Regulation of G Protein-coupled Receptor Kinases by Calmodulin and Localization of the Calmodulin Binding Domain
1997; Elsevier BV; Volume: 272; Issue: 29 Linguagem: Inglês
10.1074/jbc.272.29.18273
ISSN1083-351X
AutoresAlexey Pronin, Daulet K. Satpaev, Vladlen Z. Slepak, Jeffrey Benovic,
Tópico(s)Ion channel regulation and function
ResumoG protein-coupled receptor kinases (GRKs) specifically phosphorylate and regulate the activated form of multiple G protein-coupled receptors. Recent studies have revealed that GRKs are also subject to regulation. In this regard, GRK2 and GRK5 can be phosphorylated and either activated or inhibited, respectively, by protein kinase C. Here we demonstrate that calmodulin, another mediator of calcium signaling, is a potent inhibitor of GRK activity with a selectivity for GRK5 (IC50 ∼50 nm) > GRK6 ≫ GRK2 (IC50 ∼2 μm) ≫ GRK1. Calmodulin inhibition of GRK5 is mediated via a reduced ability of the kinase to bind to both receptor and phospholipid. Interestingly, calmodulin also activates autophosphorylation of GRK5 at sites distinct from the two major autophosphorylation sites on GRK5. Moreover, calmodulin-stimulated autophosphorylation directly inhibits GRK5 interaction with receptor even in the absence of calmodulin. Using glutathione S-transferase-GRK5 fusion proteins either to inhibit calmodulin-stimulated autophosphorylation or to bind directly to calmodulin, we determined that an amino-terminal domain of GRK5 (amino acids 20–39) is sufficient for calmodulin binding. This domain is abundant in basic and hydrophobic residues, characteristics typical of calmodulin binding sites, and is highly conserved in GRK4, GRK5, and GRK6. These studies suggest that calmodulin may serve a general role in mediating calcium-dependent regulation of GRK activity. G protein-coupled receptor kinases (GRKs) specifically phosphorylate and regulate the activated form of multiple G protein-coupled receptors. Recent studies have revealed that GRKs are also subject to regulation. In this regard, GRK2 and GRK5 can be phosphorylated and either activated or inhibited, respectively, by protein kinase C. Here we demonstrate that calmodulin, another mediator of calcium signaling, is a potent inhibitor of GRK activity with a selectivity for GRK5 (IC50 ∼50 nm) > GRK6 ≫ GRK2 (IC50 ∼2 μm) ≫ GRK1. Calmodulin inhibition of GRK5 is mediated via a reduced ability of the kinase to bind to both receptor and phospholipid. Interestingly, calmodulin also activates autophosphorylation of GRK5 at sites distinct from the two major autophosphorylation sites on GRK5. Moreover, calmodulin-stimulated autophosphorylation directly inhibits GRK5 interaction with receptor even in the absence of calmodulin. Using glutathione S-transferase-GRK5 fusion proteins either to inhibit calmodulin-stimulated autophosphorylation or to bind directly to calmodulin, we determined that an amino-terminal domain of GRK5 (amino acids 20–39) is sufficient for calmodulin binding. This domain is abundant in basic and hydrophobic residues, characteristics typical of calmodulin binding sites, and is highly conserved in GRK4, GRK5, and GRK6. These studies suggest that calmodulin may serve a general role in mediating calcium-dependent regulation of GRK activity. G protein-coupled receptor kinases (GRKs) 1The abbreviations used are: GRK(s), G protein-coupled receptor kinase(s); PKC, protein kinase C; ROS, rod outer segment(s); GST, glutathione S-transferase; SPR, surface plasmon resonance; CaM, calmodulin; MARCKS, myristoylated alanine-rich protein kinase C substrate. form a family of serine/threonine protein kinases with the unique ability to recognize specifically the agonist-activated state of G protein-coupled receptors (1Sterne-Marr R. Benovic J.L. Vitam. Horm. 1995; 51: 193-234Crossref PubMed Scopus (113) Google Scholar, 2Premont R.T. Inglese J. Lefkowitz R.J. FASEB J. 1995; 9: 175-182Crossref PubMed Scopus (474) Google Scholar). GRK-mediated phosphorylation promotes the binding of an arrestin protein, thereby uncoupling the receptor from G protein and terminating receptor signaling. Six members of the GRK family have been identified, and based on their sequence homology they have been divided into three subfamilies (2Premont R.T. Inglese J. Lefkowitz R.J. FASEB J. 1995; 9: 175-182Crossref PubMed Scopus (474) Google Scholar). GRK1 (rhodopsin kinase) forms one group; GRK2 (β-adrenergic receptor kinase) and GRK3 a second; and GRK4, GRK5, and GRK6 combine into a third subfamily. All GRKs share a similar structural organization with a poorly conserved amino-terminal domain of ∼185 residues, a conserved protein kinase catalytic domain of ∼270 residues, and a variable length carboxyl-terminal domain of 105–230 residues (3Inglese J. Freedman N.J. Koch W.J. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 23735-23738Abstract Full Text PDF PubMed Google Scholar). However, although all GRKs have a similar overall structure and function, various subfamily members also have certain unique features. For example, various GRKs utilize different mechanisms to promote membrane association, an event critical for receptor interaction. GRK1 is farnesylated (4Inglese J. Glickman J.F. Lorenz W. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 1422-1425Abstract Full Text PDF PubMed Google Scholar), GRK2 and 3 interact with phospholipids and G protein βγ subunits via pleckstrin homology domains (5Haga K. Haga T. FEBS Lett. 1990; 268: 43-47Crossref PubMed Scopus (57) Google Scholar, 6Pitcher J.A. Inglese J. Higgins J.B. Arriza J.L. Casey P.J. Kim C. Benovic J.L. Kwatra M.M. Caron M.G. Lefkowitz R.J. Science. 1992; 257: 1264-1267Crossref PubMed Scopus (573) Google Scholar, 7Kim C.M. Dion S.B. Onorato J.J. Benovic J.L. Receptor. 1993; 3: 39-55PubMed Google Scholar, 8DebBurman S.K. Ptasienski J. Boetticher E. Lomasney J.W. Benovic J.L. Hosey M.M. J. Biol. Chem. 1995; 270: 5742-5747Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), GRK4 (9Premont R.T. Macrae A.D. Stoffel R.H. Chung N. Pitcher J.A. Ambrose C. Inglese J. MacDonald M.E. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 6403-6410Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar) and GRK6 (10Stoffel R.H. Randall R.R. Premont R.T. Lefkowitz R.J. Inglese J. J. Biol. Chem. 1994; 269: 27791-27794Abstract Full Text PDF PubMed Google Scholar) are palmitoylated, and GRK5 binds to phospholipids via polybasic regions in the amino- and carboxyl-terminal domains (11Kunapuli P. Gurevich V.V. Benovic J.L. J. Biol. Chem. 1994; 269: 10209-10212Abstract Full Text PDF PubMed Google Scholar, 12Pitcher 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 (140) Google Scholar). Another characteristic that appears specific for the GRK subtype involves regulation of kinase activity. For example, in the visual system, GRK1 has been shown to be inhibited by the Ca2+-binding protein recoverin (13Chen C.-K. Inglese J. Lefkowitz R.J. Hurley J.B. J. Biol. Chem. 1995; 270: 18060-18066Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). Calcium binding to recoverin promotes its association with GRK1, inactivating the kinase and thereby reducing its ability to phosphorylate rhodopsin. Since calcium levels are decreased upon light activation of rod cells (14Yarfitz S. Hurley J.B. J. Biol. Chem. 1994; 269: 14329-14332Abstract Full Text PDF PubMed Google Scholar), recoverin binding to rhodopsin kinase might provide a mechanism for adaptation of the system to ambient light. Because recoverin has no effect on GRK2, regulation by recoverin may be specific for GRK1. Recent studies also have demonstrated that GRK2 and GRK5 are subject to regulatory phosphorylation via protein kinase C (PKC), a Ca2+/phospholipid-dependent kinase. GRK2 phosphorylation by PKC leads to an ∼2–3-fold activation of the kinase, possibly via an increased ability of GRK2 to bind to membranes (15Chuang T.T. LeVine III, H. De Blasi A. J. Biol. Chem. 1995; 270: 18660-18665Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 16Winstel R. Freund S. Krasel C. Hoppe E. Lohse M.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2105-2109Crossref PubMed Scopus (145) Google Scholar). In contrast, GRK5 is inhibited severalfold when phosphorylated by PKC due to both a decreased activity and affinity for receptor (17Pronin A.N. Benovic J.L. J. Biol. Chem. 1997; 272: 3806-3812Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). These examples illustrate how GRKs can be regulated via changes in intracellular Ca2+ concentrations. Another universal mediator of calcium signaling is calmodulin. Calmodulin is a ubiquitously expressed Ca2+-binding protein that functions as a Ca2+-dependent regulator of multiple pathways including cyclic nucleotide metabolism, ion transport, protein phosphorylation-dephosphorylation cascades, cytoskeletal function, and cell proliferation (18Gnegy M.E. Annu. Rev. Pharmacol. Toxicol. 1993; 32: 45-70Crossref Google Scholar, 19Crivici A. Ikura M. Annu. Rev. Biophys. Biomol. Struct. 1995; 24: 85-116Crossref PubMed Scopus (697) Google Scholar). In the present study we evaluated whether calmodulin can regulate GRK activity. We show that calmodulin inhibits GRK activity with a specificity of GRK5 (IC50 ∼50 nm) > GRK6 ≫ GRK2 (IC50 ∼2 μm) ≫ GRK1. The calmodulin binding domain of GRK5 was localized within the amino-terminal domain (residues 20–39). These findings suggest that calmodulin may play an important role in regulating GRK function in a subtype-specific manner. Restriction endonucleases, Vent DNA polymerase, and other molecular biology reagents were purchased from New England Biolabs or Boehringer Mannheim. SP Sepharose was obtained from Pharmacia Biotech Inc.. Calmodulin (bovine brain, >98% pure), calmodulin-agarose, and phosphatidylcholine (soybean type II-S) were from Sigma. Phosphatidylserine (bovine brain, 99% pure) was from Avanti Polar Lipids, Inc. Rat PKC-α and bovine GRK1, overexpressed and purified from Sf9 cells, were generous gifts from Dr. C. Stubbs and Drs. R. J. Lefkowitz and J. A. Pitcher, respectively. All other materials were from sources previously described (17Pronin A.N. Benovic J.L. J. Biol. Chem. 1997; 272: 3806-3812Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Expression plasmids for GRKs were constructed by cloning the coding sequences of bovine GRK2 (20Benovic J.L. De Blasi A. Stone W.C. Caron M.G. Lefkowitz R.J. Science. 1989; 246: 235-240Crossref PubMed Scopus (331) Google Scholar) and human GRK5 and GRK6 in the vector pBC12BI (21Cullen B. Methods Enzymol. 1987; 71: 684-704Crossref Scopus (662) Google Scholar). COS-1 cells were grown to ∼80–90% confluence in 60-mm dishes at 37 °C in a humidified atmosphere containing 5% CO2, 95% air in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were transfected with 4 μg of DNA/dish using LipofectAMINE following the manufacturer's instructions (Life Technologies, Inc.). Forty-eight h after transfection, cells were harvested and lysed by scraping into 1 ml of ice-cold 20 mmTris-HCl, pH 8.0, 2 mm EDTA, 200 mm NaCl, 1% Triton X-100 with protease inhibitors (5 μm aprotinin, 5 mm benzamidine, 20 μm leupeptin, 2 μm pepstatin A, 1 mm phenylmethylsulfonyl fluoride) and supernatants were prepared by centrifugation for 7 min at 100,000 × g (4 °C). GRKs were then partially purified by chromatography on SP Sepharose as described (17Pronin A.N. Benovic J.L. J. Biol. Chem. 1997; 272: 3806-3812Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Four-μl aliquots of the partially purified GRK were then assayed by incubating with rod outer segment (ROS) membranes (100 pmol of rhodopsin) in 20 μl of 20 mm Tris-HCl, pH 8.0, 4 mm MgCl2, 0.1 mm CaCl2, 0.1 mm [γ-32P]ATP (1,000 cpm/pmol) in the presence of the indicated concentration of calmodulin for 6 min at 30 °C in room light. The reactions were stopped with 200 μl of ice-cold buffer (20 mm Tris-HCl, pH 8.0, 10 mmEDTA, 100 mm NaCl) and centrifuged for 10 min at 100,000 rpm (4 °C). Pellets containing phosphorylated rhodopsin were dissolved in SDS loading buffer, and the samples were then electrophoresed on a 10% SDS-polyacrylamide gel (22Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207509) Google Scholar). Gels were stained with Coomassie Blue, dried, and autoradiographed, and the32P-labeled proteins were excised and counted to determine the pmol of phosphate transferred. Urea-treated ROS membranes containing rhodopsin were prepared from bovine retinas as described previously (23Shichi H. Somers R.L. J. Biol. Chem. 1978; 253: 7040-7046Abstract Full Text PDF PubMed Google Scholar). Bovine GRK2 and human GRK5 were overexpressed and purified from Sf9 cells as described (7Kim C.M. Dion S.B. Onorato J.J. Benovic J.L. Receptor. 1993; 3: 39-55PubMed Google Scholar, 24Kunapuli P. Onorato J.J. Hosey M.M. Benovic J.L. J. Biol. Chem. 1994; 269: 1099-1105Abstract Full Text PDF PubMed Google Scholar). GRK-mediated phosphorylation was assayed by incubating 0.8 pmol of GRK with either ROS membranes (80 pmol of rhodopsin), casein (10 μg), or phosvitin (10 μg) in 20 μl of 20 mm Tris-HCl, pH 8.0, 4 mmMgCl2, 0.1 mm CaCl2 (or 2 mm EGTA), 0.1 mm [γ-32P]ATP (1,000 cpm/pmol) in the presence of the indicated concentrations of calmodulin for 6 min at 30 °C in room light. The reactions were stopped with 5 μl of SDS sample buffer, and the samples were electrophoresed on a 10% SDS-polyacrylamide gel. Gels were stained with Coomassie Blue, dried, and autoradiographed, and the32P-labeled proteins were excised and counted. The autophosphorylation-defective mutant GRK5-DD (Ser484 and Thr485 mutated to Asp) was overexpressed and purified from Sf9 cells as described (17Pronin A.N. Benovic J.L. J. Biol. Chem. 1997; 272: 3806-3812Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Autophosphorylation reactions contained 4 pmol (0.27 μg) of either wild type GRK5 or GRK5-DD in 20 μl of 20 mm Tris-HCl, pH 8.0, 4 mm MgCl2, 0.1 mmCaCl2, 0.1 mm [γ-32P]ATP (5,000 cpm/pmol), 0.1 mg/ml ovalbumin, and either 0.85 mg/ml phospholipid vesicles or the indicated concentration of calmodulin. Reactions were incubated at 30 °C for 10 min and stopped with 5 μl of SDS sample buffer. To assess the rate of calmodulin-stimulated autophosphorylation, 100 pmol of rhodopsin was phosphorylated with 2 pmol (0.1 μm) of GRK5 in the absence or presence of 0.5 μm calmodulin. Reactions were incubated at 30 °C and at the indicated times were stopped with SDS sample buffer. Samples were electrophoresed, and the 32P-labeled proteins were excised and counted as described above. To determine the kinetics for ATP, GRK5-DD (16 pmol) was autophosphorylated in 20 mm Tris-HCl, pH 8.0, 4 mm MgCl2, 0.1 mm CaCl2, 0.1 mg/ml ovalbumin, and 2–100 μm[γ-32P]ATP (10,000 cpm/pmol) in the absence or presence of 0.8 μm calmodulin. K m andV max values were derived from double-reciprocal plots of the data. Sixty pmol of GRK5 was autophosphorylated in a 40-μl reaction at 30 °C for 15 min in the presence or absence of either 3 μmcalmodulin or 0.07 μm PKC, 1 μm phorbol 12-myristate 13-acetate, and 0.85 mg/ml phospholipid vesicles as described above and then purified by batchwise chromatography on SP Sepharose. Briefly, phosphorylation reactions were stopped on ice, mixed with an equal volume of 20 mm Tris-HCl, pH 8.0, 2 mm EDTA, 2 mm EGTA, 100 mm NaCl, 0.4% Triton X-100, and then incubated for 10 min with 50 μl of a 50% suspension of SP Sepharose in buffer A (20 mmTris-HCl, pH 8.0, 2 mm EDTA, 1 mm EGTA, 50 mm NaCl, 0.02% Triton X-100). The resin was pelleted, washed two or three times with 1 ml of buffer A, and the bound kinase was eluted with two 75-μl aliquots of 20 mm Tris-HCl, 1 mm EDTA, 600 mm NaCl, 0.02% Triton X-100. The supernatants were combined, diluted with 150 μl of 20 mmTris-HCl, pH 8.0, 4 mm MgCl2, 1 mmEDTA, and then used for further analysis. Aliquots of the phosphorylated kinase before and after SP Sepharose purification were electrophoresed on an SDS-polyacrylamide gel to enable assessment of autophosphorylation, phosphorylation by PKC, and recovery from SP Sepharose. Typically, 70–80% of the GRK5 was recovered by this procedure, whereas PKC and calmodulin did not bind to SP Sepharose. Aliquots (∼20 ng) of the SP Sepharose-purified GRK5 were also electrophoresed and subjected to Western blot analysis using antibodies raised against either amino acids 556–571 or 489–590 of human GRK5 as described (17Pronin A.N. Benovic J.L. J. Biol. Chem. 1997; 272: 3806-3812Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Four-μl aliquots (∼0.6 pmol) of the SP Sepharose-purified GRK5 were assayed by incubating with either ROS membranes (60 pmol of rhodopsin) or phosvitin (10 μg) in 20 μl of 20 mm Tris-HCl, pH 8.0, 4 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 0.1 mm [γ-32P]ATP (1,000 cpm/pmol) for 6 min at 30 °C in room light. When the effect of calmodulin was tested the reactions also included the indicated concentration of calmodulin and 0.2 mm CaCl2(with no EGTA) and were incubated for 2 min at 30 °C. Reactions were stopped with 5 μl of SDS sample buffer, the samples were electrophoresed on a 10% SDS-polyacrylamide gel, gels were stained with Coomassie Blue, dried and autoradiographed, and the32P-labeled proteins were excised and counted. To assess the kinetics of receptor phosphorylation, 25–660 pmol of rhodopsin was phosphorylated with GRK5 autophosphorylated in the presence or absence of calmodulin in 20 mm Tris-HCl, pH 8.0, 4 mmMgCl2, 1 mm EDTA, 0.1 mm[γ-32P]ATP (6,000 cpm/pmol). K m andV max values were derived from double-reciprocal plots of the data. The ability of GRK5 to associate with either receptor or phospholipid was analyzed by incubating 8-μl aliquots (∼1.2 pmol) of SP Sepharose-purified 32P-labeled autophosphorylated GRK5 in the presence or absence of the indicated concentration of phospholipid vesicles or ROS membranes (250 pmol of rhodopsin) in 60 μl of 20 mm Tris-HCl, pH 8.0, 2 mm MgCl2, 0.1 mm CaCl2, 80 mm NaCl, 0.1 mg/ml ovalbumin, and the indicated concentration of calmodulin at 30 °C for 5 min in room light. The samples were centrifuged at 100,000 rpm for 6 min, the pellets were resuspended in 60 μl of reaction buffer, and equal aliquots of the supernatant and pellet fractions were electrophoresed on a 10% SDS-polyacrylamide gel. The gels were dried, autoradiographed, and the 32P-labeled proteins were excised and counted. Pelleted GRK5 was expressed as a percentage of the total after subtracting the amount of GRK5 pelleted in the absence of phospholipids or ROS (∼10–15%). Phospholipid vesicles were prepared by sonicating 76 mg of phosphatidylcholine and 9 mg of phosphatidylserine in 5 ml of 10 mm Tris-HCl, pH 8.0, 100 mm NaCl, 0.1 mm EDTA on ice four times for 20 s. DNA sequences coding for various regions of GRK5 were generated using the polymerase chain reaction and then used to replace a BamHI/SalI fragment in the vector pGEX-4T-2 (Pharmacia). The polymerase chain reaction-derived portions of the constructs were sequenced in their entirety using the dideoxy chain termination method. The GST-GRK5 fusion proteins were expressed in Escherichia coli and purified over glutathione-agarose using standard procedures (25Smith D.B. Johnson K.S. Gene ( Amst. ). 1988; 67: 31-40Crossref PubMed Scopus (5047) Google Scholar). The purity of the proteins was >95% as determined by Coomassie Blue staining. Protein concentrations were determined by dye binding assay (Bio-Rad) using bovine serum albumin as a standard. To assess the ability of GST fusion proteins to block calmodulin-mediated activation of GRK5, 4 pmol of GRK5-DD was autophosphorylated in the presence of a 2 μm concentration of the indicated fusion protein and in the absence or presence of 0.1 μmcalmodulin. Reactions were processed by gel electrophoresis as described above, and the level of autophosphorylation was determined by excising and counting the 32P-labeled bands. None of the GST fusion proteins significantly affected the basal autophosphorylation of GRK5-DD. The binding of GRK5 and GST-GRK5 fusion proteins to calmodulin-agarose was performed in buffer B (20 mm Tris-HCl, pH 8.0, 100 mm NaCl, 1% Triton X-100, 0.01% SDS, 0.1 mg/ml ovalbumin, and either 0.1 mm CaCl2 or 2 mmEGTA). Ten pmol of GRK5 or fusion protein was incubated with 20 μl of calmodulin-agarose beads for 20 min in a total volume of 0.2 ml at 4 °C. The resin was pelleted, washed with 0.5 ml of buffer B, and bound proteins were eluted with two 100-μl aliquots of 20 mm Tris-HCl, 1 mm EDTA, 100 mmNaCl, 1% Triton X-100, 0.01% SDS, 0.1 mg/ml ovalbumin, 10 mm EGTA. The supernatants were combined, and the amount of bound and eluted protein was determined by immunoblotting using a rabbit polyclonal antibody generated against a GST fusion protein containing amino acids 98–136 of human GRK5 (17Pronin A.N. Benovic J.L. J. Biol. Chem. 1997; 272: 3806-3812Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). This antiserum recognizes both GRK5 and all GST proteins. Approximately 50–60% of GRK5 was bound to calmodulin-agarose in the presence of Ca2+ and could be eluted with EGTA, whereas no binding was detected in the presence of EGTA. Direct binding of GRK to calmodulin was also assessed by perfusing solutions of either GRK5 or GST-GRK5 fusions over the surface of a BIAcore sensor chip containing calmodulin. To immobilize calmodulin, it was first biotinylated either at lysine residues using NHS-LC-Biotin (Pierce) or at a unique cysteine residue using Iodoacetyl-LC-biotin (Pierce). The calmodulin was desalted on Sephadex G-15 to remove free biotinylation reagent and then trapped on the surface of a sensor chip containing covalently attached streptavidin (Sensor chip SA5, BIACORE, Inc). This yielded ∼2,000 relative units of calmodulin on the streptavidin chip which retained its activity for several days. For analysis of calmodulin/GRK5 interaction, solutions of the kinase were injected across chip surfaces containing either calmodulin, streptavidin only, or another calcium-binding protein, recoverin. The running buffer contained 20 mm Hepes, pH 7.4, 200 mm NaCl, 0.02% Surfactant P20 (BIACORE, Inc.), 0.01 mg/ml bovine serum albumin, 0.1 mm β-mercaptoethanol, 1 mm CaCl2. GRK5 and the GST-GRK5 fusions were diluted in this buffer to a final concentration of 200 nmprior to injection. The volume of injected sample was 40 μl, and the flow rate was 10 μl/min. Experiments were performed on a BIAcore 2000 instrument with SPR data points collected at 1 Hz and the data analyzed using BIAEvaluation 2.1 software (BIACORE, Inc.). In an effort to elucidate further the potential role of calcium in regulating GRK function, we tested whether calmodulin could modulate the activity of various GRKs. Our initial studies compared the effect of calmodulin on COS-1 cell overexpressed preparations of GRK2, GRK5, and GRK6 to phosphorylate light-activated rhodopsin. Protein extracts from control COS-1 cells displayed very low rhodopsin phosphorylation activity, whereas cells transfected with GRK2, GRK5, or GRK6 expression constructs had a much higher level of phosphorylation (Fig.1 A). In the presence of calmodulin the phosphorylation of rhodopsin by GRK5 and GRK6 was significantly inhibited with IC50 values of ∼0.25 μm for GRK5 and ∼0.7 μm for GRK6 (Fig. 1 B). In contrast, GRK2 was inhibited only at the highest concentration of calmodulin tested (IC50 > 3 μm). Thus, although all three GRKs tested were inhibited by calmodulin, GRK2 was much less sensitive than GRK5 and GRK6. Because the COS-1 cell extracts contain many other proteins that could potentially influence the assay, we also studied the effect of calmodulin on purified GRKs. Calmodulin effectively inhibited the ability of GRK5 to phosphorylate rhodopsin with an IC50∼50 nm (Fig. 2). Calmodulin also inhibited the activity of GRK2, although much less effectively (IC50∼2 μm) than GRK5, whereas the activity of GRK1 was only modestly inhibited even at 10 μm calmodulin. The effect of calmodulin on GRK5 was also completely dependent on the presence of Ca2+ (data not shown). The higher IC50 values observed for the COS-expressed GRKs versus the purified GRKs might be because of the presence of additional calmodulin-binding proteins in the cruder preparations which could bind calmodulin and reduce its effective concentration. The high sensitivity of GRK5 to inhibition by both Ca2+/calmodulin and PKC (17Pronin A.N. Benovic J.L. J. Biol. Chem. 1997; 272: 3806-3812Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) strongly suggests that GRK5 will not be involved in regulating receptors coupled to Gq/11 and phospholipase C since these receptors promote increased free calcium levels when activated, presumably leading to inhibition of GRK5. Thus, even if GRK5 can phosphorylate such receptorsin vitro, it is unlikely that this would occur in intact cells. This may explain why coexpression of α1b-adrenergic receptors with GRK5 results in enhanced basal phosphorylation but no significant agonist-induced phosphorylation of the receptor (26Diviani D. Lattion A.-L. Larbi N. Kunapuli P. Pronin A. Benovic J.L. Cotecchia S. J. Biol. Chem. 1996; 271: 5049-5058Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Similarly, recent studies have demonstrated that although GRK5 can phosphorylate myocardial type 1A angiotensin II receptors in vitro (27Opperman M. Freedman N.J. Alexander R.W. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 13266-13272Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar), desensitization of this receptor in transgenic mice overexpressing GRK5 was not affected (28Rockman H.A. Choi D.-J. Rahman N.U. Akhter S. Lefkowitz R.J. Koch W.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9954-9959Crossref PubMed Scopus (183) Google Scholar). In contrast, the lower affinity of calmodulin for GRK2 suggests that it would not be regulated by calmodulin in most cells, although calmodulin levels in brain are high (1–10 μm) (18Gnegy M.E. Annu. Rev. Pharmacol. Toxicol. 1993; 32: 45-70Crossref Google Scholar). Since GRK5 was more sensitive to inhibition by calmodulin than the other GRKs, we focused the remainder of the study on the GRK5/calmodulin interaction. To assess further the effect of calmodulin on the activity of GRK5, we utilized soluble substrates such as casein and phosvitin. Although phosphorylation of casein by GRK5 was not altered by calmodulin (data not shown), GRK5 phosphorylation of phosvitin was inhibited with an IC50 ∼0.6 μm (Fig. 2 C). The inhibition of phosvitin phosphorylation suggests that calmodulin interacts with regions of GRK5 which are likely involved in substrate binding. However, the ∼10-fold reduced sensitivity of inhibition of phosvitin phosphorylation by calmodulin relative to rhodopsin phosphorylation implies that calmodulin may either more effectively inhibit GRK5 binding to receptor substrates, and/or it may also inhibit GRK5 binding to phospholipid. Unlike the other GRKs that utilize either covalent lipid modifications (GRK1, 4, and 6) or interaction with G protein βγ subunits (GRK2 and 3) to enhance binding to phospholipid membranes, GRK5 appears to interact directly with phospholipids via regions rich in basic amino acids. GRK5 displays significant association with either phospholipid vesicles or with rhodopsin-containing ROS membranes (11Kunapuli P. Gurevich V.V. Benovic J.L. J. Biol. Chem. 1994; 269: 10209-10212Abstract Full Text PDF PubMed Google Scholar, 17Pronin A.N. Benovic J.L. J. Biol. Chem. 1997; 272: 3806-3812Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 29Premont R.T. Koch W.J. Inglese J. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 6832-6841Abstract Full Text PDF PubMed Google Scholar). When tested in a direct binding assay, calmodulin was found to inhibit GRK5 binding to ROS membranes significantly with an IC50 ∼0.3–0.4 μm (Fig. 3, A andB). However, this IC50 was some 6–8-fold higher than the IC50 for inhibition of rhodopsin phosphorylation. Indeed, at the highest calmodulin concentration tested, ∼20% of the kinase remained bound to the ROS membranes even though rhodopsin phosphorylation was reduced >99% (compare Figs. 2 B and3 B). These results suggest that calmodulin can directly inhibit GRK5 interaction with receptor. The binding of GRK5 to phospholipid vesicles was also inhibited by calmodulin (Fig. 3,C and D). However, although this inhibition was substantial at relatively low lipid concentrations (0.017 mg/ml), it could be largely overcome at higher phospholipid (0.85 mg/ml). These results imply a competitive type of inhibition and taken together with the rhodopsin studies suggest that calmodulin can directly compete for both the lipid and receptor binding sites of GRK5. GRK5 appears to be activated via a rapid phospholipid-stimulated autophosphorylation at residues Ser484 and Thr485 (11Kunapuli P. Gurevich V.V. Benovic J.L. J. Biol. Chem. 1994; 269: 10209-10212Abstract Full Text PDF PubMed Google Scholar, 29Premont R.T. Koch W.J. Inglese J. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 6832-6841Abstract Full Text PDF PubMed Google Scholar). To our surprise calmodulin significantly enhanced the autophosphorylation of GRK5 (Fig. 4 A). In an attempt to further characterize this finding we studied the effect of calmodulin on the autophosphorylation-defective mutant GRK5-DD, which has both Ser484 and Thr485 mutated to aspartate (17Pronin A.N. Benovic J.L. J. Biol. Chem. 1997; 272: 3806-3812Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Although autophosphorylation of GRK5-DD was not enhanced by phospholipids, calmodulin still significantly enhanced the autophosphorylation with an overall increase comparable to that seen for wild type GRK5. These data indicate that interaction with calmodulin results in increased autophosphorylation of GRK5 at sites distinct from Ser484 and Thr485. Interestingly, calmodulin also significantly activates autophosphorylation of GRK6, but it has no effect
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