Altered Activity of Palmitoylation-deficient and Isoprenylated Forms of the G Protein-coupled Receptor Kinase GRK6
1997; Elsevier BV; Volume: 272; Issue: 43 Linguagem: Inglês
10.1074/jbc.272.43.27422
ISSN1083-351X
Autores Tópico(s)Neuroscience and Neuropharmacology Research
ResumoG protein-coupled receptor kinases (GRKs) utilize diverse mechanisms to associate with the plasma membrane and mediate phosphorylation of agonist-occupied receptors. For example, two members of this family, GRK4 and GRK6, contain C-terminal cysteine residues that are palmitoylated. To address whether the activity and membrane association of GRK6 is regulated by palmitoylation, we overexpressed and characterized wild-type GRK6 and two GRK6 mutants, one with the palmitoylation sites mutated to serines (GRK6-pal−) and one containing a C-terminal CAAX motif to promote geranylgeranylation (GRK6-GG). Compared with wild-type GRK6, GRK6-pal− had a ∼5-fold higher K m and ∼2-fold lower V max for phosphorylating rhodopsin, whereas GRK6-GG exhibited a ∼2-fold lowerK m and ∼14-fold higherV max for rhodopsin. In contrast, wild-type GRK6 and GRK6-pal− displayed similar activity toward the nonreceptor substrate phosvitin, indicating that nonpalmitoylated GRK6 is catalytically active. Wild-type GRK6 and GRK6-GG, but not GRK6-pal−, also bound significantly to phosphatidylcholine vesicles (36 ± 3, 79 ± 4, and 4 ± 27, respectively) suggesting that GRK6 activity is dependent upon its ability to interact with the plasma membrane. When assayed in COS-1 cells GRK6-pal− promoted minimal agonist-dependent sequestration of the ॆ2-adrenergic receptor, while sequestration was significantly increased in cells expressing either wild-type GRK6 or GRK6-GG. These data demonstrate an important functional link between the ability of GRK6 to bind to the plasma membrane, a process that appears to be regulated by palmitoylation, and its activity toward receptor substrates. G protein-coupled receptor kinases (GRKs) utilize diverse mechanisms to associate with the plasma membrane and mediate phosphorylation of agonist-occupied receptors. For example, two members of this family, GRK4 and GRK6, contain C-terminal cysteine residues that are palmitoylated. To address whether the activity and membrane association of GRK6 is regulated by palmitoylation, we overexpressed and characterized wild-type GRK6 and two GRK6 mutants, one with the palmitoylation sites mutated to serines (GRK6-pal−) and one containing a C-terminal CAAX motif to promote geranylgeranylation (GRK6-GG). Compared with wild-type GRK6, GRK6-pal− had a ∼5-fold higher K m and ∼2-fold lower V max for phosphorylating rhodopsin, whereas GRK6-GG exhibited a ∼2-fold lowerK m and ∼14-fold higherV max for rhodopsin. In contrast, wild-type GRK6 and GRK6-pal− displayed similar activity toward the nonreceptor substrate phosvitin, indicating that nonpalmitoylated GRK6 is catalytically active. Wild-type GRK6 and GRK6-GG, but not GRK6-pal−, also bound significantly to phosphatidylcholine vesicles (36 ± 3, 79 ± 4, and 4 ± 27, respectively) suggesting that GRK6 activity is dependent upon its ability to interact with the plasma membrane. When assayed in COS-1 cells GRK6-pal− promoted minimal agonist-dependent sequestration of the ॆ2-adrenergic receptor, while sequestration was significantly increased in cells expressing either wild-type GRK6 or GRK6-GG. These data demonstrate an important functional link between the ability of GRK6 to bind to the plasma membrane, a process that appears to be regulated by palmitoylation, and its activity toward receptor substrates. G protein-coupled receptors mediate numerous intracellular signaling pathways upon binding extracellular agonists (e.g.hormones, neurotransmitters, odorants, chemoattractants, and light) (1Dohlman H.G. Thorner J. Caron M.G. Lefkowitz R.J. Annu. Rev. Biochem. 1991; 60: 653-688Crossref PubMed Scopus (1137) Google Scholar,2O'Dowd B.F. Lefkowitz R.J. Caron M.G. Annu. Rev. Neurosci. 1989; 2: 67-83Crossref Scopus (209) Google Scholar). Receptors of this type regulate a variety of effector molecules such as adenylyl cyclase, cGMP phosphodiesterase, phospholipases A2 and C, and numerous ion channels. Two of the best studied G protein-coupled receptors are the ॆ2-adrenergic receptor (ॆ2AR), 1The abbreviations used are: ॆ2AR, ॆ2-adrenergic receptor; ॆARK, ॆ-adrenergic receptor kinase; G protein, guanine nucleotide-binding protein; GRK, G protein-coupled receptor kinase; PH, pleckstrin homology; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; PC, phosphatidylcholine; PIP2, phosphatidylinositol 4,5-diphosphate. which mediates catecholamine stimulation of adenylyl cyclase, and the visual light receptor, rhodopsin, which facilitates phototransduction in retinal rod cells (3Gomez J. Benovic J.L. Friedlander M. Mueckler M. Molecular Biology of Receptors and Transporters: Receptors. Academic Press, New York1992: 1-34Google Scholar, 4Hargrave P.A. McDowell J.H. Friedlander M. Mueckler M. Molecular Biology of Receptors and Transporters: Receptors. Academic Press, New York1992: 49-98Google Scholar). In both systems a rapid diminution of responsiveness or desensitization occurs following receptor activation (1Dohlman H.G. Thorner J. Caron M.G. Lefkowitz R.J. Annu. Rev. Biochem. 1991; 60: 653-688Crossref PubMed Scopus (1137) Google Scholar, 3Gomez J. Benovic J.L. Friedlander M. Mueckler M. Molecular Biology of Receptors and Transporters: Receptors. Academic Press, New York1992: 1-34Google Scholar, 4Hargrave P.A. McDowell J.H. Friedlander M. Mueckler M. Molecular Biology of Receptors and Transporters: Receptors. Academic Press, New York1992: 49-98Google Scholar, 5Sterne-Marr R. Benovic J.L. Vitam. Horm. 1995; 51: 193-234Crossref PubMed Scopus (113) Google Scholar). Activation-dependent desensitization is mediated in part by specific G protein-coupled receptor kinases (GRKs) that have the unique ability to recognize and phosphorylate their receptor substrates only when they are in an active conformation (6Palczewski K. Benovic J.L. Trends Biochem. Sci. 1991; 16: 387-391Abstract Full Text PDF PubMed Scopus (187) Google Scholar, 7Lefkowitz R.J. Cell. 1993; 74: 409-412Abstract Full Text PDF PubMed Scopus (404) Google Scholar). The ॆ-adrenergic receptor kinase (ॆARK) and rhodopsin kinase have been implicated as the major kinases involved in the stimulus-dependent phosphorylation of the ॆ2AR and rhodopsin, respectively (8Benovic J.L. Strasser R.H. Caron M.G. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2797-2801Crossref PubMed Scopus (467) Google Scholar, 9Lohse M.J. Benovic J.L. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1990; 265: 3202-3211Abstract Full Text PDF PubMed Google Scholar, 10Kuhn H. Cook J.H. Dreyer W.J. Biochemistry. 1973; 12: 2495-2502Crossref PubMed Scopus (112) Google Scholar, 11Shichi H. Somers R.L. J. Biol. Chem. 1978; 253: 7040-7046Abstract Full Text PDF PubMed Google Scholar). Subsequent uncoupling of the receptor from the G protein is promoted by arrestin proteins that specifically bind to the phosphorylated and activated form of the receptor (3Gomez J. Benovic J.L. Friedlander M. Mueckler M. Molecular Biology of Receptors and Transporters: Receptors. Academic Press, New York1992: 1-34Google Scholar, 4Hargrave P.A. McDowell J.H. Friedlander M. Mueckler M. Molecular Biology of Receptors and Transporters: Receptors. Academic Press, New York1992: 49-98Google Scholar, 5Sterne-Marr R. Benovic J.L. Vitam. Horm. 1995; 51: 193-234Crossref PubMed Scopus (113) Google Scholar). Recent evidence has demonstrated that GRKs employ a variety of mechanisms that promote their localization to the cell membrane (12Inglese 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, 13Pitcher 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, 14DebBurman S.K. Ptasienski J. Boetticher L. 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, 15Onorato J.J. Gillis M.E. Liu Y. Benovic J.L. Ruoho A.E. J. Biol. Chem. 1995; 270: 21346-21353Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 16Boekhoff I. Inglese J. Schleicher S. Koch W. Lefkowitz R.J. Breer H. J. Biol. Chem. 1994; 269: 37-40Abstract Full Text PDF PubMed Google Scholar, 17Kunapuli P. Gurevich V.V. Benovic J.L. J. Biol. Chem. 1994; 269: 10209-10212Abstract Full Text PDF PubMed Google Scholar, 18Stoffel 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, 19Premont 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). Rhodopsin kinase, ॆARK, and ॆARK2 undergo stimulus-dependent translocation from the cytosol to the plasma membrane, although this is achieved through two somewhat different mechanisms (13Pitcher 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, 20Inglese J. Koch W.J. Caron M.G. Lefkowitz R.J. Nature. 1992; 359: 147-150Crossref PubMed Scopus (234) Google Scholar). Rhodopsin kinase contains a 舠CAAX舡 motif at its C terminus that directs the attachment of a C15 isoprenoid (farnesyl) moiety required for membrane binding and optimal kinase activity (12Inglese 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). Agonist-dependent translocation of ॆARK and ॆARK2 does not involve direct acylation of these kinases but, instead, appears to be facilitated by their binding to G protein ॆγ subunits that are themselves membrane-bound via isoprenylation (13Pitcher 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). ॆARK and ॆARK2 contain a pleckstrin homology (PH) domain within the kinase C terminus that is responsible for the binding of these proteins to phospholipids and Gॆγ subunits (13Pitcher 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, 14DebBurman S.K. Ptasienski J. Boetticher L. 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, 15Onorato J.J. Gillis M.E. Liu Y. Benovic J.L. Ruoho A.E. J. Biol. Chem. 1995; 270: 21346-21353Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 21Koch W.J. Inglese J. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 8256-8260Abstract Full Text PDF PubMed Google Scholar). Furthermore, binding of ॆARK and ॆARK2 to phospholipids and Gॆγ augments kinase activity toward receptor substrates indicating an important regulatory function of this complex in GRK-mediated receptor phosphorylation (14DebBurman S.K. Ptasienski J. Boetticher L. 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,15Onorato J.J. Gillis M.E. Liu Y. Benovic J.L. Ruoho A.E. J. Biol. Chem. 1995; 270: 21346-21353Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 22Pitcher J.A. Touhara K. Payne E.S. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 11707-11710Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). GRK5, which is not acylated, possesses a highly basic C-terminal domain that enables the kinase to bind to the plasma membrane (17Kunapuli P. Gurevich V.V. Benovic J.L. J. Biol. Chem. 1994; 269: 10209-10212Abstract Full Text PDF PubMed Google Scholar, 23Premont R. Koch W.J. Inglese J. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 6832-6841Abstract Full Text PDF PubMed Google Scholar). Interaction of GRK5 with membrane phospholipids enhances kinase autophosphorylation, which increases its activity toward receptor substrates (17Kunapuli P. Gurevich V.V. Benovic J.L. J. Biol. Chem. 1994; 269: 10209-10212Abstract Full Text PDF PubMed Google Scholar). Studies performed in our laboratory with GRK6 overexpressed in and purified from Sf9 insect cells revealed that GRK6 has significantly lower activity than ॆARK and GRK5 toward rhodopsin, the ॆ2AR, and the m2 muscarinic acetylcholine receptorin vitro (24Loudon R.P. Benovic J.L. J. Biol. Chem. 1994; 269: 22691-22697Abstract Full Text PDF PubMed Google Scholar). We postulated that the observed differences in GRK6, ॆARK, and GRK5 activity may be due either to distinct substrate specificities among these GRKs and/or a lack of a functionally relevant cofactor for GRK6. Recently, it was demonstrated that GRK4 and GRK6 are palmitoylated enabling these enzymes to associate with the plasma membrane (18Stoffel 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, 19Premont 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). However, the role that palmitoylation plays in the function of these kinases remains obscure. In an effort to better understand the role that palmitoylation might play in regulating GRK6 localization and activity, we generated a mutant kinase lacking the putative palmitoylation sites and assessed its function both in vitro and in intact cells. We show that nonpalmitoylated GRK6 exhibits significantly diminished activityin vitro toward rhodopsin compared with wild-type GRK6 and that this reduced activity is likely due to its inability to interact with phospholipid. In contrast, when the palmitoylation-deficient GRK6 is modified with a C-terminal isoprenoid moiety both the activity and phospholipid binding are enhanced compared with wild-type GRK6. Furthermore, we demonstrate that COS-1 cells transiently coexpressing the ॆ2AR and nonpalmitoylated GRK6 undergo significantly less agonist-dependent receptor sequestration compared with cells expressing either wild-type or isoprenylated GRK6. The chromatography resin SP(HP)-Sepharose was purchased from Pharmacia Biotech Inc. Frozen bovine retinas were from George A. Hormel & Co. COS-1 monkey kidney cells were from the American Type Culture Collection. Phosvitin was from Sigma, whereas [γ-32P]ATP was from NEN Life Science Products. Affinity-purified rabbit polyclonal antibody specific for GRK6 and control peptide containing amino acids 525–544 of GRK6 were purchased from Santa Cruz Biotechnology, Inc. Mutant GRK6 sequences were PCR-amplified from the GRK6 cDNA (25Benovic J.L. Gomez J. J. Biol. Chem. 1993; 268: 19521-19527Abstract Full Text PDF PubMed Google Scholar) in pBluescript using a forward primer corresponding to bases 1192–1209 (5′-AGATGATCGCAGGCCAGT-3′) and one of two mutant reverse primers corresponding to the 3′ end of the GRK6 sequence: 5′-CAATGGATCCCTAGAGGCGGGTGGGCAGCTCTTCCTCGCTGTCGCTGCTGTTTCCGCTGCTATCTTGGCGACTGA-3′ (GRK6-pal−, palmitoylation-deficient mutant with Cys561, Cys562, and Cys565 mutated to serine); or 5′CAATGGATCCCTACAGCAGCACGCAGAGGCGGGTGGGCAGCTCTTCCTCGCTGTCGCTGCTGTTTCCGCTGCTATCTTGGCGACTGA-3′(GRK-6GG, palmitoylation deficient mutant with Cys-Val-Leu-Leu added to the C terminus). The reverse primers also mutagenize the SacI restriction site found at base pair 1777 of the open reading frame to facilitate subcloning into the mammalian expression vector pBC12BI (see below). All PCR reactions were performed using the Expand PCR system (Boehringer Mannheim). PCR products were digested with SphI and BamHI, which yielded ∼360-base pair fragments and then subcloned into pBluescript-GRK6 digested with the same enzymes. The PCR derived portion was sequenced using an automated DNA sequencer. DNA was cut with SacI and BamHI restriction enzymes, and the ∼1600-base pair fragments subcloned into pBC-GRK6 digested with the same enzymes (26Loudon R.P. Perussia B. Benovic J.L. Blood. 1996; 88: 4547-4557Crossref PubMed Google Scholar). Oligonucleotides and DNA sequencing were provided by the Kimmel Cancer Institute DNA Facility. To overexpress wild-type and mutant GRK6, 12 ॖg each of pBC-GRK6, pBC-GRK6-pal−, and pBC-GRK6-GG were used to transiently transfect COS-1 cells in T75 tissue culture flasks by the lipofectAMINE method following the manufacturer's instructions (Life Technologies, Inc.). Cells were trypsinized 48 h after transfection, washed several times with ice-cold PBS, and then lysed in 300 ॖl of 20 mm HEPES, pH 7.5, 17 Triton X-100, 150 mmNaCl, 10 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, 20 ॖg/ml leupeptin, and 200 ॖg/ml benzamidine. Lysates were centrifuged at 15,000 × g for 5 min at 4 °C and supernatants recovered. Supernatants were diluted to 1.2 ml in 20 mm HEPES, pH 7.5, 10 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, 200 ॖg/ml benzamidine, and 20 ॖg/ml leupeptin (buffer A), and GRK6 purification was performed by batchwise SP-Sepharose chromatography using 150 ॖl of resin. After 1 h of incubation of the resin with the cell lysates at 4 °C on a rotator, the resin was washed six times with 1 ml of buffer A containing 150 mm NaCl to remove trace amounts of ॆARK present in the adsorbed lysate. A GRK6-enriched fraction was obtained by eluting the resin three times with 200 ॖl of buffer A containing 400 mm NaCl and pooling the eluants. Quantitation of GRK6 expression in COS-1 cells and recovery after SP-Sepharose chromatography was done by immunoblotting with a polyclonal rabbit antiserum raised against a glutathioneS-transferase fusion protein corresponding to the C-terminal 102 amino acids of GRK5 (immunoreactive with both GRK5 and GRK6). Equivalent amounts of lysates and partially purified GRK6 preparations (between 5 and 40 ॖg of total protein measured by Bio-Rad protein assay using bovine serum albumin) were electrophoresed on a 107 SDS-polyacrylamide gel (27Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar), transferred to nitrocellulose, washed in 0.057 Tween, Tris-buffered saline (TTBS), pH 7.5, and blocked for 1 h in TTBS with 57 (w/v) dried nonfat milk. Filters were immunoblotted for 1 h with the polyclonal antiserum (1:100 dilution) in 57 milk, TTBS and then washed five times, 10 min each, with TTBS and incubated with affinity-purified goat anti-rabbit IgG conjugated to horseradish peroxidase (Bio-Rad) in 57 milk, TTBS (1:4000 dilution). Filters were washed five times in TTBS and visualized by enhanced chemiluminescence (ECL; Amersham, Buckinghamshire, United Kingdom). Wild-type and mutant GRK6 were quantitated by comparing different amounts of the eluted preparations with known amounts of purified GRK6 (1–4 ng) (24Loudon R.P. Benovic J.L. J. Biol. Chem. 1994; 269: 22691-22697Abstract Full Text PDF PubMed Google Scholar). Typical yields from one flask of confluent COS-1 cells were 400–500 ng for wild-type GRK6, 1200–1500 ng for GRK6-pal−, and 150–200 ng for GRK6-GG. Urea-treated rod outer segments containing rhodopsin were prepared from bovine retinas, and rhodopsin phosphorylation was performed as described previously (11Shichi H. Somers R.L. J. Biol. Chem. 1978; 253: 7040-7046Abstract Full Text PDF PubMed Google Scholar, 24Loudon R.P. Benovic J.L. J. Biol. Chem. 1994; 269: 22691-22697Abstract Full Text PDF PubMed Google Scholar). For time course assays, 1.3 ng of each kinase preparation (determined by quantitative immunoblotting) was incubated with 200 ॖm[γ-32P]ATP (1 cpm/fmol), 4.6 mmMgCl2, 6.8 ॖm rhodopsin, 20 mmHEPES, pH 7.5, and 2 mm EDTA in a total volume of 30 ॖl for 0, 2.5, 5, 10, and 20 min at 30 °C. Reactions were terminated at the indicated times with 15 ॖl SDS sample buffer, and 30 ॖl of each sample were electrophoresed on a 107 SDS-polyacrylamide gel. Gels were dried, and autoradiography was performed for 2–3 h at −80 °C. Quantitation was performed by excising the phosphorylated rhodopsin bands and counting in a scintillation counter. Determination ofK m and V max values was performed by assaying 0.1–20 ॖm rhodopsin with 1.3 ng of the different GRK6 proteins in 30 ॖl of buffer (200 ॖm[γ-32P]ATP (1 cpm/fmol), 4.6 mmMgCl2, 20 mm HEPES, pH 7.5, and 2 mm EDTA) for 5 min at 37 °C. Reactions were terminated with 15 ॖl of SDS sample buffer and processed as described above. In all experiments, wild-type and mutant GRK6 preparations were stored at 4 °C and were used within 3–4 days after purification. No reduction in kinase activity was observed during this time. When tested, SP-Sepharose eluants from pBC12BI-transfected control cells exhibited no detectable levels of rhodopsin phosphorylation over time (data not shown). 5–10 × 106 COS-1 cells transfected with pBC12BI, pBC-GRK6, or pBC-GRK6-pal− were trypsinized and lysed in 100 ॖl of 17 Triton X-100 lysis buffer as described above. 3 ॖg of affinity-purified rabbit polyclonal antiserum (Santa Cruz Biotechnology, Inc.) was coupled to 10 ॖl of protein A-agarose beads (Boehringer Mannheim), washed three times with buffer B (0.27 Triton X-100, 150 mm NaCl, 20 mm HEPES, pH 7.5, 10 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, 200 ॖg/ml benzamidine, 20 ॖg/ml leupeptin), and then incubated with 90 ॖl of cell lysate for 1 h at 4 °C on a rotator. The remaining 10 ॖl of lysate was used for Western blot analysis to quantitate the levels of GRK6 expression. The beads were washed three times with buffer B followed by a single wash with 20 mm HEPES, pH 7.5, 2 mm EDTA to remove Triton X-100 and NaCl (both of which inhibit GRK activity), resuspended in 30 ॖl assay buffer (200 ॖm [γ-32P]ATP (1 cpm/fmol), 4.6 mm MgCl2, 133 ॖg/ml phosvitin (Sigma), 20 mm HEPES, pH 7.5, 2 mm EDTA), and incubated for 15 or 20 min at 30 °C. Reactions were terminated with 20 ॖl of SDS sample buffer, and the samples were centrifuged briefly and then electrophoresed on a 107 SDS-polyacrylamide gel. Gels were dried, and autoradiography was performed. Quantitation of the phosphorylated phosvitin bands was performed as described above for the rhodopsin phosphorylation assays. Recovery of kinase immunoprecipitated from cell lysates containing either wild-type or mutant GRK6 was ∼807 (data not shown). Immunoprecipitatable activity from lysates of cells transfected with the pBC vector alone revealed no significant level of phosvitin phosphorylation compared with either wild-type- or GRK6-pal−-expressing cells (data not shown). Furthermore, immunoprecipitation of GRK6 in the presence of a GRK6 peptide corresponding to the kinase C terminus reduced phosvitin phosphorylation to control levels (data not shown). Crude soybean phosphatidylcholine (PC) was sonicated on ice at a concentration of 17 mg/ml in 20 mm Tris, pH 8.0, 100 mm NaCl, 1 mm EDTA. The sonicated PC vesicles were aliquoted and stored at −80 °C, and a new aliquot was used for each experiment. Binding assays were performed using ∼15 ng of partially purified wild-type GRK6, GRK6-pal−, and GRK6-GG that was diluted in 48 ॖl of buffer containing 20 mm Tris, pH 8.0, 2 mm MgCl2, 150 mm NaCl and pre-spun at 100,000 × g for 10 min at 4 °C to remove any aggregated GRK6. The supernatants (54 ॖl) were then added to either 6 ॖl of PC vesicles (to give a final phospholipid concentration of 1.7 mg/ml) or 6 ॖl of PC buffer alone and incubated at 30 °C for 5 min. The samples were centrifuged at 100,000 × g for 10 min and the supernatants (60 ॖl) and pellets (resuspended in 60 ॖl of buffer) were dissolved in SDS sample buffer. 30 ॖl of each sample was electrophoresed on a 107 SDS-polyacrylamide gel, and the proteins were transferred to nitrocellulose for Western blot analysis as described above. Quantitation of the percent GRK6 bound to PC vesicles was done using a Molecular Dynamics personal densitometer and ImageQuant software. COS-1 cells grown to ∼907 confluence in T75 flasks were cotransfected for 48 h as described above with 8 ॖg of pBC-ॆ2AR and 8 ॖg of pBC12BI, pBC-GRK6, pBC-GRK6-pal−, or pBC-GRK6-GG. Transfected cells were trypsinized, washed several times with PBS, and resuspended in 1.1 ml of PBS, 0.1 mm ascorbate. 0.5-ml aliquots of each cell suspension were incubated with or without 10 ॖm(−)-isoproterenol for 0–45 min at 37 °C. Reactions were stopped with ice-cold PBS; the cells were centrifuged at 2,000 ×g for 5 min, washed twice with PBS, and then resuspended in 0.5 ml of PBS. Cell surface ॆ2AR levels were determined by incubating the cells with 10 nm(−)-[3H]-CGP12177 for 3 h at 14 °C followed by vacuum filtration as described (28Goodman Jr., O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1179) Google Scholar). Nonspecific binding was determined in the presence of 20 ॖm alprenolol. To assess the role of palmitoylation in GRK6 function we used two expression constructs containing either the wild-type GRK6 cDNA (26Loudon R.P. Perussia B. Benovic J.L. Blood. 1996; 88: 4547-4557Crossref PubMed Google Scholar) or a mutant GRK6 in which the three putative palmitoylated cysteines (Cys561, Cys562, and Cys565) (18Stoffel 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) were changed to serines (GRK6-pal−) (Fig.1 A). Direct experimental evidence that one or more of these residues is palmitoylated comes from [3H]palmitate labeling of COS cells expressing either wild-type or pal− GRK6 in which wild-type GRK6, but not the pal− kinase, incorporated radiolabeled palmitate (18Stoffel 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). COS-1 cells transiently expressing wild-type GRK6 or GRK6-pal− were lysed and the kinases were partially purified by SP-Sepharose chromatography. Partial purification of the expressed proteins was necessary since their activity in crude lysates was largely inhibited due to the detergent in the lysis buffer and inhibitors present in the cell lysates (data not shown). Expression of wild-type GRK6 (∼67 kDa) was ∼8-fold higher compared with endogenous levels observed in pBC control-transfected cells, whereas the GRK6-pal− mutant was expressed ∼3-fold higher than wild-type GRK6 (Fig. 1 B, lanes 1, 2, and 4). One relatively simple way to assess GRK activity toward G protein-coupled receptors involves the use of bovine rod outer segments that contain high levels of the photoreceptor rhodopsin. Using equivalent amounts of the two GRK6 proteins, time-course studies revealed a ∼5-fold reduced ability of GRK6-pal− to phosphorylate light-activated rhodopsin compared with wild-type GRK6 (Fig. 2 A). GRK6-pal− exhibited a significantly lower affinity for rhodopsin as evidenced by a ∼5-fold higher K m (12.0 ± 0.5 ॖm) compared with wild-type GRK6 (2.6 ± 0.4 ॖm) (TableI). However, mutation of the GRK6 palmitoylation site modestly reduced (∼2-fold; Table I) its apparentV max with respect to wild-type GRK6 suggesting that lack of palmitoylation does not impair kinase catalytic activity. The K m of GRK6-pal− is strikingly similar to the K m for rhodopsin previously reported for Sf9 cell expressed and purified GRK6 (24Loudon R.P. Benovic J.L. J. Biol. Chem. 1994; 269: 22691-22697Abstract Full Text PDF PubMed Google Scholar). This suggests that the purified GRK6 is not palmitoylated, which may contribute to its apparent low activity.Table IKinetic parameters for wild-type and mutant GRK6KinaseK mV maxॖmnmol P i /min/mgGRK62.6 ± 0.4231 ± 20GRK6-pal−12.0 ± 0.5110 ± 2GRK6-gg1.2 ± 0.13244 ± 209Kinetic parameters of the different GRK6 proteins for phosphorylating rhodopsin were determined by incubating 0.1–20 ॖmrhodopsin and 1.3 ng of GRK6 for 5 min at 30 °C as described under 舠Experimental Procedures.舡 All values are presented as the mean ± S.E. from three to four independent experiments. Open table in a new tab Kinetic parameters of the different GRK6 proteins for phosphorylating rhodopsin were determined by incubating 0.1–20 ॖmrhodopsin and 1.3 ng of GRK6 for 5 min at 30 °C as described under 舠Experimental Procedures.舡 All values are presented as the mean ± S.E. from three to four independent experiments. Since membrane targeting appears important for rhodopsin kinase, ॆARK, and GRK5 activity (12Inglese 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, 13Pitcher 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, 17Kunapuli P. Gurevich V.V. Benovic J.L. J. Biol. Chem. 1994; 269: 10209-10212Abstract Full Text PDF PubMed Google Scholar) and lack of palmitoylation significantly reduces GRK6 phosphorylation of rhodopsin, we next wanted to test the hypothesis that GRK6 activity toward receptor substrates is strongly influenced by its ability to interact with phospholipids. To do this we made a mutant that was still defective in palmitoylation but was also modified to include a C-terminal CAAX motif to promote protein geranylgeranylation (Fig.1 A) (29Zhang F.L. Casey P.J. Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1743) Google Scholar). In this way, we wanted to determine whether the reduced ability of GRK6-pal− to phosphorylate receptors could be overcome by enhancing its association with phospholipids by isoprenylation. Expression of this protein (GRK6-GG) was approximately 30–407 of that observed for wild-type GRK6 (Fig. 1 B,lanes 2 and 6). When expressed in COS-1 cells and p
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