Selective Regulation of Gαq/11 by an RGS Domain in the G Protein-coupled Receptor Kinase, GRK2
1999; Elsevier BV; Volume: 274; Issue: 48 Linguagem: Inglês
10.1074/jbc.274.48.34483
ISSN1083-351X
AutoresChristopher V. Carman, Jean-Luc Parent, Peter Day, Alexey Pronin, Pamela M. Sternweis, Philip Wedegaertner, Alfred G. Gilman, Jeffrey Benovic, Tohru Kozasa,
Tópico(s)Cell Adhesion Molecules Research
ResumoG protein-coupled receptor kinases (GRKs) are well characterized regulators of G protein-coupled receptors, whereas regulators of G protein signaling (RGS) proteins directly control the activity of G protein α subunits. Interestingly, a recent report (Siderovski, D. P., Hessel, A., Chung, S., Mak, T. W., and Tyers, M. (1996) Curr. Biol. 6, 211–212) identified a region within the N terminus of GRKs that contained homology to RGS domains. Given that RGS domains demonstrate AlF4−-dependent binding to G protein α subunits, we tested the ability of G proteins from a crude bovine brain extract to bind to GRK affinity columns in the absence or presence of AlF4−. This revealed the specific ability of bovine brain Gαq/11 to bind to both GRK2 and GRK3 in an AlF4−-dependent manner. In contrast, Gαs, Gαi, and Gα12/13 did not bind to GRK2 or GRK3 despite their presence in the extract. Additional studies revealed that bovine brain Gαq/11 could also bind to an N-terminal construct of GRK2, while no binding of Gαq/11, Gαs, Gαi, or Gα12/13 to comparable constructs of GRK5 or GRK6 was observed. Experiments using purified Gαq revealed significant binding of both GαqGDP/AlF4− and Gαq(GTPγS), but not Gαq(GDP), to GRK2. Activation-dependent binding was also observed in both COS-1 and HEK293 cells as GRK2 significantly co-immunoprecipitated constitutively active Gαq(R183C) but not wild type Gαq. In vitro analysis revealed that GRK2 possesses weak GAP activity toward Gαq that is dependent on the presence of a G protein-coupled receptor. However, GRK2 effectively inhibited Gαq-mediated activation of phospholipase C-β both in vitro and in cells, possibly through sequestration of activated Gαq. These data suggest that a subfamily of the GRKs may be bifunctional regulators of G protein-coupled receptor signaling operating directly on both receptors and G proteins. G protein-coupled receptor kinases (GRKs) are well characterized regulators of G protein-coupled receptors, whereas regulators of G protein signaling (RGS) proteins directly control the activity of G protein α subunits. Interestingly, a recent report (Siderovski, D. P., Hessel, A., Chung, S., Mak, T. W., and Tyers, M. (1996) Curr. Biol. 6, 211–212) identified a region within the N terminus of GRKs that contained homology to RGS domains. Given that RGS domains demonstrate AlF4−-dependent binding to G protein α subunits, we tested the ability of G proteins from a crude bovine brain extract to bind to GRK affinity columns in the absence or presence of AlF4−. This revealed the specific ability of bovine brain Gαq/11 to bind to both GRK2 and GRK3 in an AlF4−-dependent manner. In contrast, Gαs, Gαi, and Gα12/13 did not bind to GRK2 or GRK3 despite their presence in the extract. Additional studies revealed that bovine brain Gαq/11 could also bind to an N-terminal construct of GRK2, while no binding of Gαq/11, Gαs, Gαi, or Gα12/13 to comparable constructs of GRK5 or GRK6 was observed. Experiments using purified Gαq revealed significant binding of both GαqGDP/AlF4− and Gαq(GTPγS), but not Gαq(GDP), to GRK2. Activation-dependent binding was also observed in both COS-1 and HEK293 cells as GRK2 significantly co-immunoprecipitated constitutively active Gαq(R183C) but not wild type Gαq. In vitro analysis revealed that GRK2 possesses weak GAP activity toward Gαq that is dependent on the presence of a G protein-coupled receptor. However, GRK2 effectively inhibited Gαq-mediated activation of phospholipase C-β both in vitro and in cells, possibly through sequestration of activated Gαq. These data suggest that a subfamily of the GRKs may be bifunctional regulators of G protein-coupled receptor signaling operating directly on both receptors and G proteins. G protein-coupled receptor G protein-coupled receptor kinase inositol bisphosphate GTPase activating protein guanosine 5′-3-O-(thio)triphosphate phospholipase C hemagglutinin polymerase II polyacrylamide gel electrophoresis glutathioneS-transferase inositol 1,4,5-trisphosphate M1 muscarinic cholinergic receptor thromboxane A2-α receptor G protein-coupled receptors (GPCRs)1 reside at the plasma membrane where they receive diverse extracellular stimuli, in the form of light, odorants, neurotransmitters, and hormones. This information is translated into intracellular signals when agonist-bound GPCRs activate exchange of GTP for GDP on the α subunit of heterotrimeric G proteins. Activated, GTP-bound Gα (Gα(GTP)) then dissociates from Gβγ and each of these G protein components go onto regulate downstream effector molecules. In general the intracellular signal is limited by the presence of the extracellular stimuli and by the intrinsic GTPase activity of Gα. However, in order to selectively modulate the appropriate magnitude and duration of signals in diverse cellular contexts, several ubiquitous mechanisms are utilized to regulate these signaling cascades both at the level of the GPCR and at the level of the G protein. At the level of the GPCR, agonist-specific loss of receptor responsiveness involves a family of G protein-coupled receptor kinases (GRK1–6). GRKs phosphorylate the agonist-activated form of GPCRs which in turn promotes the high-affinity binding of a second family of proteins termed arrestins (1Carman C.V. Benovic J.L. Curr. Opin. Neurobiol. 1998; 8: 335-344Crossref PubMed Scopus (244) Google Scholar). These interactions function to uncouple the GPCR from further G protein activation and to promote clathrin-mediated internalization of the receptor (1Carman C.V. Benovic J.L. Curr. Opin. Neurobiol. 1998; 8: 335-344Crossref PubMed Scopus (244) Google Scholar). Initiation of this process is controlled by GRKs, which are, in turn, regulated by a variety of molecules including the activated GPCRs themselves, Gβγ subunits, PIP2, PKC, calmodulin, and caveolin (1Carman C.V. Benovic J.L. Curr. Opin. Neurobiol. 1998; 8: 335-344Crossref PubMed Scopus (244) Google Scholar, 2Stoffel III, R.H. Pitcher J.A. Lefkowitz R.J. J. Membr. Biol. 1997; 157: 1-8Crossref PubMed Scopus (44) Google Scholar, 3Carman C.V. Lisanti M.P. Benovic J.L. J. Biol. Chem. 1999; 274: 8858-8864Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). The overall topology of GRKs includes a somewhat conserved catalytic domain of ∼270 residues which is flanked by N- and C-terminal regulatory domains. The C terminus is highly variable (∼100–230 residues) and has the general function of mediating membrane localization. For example, GRK2 and GRK3 possess a C-terminal plecktrin homology domain which binds to both PIP2 and free Gβγ promoting membrane recruitment and subsequent receptor phosphorylation (2Stoffel III, R.H. Pitcher J.A. Lefkowitz R.J. J. Membr. Biol. 1997; 157: 1-8Crossref PubMed Scopus (44) Google Scholar). Interestingly, the ability of GRK2 and GRK3 to bind to Gβγ has also been implicated as playing a direct role in the regulation of G protein signaling via the sequestration of free Gβγ (4Koch W.J. Hawes B.E. Inglese J. Luttrel L.M. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 6193-6197Abstract Full Text PDF PubMed Google Scholar, 5Viard P. Exner T. Maier U. Mironneau J. Nernberg B. Macrez N. FASEB J. 1999; 13: 685-694Crossref PubMed Scopus (108) Google Scholar, 6Bunemann M. Hosey M. J. Biol. Chem. 1998; 273: 31186-31190Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The ∼190 residue N terminus of GRKs is modestly conserved and has been suggested to contain receptor binding determinants (7Palczewski K. Buczylko J. Lebioda L. Crabb J.W. Polans A.S. J. Biol. Chem. 1993; 268: 6004-6013Abstract Full Text PDF PubMed Google Scholar). Recently, calmodulin (8Pronin A.N. Satpaev D.K. Slepak V. Z. Benovic J.L. J. Biol. Chem. 1998; 272: 18273-18280Abstract Full Text Full Text PDF Scopus (139) Google Scholar), PIP2 (9Pitcher J.A. Fredericks Z.L. Stone W.C. Premont R.T. Stoffel R.H. Koch W.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 24907-24913Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), and caveolin (3Carman C.V. Lisanti M.P. Benovic J.L. J. Biol. Chem. 1999; 274: 8858-8864Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) have also been shown to interact with the N terminus. However, the overall structure and function of this domain has remained largely uncharacterized. At the level of the G protein, regulation occurs through intrinsic GTPase activity possessed by the Gα subunits which hydrolyze bound GTP and promote rebinding of Gβγ. This process has recently been found to be modulated by a ubiquitous family of proteins termed regulators of G protein signaling (RGS), which serve as GTPase-activating proteins (GAPs) that accelerate the rate of GTP hydrolysis and thereby limit the half-life of the activated species (10Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 11Zerangue N. Jan L.Y. Curr. Biol. 1998; 8: R313-R316Abstract Full Text Full Text PDF PubMed Google Scholar). RGS proteins share a ∼120-residue region of homology termed an RGS domain which folds into an α-helical module that binds preferentially to the transition state of Gα (12Tesmer J.J.G. Berman D.M. Gilman A.G. Sprang S.R. Cell. 1997; 89: 251-261Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar). This preferential binding to the transition state, which can be mimicked in vitro by the addition of GDP/AlF4−(13Coleman D.E. Berghuis A.M. Lee E. Linder M.L. Gilman A.G. Sprang S.R. Science. 1994; 265: 1405-1412Crossref PubMed Scopus (767) Google Scholar), compared with the active state, which can be stably generatedin vitro by addition of GTPγS, is thought to serve as the driving force for acceleration of GTPase activity (14Berman D.M. Kozasa T. Gilman A.G. J. Biol. Chem. 1996; 271: 27209-27212Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 15Srinivasa S.P. Watson N. Overton M.C. Blumer K.J. J. Biol. Chem. 1998; 273: 1529-1533Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). At least 18 RGS proteins have been identified. In general, these RGS proteins interact with the α subunits of the Gi and Gq families (10Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 11Zerangue N. Jan L.Y. Curr. Biol. 1998; 8: R313-R316Abstract Full Text Full Text PDF PubMed Google Scholar, 16DeVries L. Farquhar M.G. Trends Cell Biol. 1999; 9: 138-144Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). In addition, a small collection of proteins including GRKs (17Siderovski D.P. Hessel A. Chung S. Mak T.W. Tyers M. Curr. Biol. 1996; 6: 211-212Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar), axin (18Zeng L. Fagotto F. Zhang T. Hsu W. Vasicek T.J. Perry III, W.L. Lee J.J. Tilghman S.M. Gumbiner B.M. Costantini F. Cell. 1997; 90: 181-182Abstract Full Text Full Text PDF PubMed Scopus (802) Google Scholar), D-AKAP (19Huang L.J.-S. Durick K. Weiner J.A. Chun J. Taylor S.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11184-11189Crossref PubMed Scopus (202) Google Scholar), and p115 Rho-GEF (20Kozasa 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 (743) Google Scholar, 21Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (679) Google Scholar) have been identified as having somewhat less conserved RGS domains. Recently, one of these atypical RGS proteins, p115 Rho-GEF, was shown to function as a selective GAP for Gα12/13 suggesting that sequence differences in these RGS proteins may correlate with different preferences for G protein-binding partners (20Kozasa 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 (743) Google Scholar, 21Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (679) Google Scholar). To date, no functionality has been attributed to any of the other atypical RGS domains. Given that GRK2 and GRK3 represent well characterized components of GPCR regulation that are already known to bind to Gβγ subunits, we explored the possibility that these GRKs may interact with Gα subunits. These experiments revealed selective and high affinity binding of activated Gαq/11 to GRK2 and GRK3, an interaction that may function to regulate phospholipase C-β (PLC-β) activity in vivo. Hemagglutinin (HA)-specific monoclonal and polyclonal antibodies were from Roche Molecular Biochemicals and Babco, respectively. ECL reagents were from Pierce while FugeneTMwas from Life Technologies, Inc. Polymerase II (pol II)-specific polyclonal antibody was from Santa Cruz. Gαq/11-, Gαi-, Gαs-, Gα12/13-, and Gcommon-specific polyclonal antibodies were generously provided by Dr. D. Manning. CNBr-activated Sepharose 4B was from Amersham Pharmacia Biotech. ProBlott was purchased from Applied Biosystems. Phosphatidylinositol 3,4-phosphate (PIP2) and phosphatidylethanolamine were from Sigma. [γ-32P]ATP,myo-[3H]inositol, and [3H]PIP2 were from NEN Life Science Products Inc. Most other reagents were from sources previously described (3Carman C.V. Lisanti M.P. Benovic J.L. J. Biol. Chem. 1999; 274: 8858-8864Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar,20Kozasa 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 (743) Google Scholar). GRK2 and GRK3 were overexpressed in and purified from Sf9 insect cells as described previously (22Kim C.M. Dion S.B. Onorato J.J. Benovic J.L. Receptor. 1993; 3: 39-55PubMed Google Scholar). Purified GST, GST-GRK2(1–178), GST-GRK2(45–178), GST-GRK5 (1–200), and GST-GRK6 (1–192) fusion proteins and urea stripped rod outer segments were prepared as described previously (8Pronin A.N. Satpaev D.K. Slepak V. Z. Benovic J.L. J. Biol. Chem. 1998; 272: 18273-18280Abstract Full Text Full Text PDF Scopus (139) Google Scholar,23Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar). Sf9-expressed Gαq, Gαq(R183C), and Gα12 (24Hepler J.R. Kozasa T. Gilman A.G. Methods Enzymol. 1994; 237: 191-212Crossref PubMed Scopus (17) Google Scholar), as well as Escherichia coliexpressed myristoylated wild type Gαi and hexahistadine-tagged Gαs and RGS4 were purified as described previously (25Kozasa T. Gilman A.G. J. Biol. Chem. 1995; 270: 1734-1741Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 26Berman D.M. Wilkie T.M. Gilman A.G. Cell. 1996; 86: 445-452Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar). M1 muscarinic cholinergic receptor (M1AChR), PLC-β1, and PLC-β2 were purified from Sf9 cells and reconstituted into phospholipid vesicles along with purified Gαq and Gβγ1/2 as described previously (27Biddlecome G.H. Berstein G. Ross E.M. J. Biol. Chem. 1996; 271: 7999-8007Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). SDS-PAGE was performed using standard methods (28Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (218082) Google Scholar). Following electrophoresis, proteins were electroblotted onto polyvinylidene difluoride for peptide sequence analysis or nitrocellulose for immunoblotting. Immunoblotting was performed using Gαq/11-, Gαs-, Gαi-, Gα12/13-, α-tubulin-, actin-, GRK2-, Gβcommon-, HA-, or EE-specific primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies (1:2000 dilution). Immunoblots were visualized by ECL following the manufacturer's guidelines. Two g of CNBr-activated Sepharose 4B was hydrated and successively washed with 5 × 25 ml of 1 mm HCl. One mg of GRK2, GST-GRK2(1–178), GRK3, GST-GRK5(1–200), GST-GRK6(1–192), or GST was dialyzed against 3 × 500 ml of coupling buffer (0.1 mNaHCO3, pH 8.6, 500 mm NaCl). Dialyzed proteins (or an equal volume of coupling buffer for mock) were mixed with a 1-ml bed volume of CNBr-activated Sepharose 4B and rocked overnight at 4 °C. Resins were then washed with 5 × 20 ml of coupling buffer and residual unreacted sites were blocked by incubation with 0.1m Tris-HCl, pH 8.0, for 2 h at 4 °C. Resins were then washed with 2 × 20 ml of buffer A (20 mm Hepes, pH 7.4, 5 mm EDTA, 0.02% Triton X-100) containing 500 mm NaCl followed by 2 × 20 ml of buffer A containing 150 mm NaCl and finally adjusted to 50% bed volume. Coupling efficiencies ranged from 85 to 95%. Fresh bovine calf brain was stripped of connective tissue and minced in ∼1 ml of homogenization buffer (20 mm Tris-HCl, pH 7.5, 5 mm EDTA, 100 mm NaCl, 5 mmbenzamidine, 5 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 0.2% Triton X-100) per mg of tissue using a Brinkman Polytron (14,000 rpm, 30 s). The homogenate was centrifuged at 45,000 × g for 20 min and the resulting supernatant at 300,000 × g for 60 min. The final supernatant was aliquoted and stored at −70 °C until use. 250-μl aliquots (∼125 μg) of GRK-, GST-GRK-, GST-, or mock-coupled resins (50% bed volume) were incubated with 10 ml of the soluble brain extract (∼10 mg/ml total protein) and 10 ml of buffer B (20 mm Tris-HCl, pH 8.0, 2 mm MgSO4, 6 mm β-mercaptoethanol, 100 mm NaCl, 0.05% Lubrol, and 5% glycerol) with 100 μm GDP in the absence or presence of AlF4− (5 mmsodium fluoride and 30 μm AlCl3) for ∼12 h at 4 °C. The incubation mixture was then centrifuged at 1000 ×g for 1 min and the pellet washed four times with buffer B containing 100 μm GDP in the absence or presence of AlF4−. Bound proteins were released from the pelleted resin by addition of 150 μl of SDS sample buffer followed by boiling for 10 min. The eluted proteins were then subjected to 10% SDS-PAGE and transferred to polyvinylidene difluoride membrane. A specific ∼42-kDa protein band was identified by Ponceau-S staining, excised, and subjected to peptide sequence analysis. Alternatively, proteins were transferred to nitrocellulose membrane and subjected to immunoblot analysis. 0.5–5.0 μg of purified GRK2, GRK3, GST-GRK2(1–178), GST-GRK2(45–178), GST-GRK5(1–200), GST-GRK6(1–192), or GST immobilized on either CNBr-activated Sepharose 4B or glutathione-agarose beads were combined at 4 °C with 0.1–200 nm purified Gαq, Gαq (R183C), Gαs, Gαi1, or Gα12 in buffer B containing 100 μm GDP in the absence or presence of AlF4−. For binding curve experiments fixed amounts of Gαq and GRK2 affinity column were incubated in various volumes of binding buffer to produce the desired Gαq concentrations. For some experiments, Gα was preincubated in buffer B with either 1 mm GTP, 1 mm GTPγS, 1 mm GDP or 1 mmGDP/AlF4− at 25 °C for 2 h prior to addition to binding reactions. Samples were incubated at 30 °C for 60 min and chilled on ice for 5 min. The resins were then pelleted in a microcentrifuge for 10 s, washed three times with 400 μl of the appropriate binding buffers, and boiled with 50 μl of SDS sample buffer. Samples were subjected to 10% SDS-PAGE and immunoblotting using Gα-specific antibodies. Phosphorylation reactions contained, in a total volume of 20 μl, 30 nm GRK2 or GRK3, 200 nm Gαq, 100 μm[γ-32P]ATP (5 cpm/fmol), 20 mm Tris-HCl, pH 7.5, 2 mm EDTA, 7.5 mm MgCl2 in the absence or presence of one or more of the following: 100 μm GDP, AlF4−, 400 nm rhodopsin, or 60 nm Gβγ. Reactions were incubated at 37 °C for 0–60 min, stopped with SDS sample buffer, and subjected to 10% SDS-PAGE and autoradiography. Gα GTPase activity was determined in solution using a single turnover assay essentially as described previously (20Kozasa 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 (743) Google Scholar, 29Ingi T. Krumins A.M. Chidiac P. Brothers G.M. Chung S. Snown 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). Briefly, GTP-loaded Gαq(R183C), Gαs, Gαi1, Gαo, and Gα12 were generated by incubating Gα proteins in the presence of [γ-32P]GTP followed by gel filteration on G-25 Sephadex. Next, Gα([γ-32P]GTP) was incubated in the absence or presence of RGS4 (100 nm), GRK2 (300 nm), GST-GRK2(1–178) (500 nm), GST (500 nm), or control buffer. Reactions were quenched with 9 volumes of 5% (w/v) Norit A charcoal in 50 mmNaH2PO4. The charcoal was pelleted and the32Pi-containing supernatant was counted. Alternatively, the steady state GTPase activity was measured in phospholipid vesicles reconstituted with M1AChR and heterotrimeric Gq as described previously (27Biddlecome G.H. Berstein G. Ross E.M. J. Biol. Chem. 1996; 271: 7999-8007Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 29Ingi T. Krumins A.M. Chidiac P. Brothers G.M. Chung S. Snown 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). Briefly, the vesicles were equilibrated for 5 min at 20 °C in the absence or presence of carbachol (1 mm) and RGS4 (50 nm), GRK2 (300 nm), GST-GRK2(1–178) (500 nm), or GST (500 nm) in buffer containing GTP (4 μm). The experiment was initiated by addition of [γ-32P]GTP (106 cpm) followed by incubation at 30 °C. Reactions were quenched and quantitated as above. Determination of Gαq-mediated activation of PLC-β1 and PLC-β2 activity was performed in vitro essentially as described previously (30Hepler J.R. Kozasa T. Smrcka A.V. Simon M.I. Rhee S.G. Sternweis P.C. Gilman A.G. J. Biol. Chem. 1993; 268: 14367-14375Abstract Full Text PDF PubMed Google Scholar, 31Hepler J.R. Berman D.M. Giman A.G. Kozasa T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 428-432Crossref PubMed Scopus (338) Google Scholar). Briefly, lipid substrate was prepared by combining PIP2 and phosphatidylethanolamine (1:10) with [3H]PIP2 (5,000–10,000 cpm per assay) followed by sonication. Lipids were then combined with purified PLC-β1 or PLC-β2 in the absence or presence of purified Gαq(GDP)/AlF4− (150 nm) and 6 μm RGS4, GST-GRK2(1–178), or GST on ice. The incubation contained 50 μm PIP2, 50 mm Na-Hepes, pH 7.2, 3 mm EGTA, 0.2 mm EDTA, 0.83 mm MgCl2, 20 mm NaCl, 30 mm KCl, 1 mmdithiothreitol, 0.1% ultrapure albumin (bovine), 0.16% sodium cholate, and 1.5 mm CaCl2 in a total volume of 60 μl. Reactions were initiated by raising the temperature to 30 °C for 0–15 min and quenched by addition of 200 μl of 10% trichloroacetic acid and 100 μl of bovine serum albumin (10 mg/ml) and placing the reactions on ice. [3H]inositol 1,4,5-phosphate (IP3) (supernatant) was separated from unhydrolyzed [3H]PIP2 (pellet) by centrifugation at 2,000 × g for 10 min. Released [3H]IP3 was quantified by liquid scintillation counting. COS-1 and HEK293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, and 100 μg/ml streptomycin sulfate at 37 °C in a humidified atmosphere containing 5% CO2. COS-1 and HEK293 cells grown to 75–95% confluence were transfected with either 20 μg (100-mm plate) or 3 μg (12-well plate) of total DNA using FugeneTM according to the manufacturer's instructions. 100-mm plates of COS-1 or HEK293 cells were co-transfected with pcDNA3-GRK2 and pcDNA3-HA-Gαq, pcDNA3-HA-Gαq(R183C), pcDNA3-HA-Gαs, pcDNA3-HA-Gαs(R201C), pcDNA3-EE-Gαi2, or pcDNA3-EE-Gαi2(R179C) and in some experiments pcDNA3-M3AChR. At 24 h after transfection, cells were rinsed with ice-cold phosphate-buffered saline and harvested by addition of 1 ml of buffer-C (20 mm Tris-HCl, pH 7.4, 1 mm EDTA, 1 mm dithiothreitol, 100 mm NaCl, 5 mm MgCl2, 0.7% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 20 μg/ml benzamidine, and 10 μg/ml each of leupeptin, pepstatin A, and aprotinin). For cells co-transfected with pcDNA3-M3AChR, cells were incubated for 0–60 min at 37 °C in the absence or presence of 100 μm carbachol prior to harvesting. Cells were scraped and homogenized with two 15-s bursts with a Brinkman Polytron (2500 rpm) and lysates were centrifuged at 4 °C for 10 min at maximum speed in a microcentrifuge and the supernatant removed. For immunoprecipitation, 100 μl of supernatant was incubated with 4 μg of either GRK2- or pol II-specific polyclonal antibodies for 30 min at 4 °C followed by addition of 50 μl of 50% protein A-agarose pre-equilibrated in buffer C and an additional 60-min incubation at 4 °C. Samples were then centrifuged for 10 s in a microcentrifuge and the pellets were washed three times with 1 ml of buffer C each for 30 min at 4 °C. Bound proteins were eluted by addition of 50 μl of SDS sample buffer followed by boiling for 10 min. Initial supernatants, as well as elutions from immunoprecipitation reactions, were subjected to 10% SDS-PAGE and immunoblotting using GRK2- and HA-specific monoclonal antibodies. Measurement of inositol phosphate production in cells was essentially as described previously (32Parent J.-L. Labrecque P. Orsini M.J. Benovic J.L. J. Biol. Chem. 1999; 274: 8941-8948Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Briefly, HEK293 cells were seeded at a density of 80,000 cells per well in 12-well plates and transfected with the thromboxane A2-α receptor (TXA2Rα), M3AChR, or vector (pcDNA3) and a variety of GRK or RGS constructs (pcDNA3-GRK2, pcDNA3-GRK2(K220R), pcDNA3-HA-GRK2(45–178), pcDNA3-HA-GRK2(468–689), pcDNA3-GRK3, pcDNA3-GRK3(K220R), pcDNA3-GRK5, pcDNA3-GRK6, pB6-RGS4, and pB6-GAIP). The following day, cells were labeled for 18–24 h withmyo-[3H]inositol at 4 μCi/ml in Dulbecco's modified Eagle's medium (high glucose without inositol). After labeling, cells were washed once in phosphate-buffered saline and incubated in pre-warmed Dulbecco's modified Eagle's medium (high glucose, without inositol) containing 0.5% bovine serum albumin, 20 mm Hepes, pH 7.5, and 20 mm LiCl for 10 min. Cells were then stimulated for 10 min with 100 nm U46619 (TXA2Rα) or 100 μm carbachol (M3AChR). The reactions were terminated by removing the stimulation media and adding 0.8 ml of 0.4 m perchloric acid to the cells. Samples were harvested in Eppendorf tubes and 0.4 ml of 0.72 n KOH, 0.6 m KHCO3 was added. Tubes were vortexed and centrifuged for 5 min at maximum speed in a microcentrifuge. Total inositol phosphates were separated on Dowex AG1-X8 columns, and quantitated by liquid scintillation counting. Alternatively, HEK293 cells were co-transfected with pcDNA3-HA-Gαq or pcDNA3-HA-Gαq(R183C) (instead of the GPCR constructs) and various GRK or RGS constructs as stated above. For these experiments inositol phosphate measurement was as described above with the exception that these cells were not stimulated with agonist. Whereas the central catalytic and C-terminal domains of GRKs have been well characterized, the overall structure and function of the ∼190 residue N-terminal domain has remained relatively uncharacterized (1Carman C.V. Benovic J.L. Curr. Opin. Neurobiol. 1998; 8: 335-344Crossref PubMed Scopus (244) Google Scholar, 2Stoffel III, R.H. Pitcher J.A. Lefkowitz R.J. J. Membr. Biol. 1997; 157: 1-8Crossref PubMed Scopus (44) Google Scholar). Interestingly, Siderovski et al.(17Siderovski D.P. Hessel A. Chung S. Mak T.W. Tyers M. Curr. Biol. 1996; 6: 211-212Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar) identified sequence homology between RGS domains and an ∼120 residue region in the N terminus of GRKs through a BLAST search of the NCBI protein data base (17Siderovski D.P. Hessel A. Chung S. Mak T.W. Tyers M. Curr. Biol. 1996; 6: 211-212Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Indeed, both GRK2 and GRK3 (residues 51–173) are ∼20% identical and ∼30% similar to various RGS domains (Fig. 1). This compares with an average of 44% identity (∼54% similarity) shared among various RGS proteins. Importantly, the majority of the conserved hydrophobic residues shown to make up the hydrophobic core of the RGS domain (12Tesmer J.J.G. Berman D.M. Gilman A.G. Sprang S.R. Cell. 1997; 89: 251-261Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar,15Srinivasa S.P. Watson N. Overton M.C. Blumer K.J. J. Biol. Chem. 1998; 273: 1529-1533Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) are shared throughout the GRK family (Fig. 1 and data not shown). This suggests that the N terminus of GRKs may have a three-dimensional topology that is similar to RGS domains. Residues thought to be critical for Gα binding and GAP activity in most RGS proteins are only partially conserved by GRK2 and GRK3 (Fig. 1) (12Tesmer J.J.G. Berman D.M. Gilman A.G. Sprang S.R. Cell. 1997; 89: 251-261Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar, 15Srinivasa S.P. Watson N. Overton M.C. Blumer K.J. J. Biol. Chem. 1998; 273: 1529-1533Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). However, p115 Rho-GEF, a new member of the RGS family that serves as a GAP for Gα12/13, also exhibits only partial conservation of these residues compared with other RGS proteins (Fig. 1) (20Kozasa 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 (743) Google Scholar, 21Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (679) Google Scholar). Finally, it is noteworthy that residues previously defined as conserved caveolin binding determinants in GRKs (residues 60–73 in GRK2 and GRK3 (3Carman C.V. Lisanti M.P. Benovic J.L. J. Biol. Chem. 1999; 274: 8858-8864Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar)) fall within α-helix 3 of the putative RGS domain (Fig. 1). Interestingly, several RGS
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