Hamm H. Gilman A. Arshavsky V. Biochemistry. 1997; 36: 7638-7643Crossref PubMed Scopus (52) Google Scholar, 19.Natochin M. Granovsky A.E. Artemyev N.O. J. Biol. Chem. 1997; 272: 17444-17449Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 20.Wieland T. Chen C.K. Simon M.I. J. Biol. Chem. 1997; 272: 8853-8856Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). It is not known whether analogous ternary complexes are formed by other members of the Gαi family or by subunits from the Gαq family or if there are other effector/RGS combinations that are synergistic with respect to GAP activity on Gα. The Gq/11 family of G proteins is involved in an array of cellular processes that include platelet activation, cardiovascular development, and regulation of memory, appetite, motor coordination, and sleep (21.Offermanns S. Simon M.I. Oncogene. 1998; 17: 1375-1381Crossref PubMed Google Scholar, 22.Hubbard K.B. Hepler J.R. Cell. Signal. 2006; 18: 135-150Crossref PubMed Scopus (218) Google Scholar). They are also strongly implicated in pathophysiological processes such as the development of cardiac hypertrophy (23.Dorn Jr., G.W. Force T. J. Clin. Investig. 2005; 115: 527-537Crossref PubMed Google Scholar) and high blood pressure (24.Harris D.M. Cohn H.I. Pesant S. Zhou R.H. Eckhart A.D. Am. J. Physiol. Heart Circ. Physiol. 2007; 293: 3072-3079Crossref PubMed Scopus (23) Google Scholar). Although the canonical effector of Gαq is phospholipase Cβ (PLCβ) (25.Rhee S.G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1206) Google Scholar), recent structural, biochemical, and whole animal studies have confirmed that the Trio family of RhoA guanine nucleotide exchange factors (RhoGEFs), namely Trio, Duet, and p63RhoGEF, are also direct targets of Gαq (26.Lutz S. Freichel-Blomquist A. Yang Y. Rumenapp U. Jakobs K.H. Schmidt M. Wieland T. J. Biol. Chem. 2005; 280: 11134-11139Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 27.Lutz S. Shankaranarayanan A. Coco C. Ridilla M. Nance M.R. Vettel C. Baltus D. Evelyn C.R. Neubig R.R. Wieland T. Tesmer J.J. Science. 2007; 318: 1923-1927Crossref PubMed Scopus (170) Google Scholar, 28.Williams S.L. Lutz S. Charlie N.K. Vettel C. Ailion M. Coco C. Tesmer J.J. Jorgensen E.M. Wieland T. Miller K.G. Genes Dev. 2007; 21: 2731-2746Crossref PubMed Scopus (60) Google Scholar, 29.Rojas R.J. Yohe M.E. Gershburg S. Kawano T. Kozasa T. Sondek J. J. Biol. Chem. 2007; 282: 29201-29210Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), thereby linking Gαq to RhoA-mediated processes such as cell migration, proliferation, and contraction. Another putative effector target of Gαq is G protein-coupled receptor kinase 2 (GRK2) (30.Theilade J. Haunso S. Sheikh S.P. Curr. Drug Targets Immune Endocr. Metabol. Dis. 2001; 1: 139-151Crossref PubMed Google Scholar). GRK2 phosphorylates activated heptahelical receptors, which are then bound by arrestin and targeted for endocytosis (31.Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1058) Google Scholar). Furthermore, through a process known as phosphorylation-independent desensitization (32.Willets J.M. Nahorski S.R. Challiss R.A. J. Biol. Chem. 2005; 280: 18950-18958Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 33.Willets J.M. Nash M.S. Challiss R.A. Nahorski S.R. J. Neurosci. 2004; 24: 4157-4162Crossref PubMed Scopus (37) Google Scholar, 34.Iwata K. Luo J. Penn R.B. Benovic J.L. J. Biol. Chem. 2005; 280: 2197-2204Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), GRK2 is thought to sequester activated Gαq from PLCβ using its amino-terminal RGS homology (RH) domain (35.Sterne-Marr R. Tesmer J.J. Day P.W. Stracquatanio R.P. Cilente J.A. O'Connor K.E. Pronin A.N. Benovic J.L. Wedegaertner P.B. J. Biol. Chem. 2003; 278: 6050-6058Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 36.Tesmer V.M. Kawano T. Shankaranarayanan A. Kozasa T. Tesmer J.J. Science. 2005; 310: 1686-1690Crossref PubMed Scopus (232) Google Scholar). The crystal structure of the Gαq-GRK2-Gβγ complex revealed that GRK2 binds to the effector-binding site of Gαq (36.Tesmer V.M. Kawano T. Shankaranarayanan A. Kozasa T. Tesmer J.J. Science. 2005; 310: 1686-1690Crossref PubMed Scopus (232) Google Scholar), raising the possibility that GRK2 is in fact an effector that can initiate its own signaling cascades in response to the activation of Gαq. Although one obvious pathway is simply the phosphorylation of activated GPCRs, GRK2 has also recently been reported to phosphorylate insulin receptor substrate-1 (37.Usui I. Imamura T. Babendure J.L. Satoh H. Lu J.C. Hupfeld C.J. Olefsky J.M. Mol. Endocrinol. 2005; 19: 2760-2768Crossref PubMed Scopus (73) Google Scholar), p38 MAPK (38.Peregrin S. Jurado-Pueyo M. Campos P.M. Sanz-Moreno V. Ruiz-Gomez A. Crespo P. Mayor Jr., F. Murga C. Curr. Biol. 2006; 16: 2042-2047Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), and ezrin (39.Cant S.H. Pitcher J.A. Mol. Biol. Cell. 2005; 16: 3088-3099Crossref PubMed Scopus (115) Google Scholar) in response to GPCR activation. The rate of GTP hydrolysis by Gαq can be accelerated by many different RGS proteins (22.Hubbard K.B. Hepler J.R. Cell. Signal. 2006; 18: 135-150Crossref PubMed Scopus (218) Google Scholar), but two of the best characterized are RGS2 and RGS4, which are both members of the RGS B/R4 subfamily (40.Ross E.M. Wilkie T.M. Annu. Rev. Biochem. 2000; 69: 795-827Crossref PubMed Scopus (916) Google Scholar, 41.Bansal G. Druey K.M. Xie Z. Pharmacol. Ther. 2007; 116: 473-495Crossref PubMed Scopus (162) Google Scholar). These are relatively simple RGS proteins that consist of an amino-terminal membrane-targeting domain followed by a conserved ∼120-amino acid catalytic RGS domain that interacts with the three switch regions of the Gα subunit (9.Tesmer J.J.G. Berman D.M. Gilman A.G. Sprang S.R. Cell. 1997; 89: 251-261Abstract Full Text Full Text PDF PubMed Google Scholar). In cells, RGS4 inhibits both Gαi- and Gαq-mediated signaling (42.Xu X. Zeng W. Popov S. Berman D.M. Davignon I. Yu K. Yowe D. Offermanns S. Muallem S. Wilkie T.M. J. Biol. Chem. 1999; 274: 3549-3556Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar), whereas RGS2 is selective for Gαq (43.Heximer 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 (309) Google Scholar, 44.Ingi T. Krumins A.M. Chidiac P. Brothers G.M. Chung S. Snow B.E. Barnes C.A. Lanahan A.A. Siderovski D.P. Ross E.M. Gilman A.G. Worley P.F. J. Neurosci. 1998; 18: 7178-7188Crossref PubMed Google Scholar, 45.Heximer S.P. Srinivasa S.P. 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 (149) Google Scholar, 46.Scheschonka A. Dessauer C.W. Sinnarajah S. Chidiac P. Shi C.S. Kehrl J.H. Mol. Pharmacol. 2000; 58: 719-728Crossref PubMed Scopus (74) Google Scholar, 47.Hao J. Michalek C. Zhang W. Zhu M. Xu X. Mende U. J. Mol. Cell. Cardiol. 2006; 41: 51-61Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 48.Karakoula A. Tovey S.C. Brighton P.J. Willars G.B. Eur. J. Pharmacol. 2008; 587: 16-24Crossref PubMed Scopus (17) Google Scholar). Both proteins have been reported to serve as effector antagonists because they can inhibit PLCβ signaling by either GTPase-deficient Gα subunits or Gα subunits loaded with non-hydrolyzable GTP analogs (43.Heximer 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 (309) Google Scholar, 49.Hepler 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, 50.Anger T. Zhang W. Mende U. J. Biol. Chem. 2004; 279: 3906-3915Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). In this study we have used biophysical and kinetic studies to demonstrate the formation of ternary complexes of Gαq, RGS proteins, and effectors. We also show that RGS2 and RGS4 are negative allosteric modulators of proteins that bind to the effector-binding site of Gαq, providing the molecular basis for their reported roles as effector antagonists. Conversely, GRK2 and p63RhoGEF are shown to be allosteric modulators of RGS GAP activity. GRK2 is able to stimulate RGS4 GAP activity on Gαq to a similar extent as PDEγ does for RGS9 GAP activity on Gαt. These data provide important insights into the regulation of GRK2 and p63RhoGEF by both Gαq and RGS proteins in vivo. Purification of RGS2 and ΔN-RGS2—cDNA encoding human RGS2 and an amino-terminal deletion of RGS2 (ΔN-RGS2, spanning amino acid residues 72–211) were cloned into the pMALc2H10T vector (51.Kristelly R. Earnest B.T. Krishnamoorthy L. Tesmer J.J. Acta Crystallogr. D Biol. Crystallogr. 2003; 59: 1859-1862Crossref PubMed Scopus (18) Google Scholar) using the BamHI and SalI restriction sites. The proteins were expressed as tobacco etch virus protease-cleavable maltose-binding protein fusion proteins. All purification steps were performed at 4 °C or on ice. Induced Rosetta (DE3) pLys cell pellets were resuspended in lysis buffer (20 mm HEPES, pH 8.0, 500 mm NaCl, 10 mm β-mercaptoethanol) plus 1 μm leupeptin, 1 mm lima bean trypsin inhibitor, and 0.1 mm phenylmethylsulfonyl fluoride. Cells were lysed with an Avestin C3 homogenizer, ultracentrifuged at 40,000 rpm for 1 h using a Beckman Type Ti 45 rotor, and then loaded on a nickel-nitrilotriacetic acid column pre-equilibrated with lysis buffer. Maltose-binding protein-RGS2 was eluted with lysis buffer containing 150 mm imidazole, pH 8.0, and then treated with 2% (w/w) tobacco etch virus protease and dialyzed against lysis buffer overnight. The dialyzed protein was passed back over a nickel-nitrilotriacetic acid column equilibrated with lysis buffer to remove His-tagged maltose-binding protein and uncut fusion protein. RGS2 was then concentrated in a 30-kDa Centriprep (Millipore) and further purified using two tandem Superdex S200 columns (GE Healthcare) equilibrated with 20 mm HEPES, pH 8.0, 500 mm NaCl and 5 mm DTT. Some of the RGS2 used in these studies was produced from a His10-RGS2 vector (a gift from Scott Heximer, University of Toronto). Purification of His10-RGS2 was as previously described (52.Heximer S.P. Methods Enzymol. 2004; 390: 65-82Crossref PubMed Scopus (34) Google Scholar). Purification of Other Proteins—A Gαi/q chimera, in which the amino-terminal helix of Gαq is replaced with that of Gαi (36.Tesmer V.M. Kawano T. Shankaranarayanan A. Kozasa T. Tesmer J.J. Science. 2005; 310: 1686-1690Crossref PubMed Scopus (232) Google Scholar), a fragment of human p63RhoGEF spanning residues 149–502 (henceforth referred to as p63RhoGEF), GRK2, and RGS4 were purified as previously described (9.Tesmer J.J.G. Berman D.M. Gilman A.G. Sprang S.R. Cell. 1997; 89: 251-261Abstract Full Text Full Text PDF PubMed Google Scholar, 27.Lutz S. Shankaranarayanan A. Coco C. Ridilla M. Nance M.R. Vettel C. Baltus D. Evelyn C.R. Neubig R.R. Wieland T. Tesmer J.J. Science. 2007; 318: 1923-1927Crossref PubMed Scopus (170) Google Scholar, 53.Lodowski D.T. Barnhill J.F. Pitcher J.A. Capel W.D. Lefkowitz R.J. Tesmer J.J. Acta Crystallogr. D Biol. Crystallogr. 2003; 59: 936-939Crossref PubMed Scopus (21) Google Scholar). A construct expressing a fragment of RGS4 analogous to ΔN-RGS2 (ΔN-RGS4, spanning amino acid residues 51–205) was created in pMALc2H10T using the EcoRI and HindIII restriction sites. The overexpressed protein was purified essentially as described for RGS2 except using 100 instead of 500 mm NaCl. Point mutants RGS2-N149D, RGS4-N128G, GRK2-D110A, and p63RhoGEF-F471E were purified as described for their respective wild-type proteins. Purification of Gαi/qR183C was performed as described for Gαi/q with the following modifications. The nickel-column eluate was supplemented with 10% glycerol, and proteins were dialyzed overnight against dialysis buffer (20 mm HEPES, pH 8.0, 100 mm NaCl, 2 mm DTT, 1 mm MgCl2, 50 μm GDP, and 10% glycerol) and concentrated to ∼8 mg/ml. Gαi/qR183C was purified on two tandem S200 gel filtration columns pre-equilibrated with 20 mm HEPES, pH 8.0, 200 mm NaCl, 5 mm DTT, 5 mm MgCl2, 50 μm GDP, and 5% glycerol. Gαi/qR183C was purified to greater than 90% homogeneity as judged by SDS-PAGE and was concentrated to ∼3 mg/ml and then frozen in tubes on liquid nitrogen in volumes of 5 μl. Flow Cytometry Protein Interaction Assay—Equilibrium binding of either RGS2, RGS4, GRK2, or p63RhoGEF to Gαi/q was measured by flow cytometry protein interaction assay (FCPIA). RGS2 and GRK2 were fluorescently labeled either with amine reactive Alexa Fluor (AF) 532 carboxylic acid succinimidyl ester or with the thiol-reactive AF 532 C5-maleimide (Invitrogen). Both probes gave similar results in binding assays. RGS4 and p63RhoGEF were labeled only with the thiol-reactive fluorophore. Gαi/q was initially biotinylated using biotinamidohexanoic acid N-hydroxysuccinimide ester (Sigma) in the form of a Gαi/qβγ heterotrimer, as previously described (27.Lutz S. Shankaranarayanan A. Coco C. Ridilla M. Nance M.R. Vettel C. Baltus D. Evelyn C.R. Neubig R.R. Wieland T. Tesmer J.J. Science. 2007; 318: 1923-1927Crossref PubMed Scopus (170) Google Scholar). Gαi/q was later biotinylated directly as a monomer because it behaved similarly in binding assays and had the advantage of not requiring separation from Gβγ. Biotinylated Gαi/q (b-Gαi/q, 5 nm) was linked to xMap LumAvidin microspheres (Luminex) and washed 3 times with 20 mm HEPES, pH 8.0, 100 mm NaCl, 5 mm MgCl2, 0.1% lubrol, 2 mm DTT, 1% bovine serum albumin, 50 μm GDP plus other additions as indicated. The indicated concentrations of AF 532-labeled protein were then added to bead-bound b-Gαi/q and then allowed to equilibrate for at least 30 min before being processed on a Luminex 96-well plate bead analyzer. For competition studies, unlabeled proteins were added at the concentrations indicated. Longer incubation times (e.g. overnight) did not alter the results, indicating that equilibrium was attained under our assay conditions. The association of AF-labeled protein with beads is reported as the median fluorescence intensity (MFI) for each sample. Each data point was typically measured in duplicate. Direct binding and competition data were fit by nonlinear regression either to one-site binding equations or to an allosteric model using GraphPad Prism (Version 5.0a). Allosteric modulation of AF-GRK2 binding to Gαi/q by RGS proteins was fit using Equations 1 and 2,Y=Y0+NS×[GRK2]+[GRK2]×Bmax[GRK2]+Kd(Eq. 1) where Y is the total fluorescence measured, Y0 is the background fluorescence, NS is the linear increase in fluorescence due to nonspecific binding of AF-GRK2 to beads, and Bmax is the maximum fluorescence change due to specific binding. For all but one of the RGS2 dose-response curves (Fig. 5B), Y0 and NS were directly measured and subtracted from the data to obtain specific binding. For these corrected sets, Y0 and NS were fixed with values of 0. Kd′ is the apparent dissociation constant for AF-GRK2,Kd'=Kd×(KA+[A])(KA+[A]/α)(Eq. 2) where Kd is the dissociation constant of AF-GRK2 in the absence of allosteric modulation, KA is the dissociation constant of allosteric modulator A (i.e. RGS2 or RGS4) in the absence of AF-GRK2, and α is the cooperativity factor (54.Ehlert F.J. Mol. Pharmacol. 1988; 33: 187-194PubMed Google Scholar). An α value greater than 1 corresponds to negative allostery. Kd, KA, and α were fit globally from 2–5 separate series of binding saturation curves with automatic outlier rejection as implemented by GraphPad Prism. The dose-response curves were also analyzed using a competitive model wherein the [A]/α term in Equation 2 was deleted. Model comparisons used the F test as implemented by GraphPad Prism. Dissociation Rate of GRK2—To determine koff for GRK2 from b-Gαi/q, 10 nm AF-GRK2 was incubated with bead bound b-Gαi/q for 1 or 24 h at 4 °C. Plates were then allowed to equilibrate at room temperature for 30 min, and the dissociation of AF-GRK2 was initiated by adding either unlabeled GRK2 (final concentration 1 μm), GRK2 plus RGS2 (both 1 μm final), or GRK2 plus RGS4 (both 1 μm final). The loss of fluorescence was measured by FCPIA at the indicated time points. Data were fit to a one-phase exponential decay model. Purification of an RGS4-Gαi/q-p63RhoGEF-RhoA Complex—Gαi/q and a 1.25 m excess of p63RhoGEF and a 2 m excess of RGS4 were incubated on ice for 30 min in the presence of 20 μm AlCl3 and 10 mm NaF. Total protein concentration was greater than 5 mg/ml. RGS4-Gαi/q-p63RhoGEF complexes thus formed were gel-filtered through a 2-ml desalting spin column (Zeba™) to remove excess GDP. RhoA was incubated with 10 mm EDTA on ice for 30 min, and buffer was exchanged with a 0.5-ml spin column to form GDP-free RhoA. The RGS4-Gαi/q-p63RhoGEF complex was then incubated with 1.5 m excess of GDP-free RhoA on ice for 15 min and then resolved on two tandem Superdex 200 10/300 gel filtration columns pre-equilibrated with 20 mm HEPES, pH 8.0, 50 mm NaCl, 1 mm DTT, 1 μm EDTA, 20 μm AlCl3, and 10 mm NaF. RGS Protein Pulldown Assays—Mutations in Gαq were generated in mouse Gαq cDNA in pCMV5, and the mutants were expressed in HEK293 cells as previously described (36.Tesmer V.M. Kawano T. Shankaranarayanan A. Kozasa T. Tesmer J.J. Science. 2005; 310: 1686-1690Crossref PubMed Scopus (232) Google Scholar). RGS2/RGS4 was biotinylated by incubating with equimolar amounts of biotinamidohexanoyl-6-amino-hexanoic acid N-hydroxysuccinimide ester (Sigma) on ice for 1 h and then filtering the sample through a 0.5-ml spin column (Zeba™). Gαq (wild type or the indicated mutant) cell lysates (100 μl) were incubated with 1 μg of biotinylated RGS2/RGS4 and streptavidin beads (Invitrogen) for 3 h at 4 °C in the presence or absence of 30 μm AlCl3, 10 mm NaF. The beads were then washed 3 times with 500 μl of the lysis buffer (±30 μm AlCl3 and 10 mm NaF as appropriate) and then treated with 5 μl of 4× SDS-PAGE loading buffer. Gαq was detected by Western analysis. GAP Assays—GTPase activation assays were conducted as previously described (55.Chidiac P. Ross E.M. J. Biol. Chem. 1999; 274: 19639-19643Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Gαi/qR183C (final concentration 1–3 μm) was incubated in a 120-μl GTP mixture (6.25 μm [γ-32P]GTP (30–100 cpm/fmol), 50 mm HEPES, pH 7.4, 1 mm EDTA, 1 mm DTT, 0.9 mm MgSO4, 5.5 mm CHAPS, 0.1 mg/ml bovine serum albumin, 5% glycerol, and 37.5 μm (NH4)2SO4) for ∼3 h at 20 °C. [γ-32P]GTP-bound Gαi/qR183C was purified from unbound nucleotide at 4 °C using a 0.5-ml spin column (Zeba™). A sample of this protein was reserved for liquid scintillation counting to calculate specific activity. GTP hydrolysis was performed in an assay buffer containing 20 mm HEPES, pH 7.4, 80 mm NaCl, 1 mm EDTA, 1 mm DTT, 0.9 mm MgSO4, 1 mm GTP, 0.20% (w/v) cholate, and 10 μg/ml bovine serum albumin. The reaction was initiated by the addition of 30 μl of the [γ-32P]GTP-loaded Gαi/qR183C to 270 μl of assay buffer at 20 °C either alone or in the presence of additional proteins as indicated. The reaction was terminated at each time point by adding 50 μl of the reaction mix to 750 μl of quench buffer (5% activated charcoal in 50 mm NaH2PO4, pH 2) on ice. The radioactivity remaining in the supernatant was quantified by liquid scintillation counting of 300 μl of the supernatant. In each assay typically ∼5 nm Gαi/qR183C was estimated as being loaded with [γ-32P]GTP. Nucleotide Exchange Assay—Nucleotide exchange on RhoA was measured as previously described (27.Lutz S. Shankaranarayanan A. Coco C. Ridilla M. Nance M.R. Vettel C. Baltus D. Evelyn C.R. Neubig R.R. Wieland T. Tesmer J.J. Science. 2007; 318: 1923-1927Crossref PubMed Scopus (170) Google Scholar) except that the reaction mix (2 μm RhoA, 200 nm p63RhoGEF, 400 nm Gαi/q) and the indicated concentrations of RGS proteins were first equilibrated for 2 h at 4 °C before the addition of 1 μm BODIPY FL GTPγS (Invitrogen). Crystallographic studies demonstrated that GRK2 and p63RhoGEF both engage Gαq in a manner that would appear to allow the binding of the RGS domain of either RGS4 (9.Tesmer J.J.G. Berman D.M. Gilman A.G. Sprang S.R. Cell. 1997; 89: 251-261Abstract Full Text Full Text PDF PubMed Google Scholar) or RGS2 (56.Soundararajan M. Willard F.S. Kimple A.J. Turnbull A.P. Ball L.J. Schoch G.A. Gileadi C. Fedorov O.Y. Dowler E.F. Higman V.A. Hutsell S.Q. Sundstrom M. Doyle D.A. Siderovski D.P. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 6457-6462Crossref PubMed Scopus (130) Google Scholar) to Gαq without steric overlap (Fig. 1, A and B). Models of these RGS-Gαi/q-effector complexes, thus, resemble the structure of the PDEγ-Gαt/i1-RGS9 complex (Fig. 1C). The positions of the modeled RGS box domains in these complexes are also consistent with the predicted orientation of these complexes at the cell surface in that the membrane binding elements of the RGS proteins are juxtaposed with the phospholipid bilayer. We, therefore, initiated in vitro experiments to confirm the formation of these complexes and to better" @default.
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• W2041361220 date "2008-12-01" @default.
• W2041361220 modified "2023-09-28" @default.
• W2041361220 title "Assembly of High Order Gαq-Effector Complexes with RGS Proteins" @default.
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• W2041361220 doi "https://doi.org/10.1074/jbc.m805860200" @default.
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