G Protein-coupled Receptor Kinase 2/Gαq/11 Interaction
2003; Elsevier BV; Volume: 278; Issue: 8 Linguagem: Inglês
10.1074/jbc.m208787200
ISSN1083-351X
AutoresRachel Sterne‐Marr, J.J.G. Tesmer, Peter Day, RoseAnn P. Stracquatanio, Jill-Ann E. Cilente, Katharine E. O'Connor, Alexey Pronin, Jeffrey Benovic, Philip Wedegaertner,
Tópico(s)Cell Adhesion Molecules Research
ResumoG protein-coupled receptors (GPCRs) transduce cellular signals from hormones, neurotransmitters, light, and odorants by activating heterotrimeric guanine nucleotide-binding (G) proteins. For many GPCRs, short term regulation is initiated by agonist-dependent phosphorylation by GPCR kinases (GRKs), such as GRK2, resulting in G protein/receptor uncoupling. GRK2 also regulates signaling by binding Gαq/ll and inhibiting Gαq stimulation of the effector phospholipase Cβ. The binding site for Gαq/ll resides within the amino-terminal domain of GRK2, which is homologous to the regulator of G protein signaling (RGS) family of proteins. To map the Gαq/llbinding site on GRK2, we carried out site-directed mutagenesis of the RGS homology (RH) domain and identified eight residues, which when mutated, alter binding to Gαq/ll. These mutations do not alter the ability of full-length GRK2 to phosphorylate rhodopsin, an activity that also requires the amino-terminal domain. Mutations causing Gαq/ll binding defects impair recruitment to the plasma membrane by activated Gαq and regulation of Gαq-stimulated phospholipase Cβ activity when introduced into full-length GRK2. Two different protein interaction sites have previously been identified on RH domains. The Gα binding sites on RGS4 and RGS9, called the "A" site, is localized to the loops between helices α3 and α4, α5 and α6, and α7 and α8. The adenomatous polyposis coli (APC) binding site of axin involves residues on α helices 3, 4, and 5 (the "B" site) of its RH domain. We demonstrate that the Gαq/ll binding site on the GRK2 RH domain is distinct from the "A" and "B" sites and maps primarily to the COOH terminus of its α5 helix. We suggest that this novel protein interaction site on an RH domain be designated the "C" site. G protein-coupled receptors (GPCRs) transduce cellular signals from hormones, neurotransmitters, light, and odorants by activating heterotrimeric guanine nucleotide-binding (G) proteins. For many GPCRs, short term regulation is initiated by agonist-dependent phosphorylation by GPCR kinases (GRKs), such as GRK2, resulting in G protein/receptor uncoupling. GRK2 also regulates signaling by binding Gαq/ll and inhibiting Gαq stimulation of the effector phospholipase Cβ. The binding site for Gαq/ll resides within the amino-terminal domain of GRK2, which is homologous to the regulator of G protein signaling (RGS) family of proteins. To map the Gαq/llbinding site on GRK2, we carried out site-directed mutagenesis of the RGS homology (RH) domain and identified eight residues, which when mutated, alter binding to Gαq/ll. These mutations do not alter the ability of full-length GRK2 to phosphorylate rhodopsin, an activity that also requires the amino-terminal domain. Mutations causing Gαq/ll binding defects impair recruitment to the plasma membrane by activated Gαq and regulation of Gαq-stimulated phospholipase Cβ activity when introduced into full-length GRK2. Two different protein interaction sites have previously been identified on RH domains. The Gα binding sites on RGS4 and RGS9, called the "A" site, is localized to the loops between helices α3 and α4, α5 and α6, and α7 and α8. The adenomatous polyposis coli (APC) binding site of axin involves residues on α helices 3, 4, and 5 (the "B" site) of its RH domain. We demonstrate that the Gαq/ll binding site on the GRK2 RH domain is distinct from the "A" and "B" sites and maps primarily to the COOH terminus of its α5 helix. We suggest that this novel protein interaction site on an RH domain be designated the "C" site. G protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCR, G protein-coupled receptor; PLCβ, phospholipase Cβ; GRK, GPCR kinase; RGS, regulator of G protein signaling; GAP, GTPase activating protein; RH, RGS homology; AlF 4−, aluminum fluoride; GFP, green fluorescent protein; WT, wild type; GST, glutathione S-transferase; GEF, guanine-nucleotide exchange factor; APC, adenomatous polyposis coli; LARG, leukemia-associated RhoGEF; DMEM, Dulbecco's modified Eagle's medium; IP, inositol phosphate; CaM, calmodulin; PIP3, phosphatidylinositol 3,4,5-trisphosphate; GTPγS, guanosine 5′-3-O-(thio)triphosphate 1The abbreviations used are: GPCR, G protein-coupled receptor; PLCβ, phospholipase Cβ; GRK, GPCR kinase; RGS, regulator of G protein signaling; GAP, GTPase activating protein; RH, RGS homology; AlF 4−, aluminum fluoride; GFP, green fluorescent protein; WT, wild type; GST, glutathione S-transferase; GEF, guanine-nucleotide exchange factor; APC, adenomatous polyposis coli; LARG, leukemia-associated RhoGEF; DMEM, Dulbecco's modified Eagle's medium; IP, inositol phosphate; CaM, calmodulin; PIP3, phosphatidylinositol 3,4,5-trisphosphate; GTPγS, guanosine 5′-3-O-(thio)triphosphate are a large family of integral membrane proteins that form seven transmembrane helices and couple to heterotrimeric guanine nucleotide (G)-binding proteins on their cytoplasmic surface. They transmit the signals from light and odorant receptors as well as the signals initiated by numerous hormones and neurotransmitters. In their inactive state, heterotrimeric G proteins are complexes of three polypeptide chains (Gαβγ). Upon activation, GPCRs catalyze the exchange of GTP for GDP on the Gα subunit resulting in dissociation of the GTP-bound Gα subunit from the Gβγ dimer (1Bourne H.R. Curr. Opin. Cell Biol. 1997; 9: 134-142Google Scholar). Gα and Gβγ are then free to regulate effectors such as adenylyl cyclase, phospholipase Cβ (PLCβ), cGMP phosphodiesterase, ion channels, Rho family guanine-nucleotide exchange factors (RhoGEF), and activate mitogen-activated protein kinase signal transduction pathways (2Birnbaumer L. Annu. Rev. Pharmacol. Toxicol. 1990; 30: 675-705Google Scholar, 3Hepler J.R. Gilman A.G. Trends Biochem. Sci. 1992; 17: 383-387Google Scholar, 4Neer E.J. Cell. 1995; 80: 249-257Google Scholar, 5Marinissen M.J. Gutkind J.S. Trends Pharmacol. 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This occludes Gαβγ interaction with receptor and, in some nonvisual cells, leads to sequestration of the receptor away from the plasma membrane into endocytic vesicles (8Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Google Scholar, 9Ferguson S.S.G. Downey W.I. Colapietro A. Barak L. Menard L. Caron M.G. Science. 1996; 271: 363-366Google Scholar, 10Goodman Jr., O. Krupnick J. Santini F. Gurevich V. Penn R. Gagnon A. Keen J. Benovic J.L. Nature. 1996; 383: 447-450Google Scholar, 11Buneman M. Lee K. Pals-Rylaarsdam R. Roseberry A. Hosey M. Annu. Rev. Physiol. 1999; 61: 169-192Google Scholar). GRKs are found in metazoans and, in mammals, the GRK family has seven members (7Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Google Scholar, 12Weiss E.R. Raman D. Shirakawa S. Ducceschi M.H. Bertram P.T. Wong F. Kraft T.W. Osawa S. Mol. Vision. 1998; 4: 27Google Scholar). GRKs are serine/threonine kinases with a tripartite modular structure. A central ∼350 amino acid kinase domain is closely related by sequence identity to those of cAMP-dependent protein kinases, protein kinase C, and ribosomal S6 kinases (13Hanks S. Hunter T. FASEB J. 1995; 9: 576-596Google Scholar). At the carboxyl terminus of the catalytic core (14Hanks S. Quinn A. Methods Enzymol. 1991; 200: 38-62Google Scholar) homology to cAMP-dependent protein kinase predicts a putative "nucleotide gate" (15Narayana N. Cox S. Nguyen-huu X. Ten Eyck L. Taylor S. Structure. 1997; 5: 921-935Google Scholar). The catalytic domain is flanked by an amino-terminal domain of 178 residues and a carboxyl-terminal domain that varies in structure among members of the family. Using distinct mechanisms, the carboxyl-terminal domains of GRKs direct the membrane association of these kinases (16Sterne-Marr R. Benovic J.L. Vitam. Horm. 1995; 51: 193-234Google Scholar, 17Pitcher J.A. Hall R.A. Daaka Y. Zhang J. Ferguson S.S. Hester S. Miller S. Caron M.G. Lefkowitz R.J. Barak L.S. J. Biol. Chem. 1998; 273: 12316-12324Google Scholar). The amino-terminal domains of all GRK family members are homologous to the regulator of G-protein signaling (RGS) family of proteins (18Siderovski D.P. Hessel A. Chung S. Mak T.W. Tyers M. Curr. Biol. 1996; 6: 211-212Google Scholar). RGS proteins are a multifunctional family of proteins of variable length that share a ∼120-amino acid "RGS domain." In this paper, we refer to this domain as the RGS homology (RH) domain. RGS proteins act as GTPase activating proteins (GAPs) for the Gαi/o(including Gαt) and Gαq family of Gα subunits (19Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Google Scholar, 20Hepler J.R. Trends Pharmacol. Sci. 1999; 20: 376-382Google Scholar, 21Ross E.M. Wilkie T.M. Annu. Rev. Biochem. 2000; 69: 795-827Google Scholar) and as antagonists of Gα/effector interaction (19Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Google Scholar,22Hepler J.R. Berman D.M. Gilman A.G. Kozasa T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 428-432Google Scholar). In general, these proteins bind preferentially to the GDP-aluminum fluoride (GDP·AlF 4−) > GTPγS ≫ GDP-bound form of Gα (21Ross E.M. Wilkie T.M. Annu. Rev. Biochem. 2000; 69: 795-827Google Scholar). The crystal structures of the RGS4·Gαi1 complex and the RGS9·Gαt/i chimera·cGMP phosphodiesterase γ complex show that the RGS proteins contact switch regions I, II, and III of Gα, which are polypeptide loops that undergo conformational changes in the transformation between the GDP-bound (inactive) and the GTP-bound (active) states of the G protein. In these examples, the binding of the RGS protein appears to stabilize the three switch regions in a conformation that preferentially binds the transition state for GTP hydrolysis (23Tesmer J.J.G. Berman D.M. Gilman A.G. Sprang S.R. Cell. 1997; 89: 251-261Google Scholar, 24Slep K.C. Kercher M.A. He W. Cowan C.W. Wensel T.G. Sigler P.B. Nature. 2001; 409: 1071-1077Google Scholar). RH domains can be grouped into five subfamilies based on their evolutionary relatedness: R4, R7, R12, RZ, and RA (axin) families (21Ross E.M. Wilkie T.M. Annu. Rev. Biochem. 2000; 69: 795-827Google Scholar). Members of the R4, R7, R12, and RZ families are negative regulators of G protein signaling as described above. The newly described Gαs-specific RGS, RGS-PX1 (25Zheng B. Ma Y.-C. Ostrom R.S. Lavoie C. Gill G.N. Insel P.A. Huang X.-Y. Farquhar M.G. Science. 2001; 294: 1939-1942Google Scholar), likely defines another RGS subfamily as this protein is similarly related to all 5 RGS subfamilies (∼24% amino acid identity). Other families of proteins have RH domains but their roles in regulating heterotrimeric G protein signaling are either distinct from RGS proteins, not well characterized, or do not regulate heterotrimeric G protein signaling. Axin plays a role in the wnt/embryonic development signaling pathway (26Zeng L. Fagotto F. Zhang T. Hsu W. Vasicek T. Perry III, W. Lee J. Tilgfhman S. Gumbiner B. Costantini F. Cell. 1997; 90: 181-192Google Scholar) and shares ∼30% amino acid identity with RGS proteins of other subfamilies. This RH domain has never been demonstrated to bind or GAP a Gα subunit (27Mao J. Yuan H. Xie W. Simon M.I. Wu D. J. Biol. Chem. 1998; 273: 27118-27123Google Scholar). Instead the RH domain of axin binds the tumor suppressor protein, APC (28Kishida S. Yamamoto H. Ikeda S. Kishida M. Sakamoto I. Koyama S. Kikuchi A. J. Biol. Chem. 1998; 273: 10823-10826Google Scholar), a downstream target in the wnt signaling pathway. The APC binding site of axin is distinct from the Gα binding site of RGS proteins. A family of guanine nucleotide exchange factors for the monomeric G protein Rho (RhoGEFs) also has RH domains that share <20% identity to the RGS family. p115RhoGEF, PDZRhoGEF, and LARG (leukemia-associated RhoGEF), bind and in some cases serve as GAPs for Gα12, Gα13, and Gαq, via their RH domains yet they are also downstream effectors of Gα12 and Gα13 (29Kozasa T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. Gilman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Google Scholar, 30Fukuhara S. Murga C. Zohar M. Igishi T. Gutkind J.S. J. Biol. Chem. 1999; 274: 5868-5879Google Scholar, 31Fukuhara S. Chikumi H. Gutkind J.S. FEBS Lett. 2000; 485: 183-188Google Scholar, 32Booden M. Siderovski D.P. Der C. Mol. Cell. Biol. 2002; 22: 4053-4061Google Scholar). d-AKAP2, dual specificity A kinase anchoring protein 2, binds the regulatory subunit of cAMP-dependent protein kinase and has 2 RH domains. However, no G protein interaction has been reported for this protein (33Wang L. Sunahara R.K. Krumins A. Perkins G. Crochiere M.L. Mackey M. Bell S. Ellisman M.H. Taylor S.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3220-3225Google Scholar). The RH domain of GRK2 is most closely related to that of axin (26% amino acid identity) and RGS12 (24% amino acid identity) and binds to Gαq and Gα11 in an AlF 4−-dependent fashion, but not to Gαs, Gαi, or Gα12/13 (34Carman C.V. Parent J.L. Day P.W. Pronin A.N. Sternweis P.M. Wedegaertner P.B. Gilman A.G. Benovic J.L. Kozasa T. J. Biol. Chem. 1999; 274: 34483-34492Google Scholar, 35Sallese M. Mariggio S. D'Urbano E. Iacovelli L. De Blasi A. Mol. Pharmacol. 2000; 57: 826-831Google Scholar, 36Usui H. Nishiyama M. Moroi K. Shibasaki T. Zhou J. Ishida J. Fukamizu A. Haga T. Sekiya S. Kimura S. Int. J. Mol. Med. 2000; 5: 335-340Google Scholar). Whereas all GRKs have putative amino-terminal RH domains, Gα interaction has only been observed for GRK2 and GRK3. Unlike other Gαq-binding RGS proteins such as RGS2 (37Ingi 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: 7178-7188Google Scholar), RGS3 (38Scheschonka A. Dessauer C. Sinnaraja S. Chidiac P. Shi C.-S. Kehrl J.H. Mol. Pharmacol. 2000; 58: 719-728Google Scholar), RGS4 (22Hepler J.R. Berman D.M. Gilman A.G. Kozasa T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 428-432Google Scholar), and RGS18 (39Nagata Y. Oda M. Nakata H. Shozaki Y. Kozasa T. Todokoro K. Blood. 2001; 97: 3051-3060Google Scholar), the GRK2 RH domain does not stimulate the GTPase activity of Gαq in a single turnover GAP assay and only weakly stimulates GTPase when Gαq is reconstituted with M1 muscarinic receptor and assayed in an agonist-induced steady-state GTPase assay (34Carman C.V. Parent J.L. Day P.W. Pronin A.N. Sternweis P.M. Wedegaertner P.B. Gilman A.G. Benovic J.L. Kozasa T. J. Biol. Chem. 1999; 274: 34483-34492Google Scholar). Because the GRK2 RH domain inhibits Gαq-stimulated PLCβ activity both in vivo and in vitro yet lacks significant GAP activity in vitro, it has been postulated that GRK2 RGS acts by sequestration of Gαq. It is also possible that GRK2 is an effector of Gαq. In this scenario, activation of Gαq would recruit GRK2 to the site of an activated receptor. To investigate the role of GRK2/Gαq interaction in the regulation of Gqsignaling, we have created mutations in the RH domain of GRK2 that result in altered binding to Gαq/11. Surprisingly, we found that the surface of GRK2 used to bind Gαq/11 is distinct from the interaction site utilized by other RGS proteins to bind Gα subunits and from the site used by axin to bind APC. Human embryonic kidney cells (HEK293) and African Green monkey kidney cells (COS-1) were from the American Tissue Culture Collection. Gαq/11-specific polyclonal antibodies were generously provided by Dr. D. Manning or purchased from Santa Cruz Antibodies, and EE-specific monoclonal antibody was provided by Dr. H. Bourne. RGS2-GFP (40Heximer S.P. Lim H. Bernard J.L. Blumer K.J. J. Biol. Chem. 2001; 276: 14195-14203Google Scholar) and GRK2-(45–178)-GFP were expressed from the plasmid pEGFP (Clontech, Palo Alto, CA) and were generously provided by Dr. S. Heximer and Dr. R. Penn, respectively.myo-[3H]Inositol was from AmershamBiosciences, Dowex AG1-X8 resin was from Bio-Rad, and scintillation fluid was from Packard. Molecular biologicals were from Roche Molecular Biochemicals unless otherwise indicated, immunoblotting detection reagents were from Pierce, and all other biochemicals were from Sigma or Fisher. Nucleotides encoding residues 45–178 of bovine GRK2 cDNA were amplified by the polymerase chain reaction using primers that incorporated BamHI and EcoRI restriction sites at the 5′ and 3′ ends of the coding region, respectively. The resulting PCR fragment was subcloned into BamHI and EcoRI sites of the glutathione S-transferase fusion protein vector, pGEX-2T (Amersham Biosciences) to generate pGEX-GRK2-(45–178). Sequential PCR (41Cormack B. Current Protocols in Molecular Biology. John Wiley and Sons, New York1991Google Scholar) was used to produce the E78K, V83A, and D160K derivatives of pGEX-GRK2-(45–178), and Quik-Change mutagenesis (Stratagene) was used to generate all other mutations. The GRK2 portion of each construct was sequenced to verify that only the intended mutation had occurred. GST-GRK2 fusion proteins were expressed and purified by modifications of the procedures of Smith and Johnson (42Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Google Scholar) and Frangioni and Neel (43Frangioni J.V. Neel B.G. Anal Biochemistry. 1993; 210: 179-187Google Scholar). Briefly, 40-ml cultures in Luria broth containing 5 μg/ml carbenicillin were grown at 37 °C to an optical density of 0.5, fusion protein expression was induced by the addition of isopropyl-1-thio-β-d-galactopyranoside to 0.5 mm, and incubation was continued for 3 h at 25 °C. Cells were pelleted, washed in STE (20 mm Tris, 150 mm NaCl, 1 mm EDTA, pH 8), and frozen at −70 °C. Pellets were resuspended on ice in STE containing 100 μg/ml lysozyme and incubated on ice for 15 min before the addition of β-mercaptoethanol to 10 mm, phenylmethylsulfonyl fluoride to 100 μm, leupeptin to 1 μg/ml, benzamidine to 20 μg/ml, and Sarkosyl to 1.5%. Lysates were sonicated in 10-s bursts followed by 15-s rest periods to reduce viscosity. Insoluble protein was removed by centrifugation at 12,000 rpm for 10 min at 4 °C and Triton X-100 was added to a final concentration of 2%. The lysate was adjusted to 25 mg/ml protein as determined by the Bradford assay using γ-globulin as a standard (Bio-Rad) and fusion proteins were bound to glutathione-agarose beads (3 ml of packed beads/100 mg of protein) by mixing for 1 h at 4 °C. The beads were washed once with STE, 1.5% Sarkosyl, 2% Triton X-100, three times with STE, and stored in STE, 25% glycerol, 10 mm β-mercaptoethanol at −20 °C. To determine the amount of GRK2 associated with glutathione-agarose beads, fusion proteins were eluted in 50 mm Tris-HCl, pH 8, 10 mm glutathione, 10 mm β-mercaptoethanol at room temperature for 1 h. Bradford assays were then carried out on the eluates. Bovine brain extract was used as a source of Gαq/11 for in vitro binding assays. For 1-ml binding assays, 8 μg of fusion protein was incubated overnight at 4 °C with 200 μg of brain extract protein, prepared as described by Carman et al. (34Carman C.V. Parent J.L. Day P.W. Pronin A.N. Sternweis P.M. Wedegaertner P.B. Gilman A.G. Benovic J.L. Kozasa T. J. Biol. Chem. 1999; 274: 34483-34492Google Scholar), in 20 mm Tris-HCl, pH 8, 2 mm MgSO4, 6 mmβ-mercaptoethanol, 100 mm NaCl, 0.05% C12E10, 5% glycerol, 100 μm GDP in the absence or presence of 30 μm aluminum chloride and 5 mm sodium fluoride (AlF 4−). Glutathione-agarose beads were washed 4 times with the buffer described above (in the absence or presence of AlF 4− as appropriate), proteins were eluted with SDS-PAGE sample buffer, and separated on 12% polyacrylamide gels. The proteins were transferred to nitrocellulose, probed with anti-Gαq/11-specific polyclonal antibodies, incubated with peroxidase-conjugated secondary antibody, and Gαq/11 was visualized by chemiluminescence using SuperSignal West Pico (Pierce). COS-1 cells were grown at 37 °C to 50–90% confluence on 10-cm dishes in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. The cells were transfected with 10 μg of total DNA (pcDNA3 alone, pcDNA3-GRK2, or pcDNA3-mutant GRK2) using FuGENE 6 (Roche Molecular Biochemicals) following the manufacturer's recommendations. The cells were harvested after 48 h, washed twice in ice-cold phosphate-buffered saline, and lysed in 1 ml of buffer (20 mm HEPES, pH 7.2, 150 mm NaCl, 10 mm EDTA, 0.02% Triton X-100, 0.5 mmphenylmethylsulfonyl fluoride, 20 μg/ml leupeptin, and 100 μg/ml benzamidine) by Polytron homogenization (two 15-s bursts at 2500 rpm). Lysates were centrifuged for 10 min at 40,000 × g to remove particulate matter and supernatants were then assayed. To test for GRK activity, lysates containing wild type (WT) or mutant GRK2 protein were assayed for their ability to phosphorylate light-activated rhodopsin. Two microliters of COS-1 cell lysate were incubated with 20 mm Tris-HCl, pH 7.5, 2 mmEDTA, 5 mm MgCl2, 100 μm ATP, ∼1 μCi of [γ-32P]ATP, and ∼3.5 μmrhodopsin for 10 min at 30 °C in room light. Reactions were quenched by addition of SDS sample buffer followed by 30 min incubation at room temperature. Rhodopsin was separated by electrophoresis on a 10% SDS-polyacrylamide gel, and gels were fixed in 0.7 mtrichloroacetic acid, 0.14 m 5′-sulfosalicylic acid for 10 min to remove unincorporated radionucleotide, washed twice in 50% ethanol, 16% acetic acid for 10 min, dried, and then subjected to autoradiography. Rhodopsin bands were excised and counted in a liquid scintillation counter. Repeated measures analysis of variance was used to test the statistical significance. A homology model of the RH domain of GRK2 (residues 42–178) was based on the structure of the RH domain of axin (PDB code 1EMU), which is the closest homolog based on a BLAST (44Altschul S. Gish W. Miller W. Myers E. Lipman D. J. Mol. Biol. 1990; 215: 403-410Google Scholar) search of the protein data bank (26% identity within residues 64–174 of GRK2). The GRK2 model was built manually using the program O (45Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Google Scholar) by choosing appropriate and reasonable rotamers for nonidentical residues (46Kleywegt G. Jones T. Acta Crystallogr. Sect. D. 1998; 54: 1119-1131Google Scholar). In regions that appeared to have higher sequence identity with other RH domains of known structure, the GRK2 model was adjusted locally according to those models. The α5/α6 loop of the GRK2 RH domain has no obvious sequence homology to RH domains of known structure and was ultimately modeled based on the axin structure because the fit of the side chains appeared to be reasonable and because the α5/α6 loops of GRK2 and axin are identical in length (the loop is one amino acid shorter in the RGS family of proteins). The overall model was refined in O to idealize its stereochemistry. To measure in vivo synthesized inositol phosphate (IP), 3.3 × 105 COS-1 cells were plated on 6-cm dishes in DMEM (Mediatech, Herndon, VA) containing penicillin (100 units/ml), streptomycin (100 μg/ml), and 10% fetal bovine serum. After 24 h, cells were transfected using FuGENE 6 (Roche Molecular Biochemicals) with 1 μg of total DNA at a ratio of 3:1 pcDNA3-HA-Gαq-R183C:pcDNA3-GRK2 (or mutant derivatives of GRK2). Following a 24-h incubation, transfected cells were replated (∼7 × 104 cell/well) in triplicate on 24-well plates and incubated in complete DMEM. The media was removed and cells were labeled with myo-[3H]inositol (Amersham Biosciences) for 13–18 h in DMEM, without sodium pyruvate, with high glucose, with l-glutamine, and with pyridoxine hydrochloride. In early experiments, labeling was carried out in inositol-free DMEM (Invitrogen), whereas later experiments utilized complete DMEM. Cells were washed in the same media lacking radiolabel but containing 5 mm LiCl for 1 h at 37 °C. The media was removed and cells were lysed with 0.75 ml of 20 mm formic acid for 30 min at 4 °C before 0.1 ml of 3% ammonium hydroxide was added. Inositol was separated from IP by sequential elution from 1-ml Dowex AG1-X8 (100–200 mesh) columns. The inositol fraction was eluted with 0.18% ammonium hydroxide, whereas IPs were eluted with 4 m ammonium formate, 0.2m formic acid. The inositol and IP fractions were mixed with Ultima Gold and Ultima Flo AF scintillation fluid (Packard), respectively, and subjected to scintillation counting. To compare experiments with differing levels ofmyo-[3H]inositol incorporation, IP production was determined as a fraction, IP/(IP + inositol), and plotted relative to the control Gαq-R183C-stimulated IP production. Statistical significance was assessed using repeated measures analysis of variance with a Dunnett's post-test. IP accumulation experiments shown in Fig. 7 were carried out as described above except that HEK293 cells were utilized. In addition, 250 ng of plasmids encoding Gαq-R183C or Gαq-R183C/G188S were transfected with increasing amounts of pcDNA3-GRK2, pcDNA3-RGS2, and pB6-GAIP plasmids as indicated in the figure. pcDNA3 vector was used as carrier DNA such that 1 μg of DNA was transfected in each well of the 6-well plate. An unpaired ttest was used to assess statistical significance. HEK293 cells were transfected in 6-well plates with the indicated amounts of expression plasmids for GRK2-(45–178)-GFP, RGS2-GFP, and/or Gαq using FuGENE 6 reagent (Roche Molecular Biochemicals). After 24 h, transfected cells were replated onto glass coverslips and grown for an additional 24 h before fixing in 3.7% formaldehyde for 20 min. Cells were washed with phosphate-buffered saline and then incubated in blocking buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 1% Triton X-100, and 2.5% nonfat milk). Coverslips were then incubated in blocking buffer containing a 1:100 dilution of anti-Gαqpolyclonal antibody (Santa Cruz) for 1 h. Following washes with blocking buffer, cells were incubated in a 1:100 dilution of Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (Molecular Probes) for 30 min. The coverslips were washed and mounted on glass slides with Prolong Antifade reagent. Representative images were recorded by confocal microscopy at the Kimmel Cancer Center Bioimaging Facility using a Bio-Rad MRC-600 laser scanning confocal microscope running CoMos 7.0a software and interfaced to a Zeiss Axiovert 100 microscope with Zeiss Plan-Apo 63× 1.40 NA oil immersion objective. Dual-labeled samples were analyzed using simultaneous excitation at 488 and 568 nm. Images of "x-y" sections through the middle of a cell were recorded. For a growing list of Gq-coupled receptors, it has been reported that desensitization can occur in a GRK2-dependent but phosphorylation-independent fashion. For example, hormone-mediated PLCβ activation via the metabotropic glutamate receptor (mGluR1a) (47Dale L.B. Bhattacharya M. Anborgh P.H. Murdoch B. Bhatia M. Nakanishi S. Ferguson S.S.G. J. Biol. Chem. 2000; 275: 38213-38220Google Scholar), the parathyroid hormone receptor (48Dicker F. Quitterer U. Winstel R. Honold K. Lohse M.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5476-5481Google Scholar), the thromboxane A2 receptor (34Carman C.V. Parent J.L. Day P.W. Pronin A.N. Sternweis P.M. Wedegaertner P.B. Gilman A.G. Benovic J.L. Kozasa T. J. Biol. Chem. 1999; 274: 34483-34492Google Scholar), the endothelin receptor (49Freedman N.J. Ament A.S. Oppermann M. Stoffel R.H. Exum S.T. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 17734-17743Google Scholar), and the angiotensin II-1A receptor (50Oppermann M. Freedman N.J. Alexander R.W. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 13266-13272Google Scholar), is inhibited by overexpression of kinase-deficient GRK2-K220R. The parathyroid receptor and mGluR1a interact with full-length GRK2 (47Dale L.B. Bhattacharya M. Anborgh P.H. Murdoch B. Bhatia M. Nakanishi S. Ferguson S.S.G. J. Biol. Chem. 2000; 275: 38213-38220Google Scholar, 48Dicker F. Quitterer U. Winstel R. Honold K. Lohse M.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5476-5481Google Scholar) and the RH domain of GRK2 co-immunoprecipitates with mGluR1a (51Dhami G.K. Anborgh P.H. Dale L.B. Sterne-Marr R. Ferguson S.S.G. J. Biol. Chem. 2002; 277: 25266-25272Google Scholar). For the mGluR1a (51Dhami G.K. Anborgh P.H. Dale L.B. Sterne-Marr R. Ferguson S.S.G. J. Biol. Chem. 2002; 277: 25266-25272Google Scholar), the endothelin receptor (49Freedman N.J. Ament A.S. Oppermann M. Stoffel R.H. Exum S.T. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 17734-17743Google Scholar), and the thromboxane A2 receptor (34Carman C.V. Parent J.L. Day P.W. Pronin A.N. Sternweis P.M. Wedegaertner P.B. Gilman A.G. Benovic J.L. Kozasa T. J. Biol. Chem. 1999; 274: 34483-34492Google Scholar), overexpression of the RH domain of GRK2 inhibits Gq-stimulated phosphoinositide hydrolysis. Thus, phosphorylation-independent regulation of these receptors may be due either to GRK2/receptor interaction or to GRK2·Gαqcomplex formation (or both). As a first step to determine the extent to which Gαq binding by
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