Title: Regulation of Membrane Targeting of the G Protein-coupled Receptor Kinase 2 by Protein Kinase A and Its Anchoring Protein AKAP79
Abstract: The β2 adrenergic receptor (β2AR) undergoes desensitization by a process involving its phosphorylation by both protein kinase A (PKA) and G protein-coupled receptor kinases (GRKs). The protein kinase A-anchoring protein AKAP79 influences β2AR phosphorylation by complexing PKA with the receptor at the membrane. Here we show that AKAP79 also regulates the ability of GRK2 to phosphorylate agonist-occupied receptors. In human embryonic kidney 293 cells, overexpression of AKAP79 enhances agonist-induced phosphorylation of both the β2AR and a mutant of the receptor that cannot be phosphorylated by PKA (β2AR/PKA−). Mutants of AKAP79 that do not bind PKA or target to the β2AR markedly inhibit phosphorylation of β2AR/PKA−. We show that PKA directly phosphorylates GRK2 on serine 685. This modification increases Gβγ subunit binding to GRK2 and thus enhances the ability of the kinase to translocate to the membrane and phosphorylate the receptor. Abrogation of the phosphorylation of serine 685 on GRK2 by mutagenesis (S685A) or by expression of a dominant negative AKAP79 mutant reduces GRK2-mediated translocation to β2AR and phosphorylation of agonist-occupied β2AR, thus reducing subsequent receptor internalization. Agonist-stimulated PKA-mediated phosphorylation of GRK2 may represent a mechanism for enhancing receptor phosphorylation and desensitization. The β2 adrenergic receptor (β2AR) undergoes desensitization by a process involving its phosphorylation by both protein kinase A (PKA) and G protein-coupled receptor kinases (GRKs). The protein kinase A-anchoring protein AKAP79 influences β2AR phosphorylation by complexing PKA with the receptor at the membrane. Here we show that AKAP79 also regulates the ability of GRK2 to phosphorylate agonist-occupied receptors. In human embryonic kidney 293 cells, overexpression of AKAP79 enhances agonist-induced phosphorylation of both the β2AR and a mutant of the receptor that cannot be phosphorylated by PKA (β2AR/PKA−). Mutants of AKAP79 that do not bind PKA or target to the β2AR markedly inhibit phosphorylation of β2AR/PKA−. We show that PKA directly phosphorylates GRK2 on serine 685. This modification increases Gβγ subunit binding to GRK2 and thus enhances the ability of the kinase to translocate to the membrane and phosphorylate the receptor. Abrogation of the phosphorylation of serine 685 on GRK2 by mutagenesis (S685A) or by expression of a dominant negative AKAP79 mutant reduces GRK2-mediated translocation to β2AR and phosphorylation of agonist-occupied β2AR, thus reducing subsequent receptor internalization. Agonist-stimulated PKA-mediated phosphorylation of GRK2 may represent a mechanism for enhancing receptor phosphorylation and desensitization. G protein-coupled receptor A kinase-anchoring protein protein kinase A protein kinase C β2 adrenergic receptor human embryonic kidney G protein-coupled receptor kinase glutathione S-transferase polyacrylamide gel electrophoresis extracellular signal-regulated kinase 1 and/or 2 Hormonal signaling through G protein-coupled receptors (GPCRs)1 is attenuated during prolonged exposure to agonist, a process known as desensitization (1Freedman N.J. Lefkowitz R.J. Recent Prog. Horm. Res. 1996; 51: 319-353PubMed Google Scholar). One of the initial events in this multistep process is the phosphorylation of agonist-occupied receptor molecules. Two families of kinases are responsible for the phosphorylation of GPCRs, the second messenger-activated kinases (PKA and PKC) and the GPCR kinases (GRKs 1–7) (2Benovic J.L. Pike L.J. Cerione R.A. Staniszewski C. Yoshimasa T. Codina J. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1985; 260: 7094-7101Abstract Full Text PDF PubMed Google Scholar, 3Bouvier M. 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The mechanisms by which different GRKs are recruited to the receptor varies, but for both GRK2 and GRK3, recruitment is achieved through the binding of phospholipids and Gβγ subunits to the COOH-terminal pleckstrin homology domain of the kinase (10Daaka Y. Pitcher J.A. Richardson M. Stoffel R.H. Robishaw J.D. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2180-2185Crossref PubMed Scopus (156) Google Scholar, 11DebBurman S.K. Ptasienski J. Benovic J.L. Hosey M.M. J. Biol. Chem. 1996; 271: 22552-22562Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 12DebBurman 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, 13Koch W.J. Inglese J. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 8256-8260Abstract Full Text PDF PubMed Google Scholar, 14Pitcher J.A. Inglese J. Higgins J.B. Arriza J.L. Casey P.J. Kim C. Benovic J.L. Kwatra M.M. 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It has been demonstrated that the overexpression of AKAP79 with the β2AR enhances receptor phosphorylation and, furthermore, that mutants of AKAP79, which fail to bind to the β2AR or to PKA, are effective at reducing phosphorylation. In this manner, AKAP79 may be acting as a scaffold which coordinates the events involved in receptor desensitization. To investigate this novel function of AKAP79 further, we set out to test whether another event involved in β2AR desensitization, i.e. receptor phosphorylation by GRK2, is modulated by PKA scaffolding. GRK2 was purified from baculovirus-infected Sf9 cells as described previously (38Kim C.M. Dion S.B. Onorato J.J. Benovic J.L. Receptor. 1993; 3: 39-55PubMed Google Scholar). Bovine GRK2ct (residues 467–689) was expressed as a glutathione S-transferase (GST) fusion protein in bacteria and purified as described previously (13Koch W.J. Inglese J. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 8256-8260Abstract Full Text PDF PubMed Google Scholar). Purification of rod outer segment membranes (39Papermaster D. Dreyer W. Biochemistry. 1974; 13: 2438-2444Crossref PubMed Scopus (580) Google Scholar), Gβγ subunits, (40Casey P. Graziano M. Gilman A. Biochemistry. 1989; 28: 611-616Crossref PubMed Scopus (91) Google Scholar) and tubulin (41Simon J. Parsons S. Salmon E. Micron Microsc. Acta. 1991; 22: 405-412Crossref Scopus (10) Google Scholar) was described previously. AKAP79, AKAP79pro, and AKAP79108–427 mammalian expression constructs were described previously (27Fraser I.D. Cong M. Kim J. Rollins E.N. Daaka Y. Lefkowitz R.J. Scott J.D. Curr. Biol. 2000; 10: 409-412Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 37Klauck T.M. Faux M.C. Labudda K. Langeberg L.K. Jaken S. Scott J.D. Science. 1996; 271: 1589-1592Crossref PubMed Scopus (483) Google Scholar). Mammalian expression constructs of FLAG epitope-tagged β2 adrenergic receptor (β2AR) and β2AR/PKA− mutant receptor (PKA phosphorylation sites serines 261, 262, 345, and 346 all mutated to alanines) were described previously (42Oppermann M. Diverse-Pierluissi M. Drazner M.H. Dyer S.L. Freedman N.J. Peppel K.C. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7649-7654Crossref PubMed Scopus (82) Google Scholar). The PKA catalytic subunit was purchased from Promega, and the anti-Gβ antibody was from Perkin Elmer Life Sciences. The anti-GRK2 monoclonal and AKAP79 polyclonal antibodies were described previously (20Carr D.W. Stofko-Hahn R.E. Fraser I.D. Cone R.D. Scott J.D. J. Biol. Chem. 1992; 267: 16816-16823Abstract Full Text PDF PubMed Google Scholar, 42Oppermann M. Diverse-Pierluissi M. Drazner M.H. Dyer S.L. Freedman N.J. Peppel K.C. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7649-7654Crossref PubMed Scopus (82) Google Scholar). M2 anti-FLAG antibody conjugated to Sepharose beads and M2 antibody were from Sigma. Unless otherwise stated, the chemicals were from Sigma. pcDNA1-bovine GRK2 (43Freedman N.J. Liggett S.B. Drachman D.E. Pei G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17953-17961Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar) was restriction digested with HindIII and XbaI, and the insert was ligated into the pcDNA3 vector (Invitrogen). A single point mutation changing serine 685 to alanine was introduced by the polymerase chain reaction using the primers 5′-TCCCCAACCGCCTCGAGTGGC-3′ and 5′-CTAGTCTAGATCAGAGGCCGTTGGCGGCGCCGCGC-3′. The polymerase chain reaction product was restriction digested with XhoI andXbaI and used to replace the equivalent fragment in the pcDNA3 construct. The sequence and orientation of the clone were confirmed by automated DNA sequence analysis. The RsrII/BamHI restriction fragment from a GRK2Δ19 construct (construct 2 in Ref. 13Koch W.J. Inglese J. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 8256-8260Abstract Full Text PDF PubMed Google Scholar encoding full-length GRK2 with a stop codon inserted at codon 671) was used to replace the equivalent fragment in pVL1392-GRK2(S670A) (44Pitcher J.A. Tesmer J.J. Freeman J.L. Capel W.D. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1999; 274: 34531-34534Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). The orientation and sequence of the clone were confirmed by automated DNA sequence analysis. Purification of the expressed protein from Sf9 cells was identical as that described for full-length GRK2 (38Kim C.M. Dion S.B. Onorato J.J. Benovic J.L. Receptor. 1993; 3: 39-55PubMed Google Scholar). HEK293 cells were grown at 37 °C in minimal essential medium containing 10% fetal bovine serum and 1× penicillin/streptomycin (Life Technologies, Inc.) under 5% CO2. Cells at 60% confluence were transfected with up to 5 μg of plasmid DNA and 15 μl of Fugene 6 (Roche Molecular Biochemicals). Two days after transfection, cells were lysed in radioimmune precipitation buffer (150 mm NaCl, 50 mm Tris, pH 8.0, 5 mm EDTA, 1% v/v Nonidet P-40, 0.5% w/v sodium deoxycholate, 10 mm NaF, 10 mm Na2-pyrophosphate, 0.1% w/v SDS, 5 μg/ml aprotinin, 150 μg/ml benzamidine, 5 μg/ml leupeptin, 4 μg/ml pepstatin, and 20 μg/ml phenylmethylsulfonyl fluoride). If cross-linking of proteins was necessary before immunoprecipitation, cells were incubated at room temperature for 20 min in phosphate-buffered saline containing 10 mm HEPES, pH 7.4, and 1 mg/ml dithiobis(succinimidyl propionate) before lysis in radioimmune precipitation buffer (45Freedman N.J. Ament A.S. Oppermann M. Stoffel R.H. Exum S.T. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 17734-17743Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). After removal of insoluble cell debris by centrifugation, protein concentrations were equalized in all samples, and FLAG epitope-tagged proteins were immunoprecipitated for 15 h with 40 μl of a 1:1 slurry of M2 anti-FLAG antibody covalently coupled to Sepharose beads. The beads were washed four times with radioimmune precipitation buffer, and bound proteins were eluted in 50 μl of 2× SDS-PAGE sample buffer (100 mm Tris, pH 7.2, 4% w/v SDS, 200 mm dithiothreitol, 20% v/v glycerol, 20 μg/ml bromphenol blue with 5% v/v β-mercaptoethanol for cross-linked samples) for 10 min at 95 °C. Samples were resolved on 10% or 4–20% polyacrylamide gels (Novex) and transferred to nitrocellulose filters for immunoblotting. Filters were blocked with 5% w/v fat-free milk powder in Tris-buffered saline with Tween 20 (20 mm Tris, pH 7.4, 500 mm NaCl, 0.1% v/v Tween 20) and incubated overnight at 4 °C with appropriate primary antiserum. After thorough washing in Tris-buffered saline with Tween 20, filters were incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit or mouse secondary antibody (Amersham Pharmacia Biotech), washed again with Tris-buffered saline with Tween 20, immersed in ECL reagent (Amersham Pharmacia Biotech), and exposed to x-ray film. COS7 cells expressing FLAG-β2AR/PKA− alone or coexpressing AKAP79 with GRK2 or GRK2S685A were stimulated with 10 μm isoproterenol for 30 min. Agonist-induced receptor internalization was measured as the loss of cell surface FLAG epitopes available for M2 antibody binding by detection of a fluorescently labeled secondary antibody as described previously (46Barak L.S. Tiberi M. Freedman N.J. Kwatra M.M. Lefkowitz R.J. Caron M.G. J. Biol. Chem. 1994; 269: 2790-2795Abstract Full Text PDF PubMed Google Scholar). Receptor phosphorylation was assessed after the labeling of the intracellular ATP pool of HEK293 cells stably transfected with FLAG-β2AR or FLAG-β2AR/PKA− with [32P]orthophosphate (PerkinElmer Life Sciences) as described previously (43Freedman N.J. Liggett S.B. Drachman D.E. Pei G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17953-17961Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). Cells were labeled for 1 h at 37 °C in phosphate-free Dulbecco's modified Eagle's medium containing 10 mm HEPES, pH 7.4, 100 μCi/ml [32P]orthophosphate, and 1 μg/ml microcystin L-R (Calbiochem). Cells were stimulated with 10 μmisoproterenol for 5 min, washed twice with ice-cold phosphate-buffered saline buffer, solubilized in 750 μl of radioimmune precipitation buffer, and equivalent protein amounts were subjected to immunoprecipitation. Samples were resolved on 10% polyacrylamide gels and dried under vacuum. Radioactive bands were visualized and quantified using a PhosphorImager (Molecular Dynamics) and by exposure to x-ray film. The levels of receptor expression were measured by flow cytometry by detecting cell surface-bound anti-epitope tag M2 antibodies and fluorescein-conjugated secondary antibodies (46Barak L.S. Tiberi M. Freedman N.J. Kwatra M.M. Lefkowitz R.J. Caron M.G. J. Biol. Chem. 1994; 269: 2790-2795Abstract Full Text PDF PubMed Google Scholar). All phosphorylation levels were normalized to receptor expression and are shown as stimulated-basal values. One microgram of GRK2 or GRK2Δ19 purified from baculovirus-infected Sf9 cells or GRK2ct purified by cleavage with thrombin from bacterially expressed GST fusion protein (13Koch W.J. Inglese J. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 8256-8260Abstract Full Text PDF PubMed Google Scholar) was incubated with 1 unit of PKA catalytic subunit (Promega) in phosphorylation reaction buffer containing 20 mm Tris, pH 7.5, 10 mm MgCl2, 2 mm EDTA, 1 mm dithiothreitol, 60 μm[γ32P]ATP (∼1000 cpm/pmol), 1 μg/ml phospholipids (crude preparation containing 20% w/vl-α-phosphatidylcholine, Sigma), and 0.8 μmGβγ subunits. After 30 min of incubation at 30 °C, reactions were stopped by the addition of an equal volume of 2× SDS-PAGE sample buffer. Samples were boiled for 10 min and resolved on 4–20% gradient polyacrylamide gels. Radioactive bands in dried gels were visualized and quantified using a PhosphorImager and by exposure to x-ray film. In vitro phosphorylation of rhodopsin, tubulin, and the peptide substrate RRRREEEEESAAA by GRK2 was performed as described previously (47Pitcher 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: 12316-12324Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The reactions used 25 ng of GRK2 to phosphorylate 2 μg of rhodopsin or 100 ng of GRK2 to phosphorylate either 0.2 μg of tubulin or 1 mm peptide substrate. One microgram of GST-GRK2ct (residues 467–689 of bovine GRK2) bound to glutathione-conjugated Sepharose 4B beads (Calbiochem) was incubated with or without PKA as described above. After 30 min of incubation at 30 °C, beads were washed extensively with phosphate-buffered saline containing 0.01% v/v lubrol to remove all the PKA and ATP. Four micrograms of Gβγ from bovine brain was then added and incubated at 4 °C for 2 h. After extensive washes with phosphate-buffered saline containing 0.01% lubrol, 2× sample buffer was added, and proteins were eluted by boiling for 10 min. Samples were resolved on 4–20% polyacrylamide gels, transferred to nitrocellulose filters, and immunoblotted with an antibody recognizing Gβ subunits. It has been shown previously that the protein kinase A-anchoring protein AKAP79 can directly interact with and regulate the phosphorylation of the β2AR (27Fraser I.D. Cong M. Kim J. Rollins E.N. Daaka Y. Lefkowitz R.J. Scott J.D. Curr. Biol. 2000; 10: 409-412Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). In HEK293 cells overexpressing FLAG epitope-tagged β2AR, the coexpression of AKAP79 increases β2AR phosphorylation by 40%, whereas the coexpression of the AKAP79 mutant AKAP79108–427, which does not bind to the receptor or localize PKA to the membrane, impairs receptor phosphorylation by 50% (Fig. 1 A). To determine whether this enhancement of β2AR phosphorylation is a result of AKAP79 directly facilitating phosphorylation of the receptor by PKA, agonist-induced phosphorylation of a FLAG epitope-tagged β2AR mutant lacking all PKA phosphorylation sites (β2AR/PKA−) was measured. Surprisingly, the coexpression of wild type AKAP79 enhances agonist-induced phosphorylation of β2AR/PKA− by 40% (Fig.1 B). In contrast, cells transfected with either of two AKAP79 mutants that are unable to target PKA to the plasma membrane, AKAPpro and AKAP108–427, exhibit a 55–70% decrease in β2AR/PKA− phosphorylation. AKAP79pro is targeted to the plasma membrane and can bind to β2AR but is unable to bind to the PKA regulatory subunit, whereas AKAP79108–427 binds to PKA but lacks the receptor and membrane-targeting domain (27Fraser I.D. Cong M. Kim J. Rollins E.N. Daaka Y. Lefkowitz R.J. Scott J.D. Curr. Biol. 2000; 10: 409-412Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 37Klauck T.M. Faux M.C. Labudda K. Langeberg L.K. Jaken S. Scott J.D. Science. 1996; 271: 1589-1592Crossref PubMed Scopus (483) Google Scholar). Although GRK2 and AKAP79 do not interact directly (data not shown), when GRK2 and AKAP79 are overexpressed together a 2.3-fold increase in β2AR/PKA− phosphorylation is observed compared with a 1.4-fold increase by overexpression of GRK2 alone (Fig. 1 C). Overexpression of either of the two AKAP79 mutants with GRK2 inhibits receptor phosphorylation by 40–60% compared with control cells expressing only endogenous levels of GRK2 (Fig. 1 C). These results suggest that AKAP79 does not affect β2AR phosphorylation by simply enhancing direct receptor phosphorylation by PKA. Rather, they suggest that AKAP79 enhances GRK2-mediated phosphorylation of the β2AR. To ensure that PKA is not capable of phosphorylating the β2AR/PKA− mutant, the phosphorylation of the wild type receptor and β2AR/PKA− was compared following activation of PKA by stimulation of a coexpressed Gs-coupled receptor (Fig.1 D). In the absence of its ligand, the β2AR can be phosphorylated by PKA but not by the GRKs as they specifically phosphorylate only agonist-occupied receptors. Stimulation of endogenous vasoactive intestinal peptide receptors leads to the phosphorylation of wild type β2AR to 20% of the level observed with isoproterenol stimulation (Fig. 1 D). This level is consistent with previous reports for PKA phosphorylation of the β2AR in HEK293 cells (48Ferguson S.S. Menard L. Barak L.S. Koch W.J. Colapietro A.M. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). The β2AR/PKA− mutant, however, shows no vasoactive intestinal peptide-induced phosphorylation (Fig.1 D), confirming that the removal of the PKA phosphorylation sites renders the receptor incapable of being further phosphorylated by PKA. Taken together, the regulation of β2AR/PKA− phosphorylation by wild type and mutants of AKAP79 indicates that AKAP79 may indirectly regulate GRK2 activity by a mechanism requiring the anchoring of PKA to the plasma membrane. To further test the ability of PKA to affect GRK2 activity against receptor substrates, we measured the effect of PKA on GRK2-mediated phosphorylation of rhodopsin in vitro. Incubation with PKA and 167 nm Gβγ subunits or with GRK2 alone results in little or no phosphorylation of rhodopsin (Fig.2). However, with the addition of increasing concentrations of Gβγ subunits, GRK2 phosphorylation of rhodopsin is greatly increased and is further enhanced with the addition of PKA. As PKA does not significantly phosphorylate rhodopsin nor is its activity regulated by Gβγ subunits, the observed increase in phosphorylation is attributed to enhanced GRK2 activity toward rhodopsin. It has been reported that PKC can directly phosphorylate GRK2 at a site within the carboxyl terminus and that this facilitates GRK2 translocation to the plasma membrane (49Chuang 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, 50Winstel 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). To investigate whether a similar mechanism exists for the phosphorylation of GRK2 by PKA, we tested the ability of PKA to phosphorylate purified full-length GRK2 and fragments of GRK2 (Fig.3 A). In the presence of phospholipids that activate GRK2, bovine GRK2 purified from Sf9 cells shows weak autophosphorylation (a stoichiometry of 0.03 mol of phosphate/mol of protein, Fig. 3 B). Incubation of GRK2 with PKA leads to a significant increase in phosphorylation (0.25 mol of phosphate/mol of protein), which is further enhanced by the addition of 0.8 μm Gβγ subunits to 0.7 mol of phosphate/mol of protein. A fragment of the carboxyl terminus of GRK2 (residues 467–689, GRK2ct), which encompasses the pleckstrin homology domain and Gβγ binding domain, also acts as a good PKA substrate (0.4 mol of phosphate/mol of protein), and this phosphorylation is markedly enhanced by the addition of Gβγ subunits (0.7 mol of phosphate/mol of protein, Fig. 3 B). Furthermore, a GRK2 mutant that lacks 19 residues at the carboxyl terminus is unable to be phosphorylated by PKA (Fig. 3 B). Examination of the extreme COOH-terminal sequence of GRK2 reveals three potential sites of PKA phosphorylation, serines 670, 676, and 685. Although the phosphorylation of serine 670 by Erk1/Erk2 was reported previously to inactivate GRK2 (44Pitcher J.A. Tesmer J.J. Freeman J.L. Capel W.D. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1999; 274: 34531-34534Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), no studies have investigated whether phosphorylation of serines 676 and 685 can affect GRK2 activity. The site of PKA phosphorylation on GRK2 was mapped by incubating the GRK2 carboxyl terminus fragment with PKA and [γ-32P]ATP followed by tryptic digestion and subsequent high pressure liquid chromatography fractionation and sequencing of radiolabeled peptides. This yields one major phosphopeptide corresponding to residues 678–689 of GRK2 containing a