Phosphorylation of Phosducin and Phosducin-like Protein by G Protein-coupled Receptor Kinase 2

Abstract

G protein-coupled receptor kinase 2 (GRK2) is able to phosphorylate a variety of agonist-occupied G protein-coupled receptors (GPCR) and plays an important role in GPCR modulation. However, recent studies suggest additional cellular functions for GRK2. Phosducin and phosducin-like protein (PhLP) are cytosolic proteins that bind Gβγ subunits and act as regulators of G-protein signaling. In this report, we identify phosducin and PhLP as novel GRK2 substrates. The phosphorylation of purified phosducin and PhLP by recombinant GRK2 proceeds rapidly and stoichiometrically (0.82 ± 0.1 and 0.83 ± 0.09 mol of Pi/mol of protein, respectively). The phosphorylation reactions exhibit apparent Km values in the range of 40–100 nm, strongly suggesting that both proteins could be endogenous targets for GRK2 activity. Our data show that the site of phosducin phosphorylation by GRK2 is different and independent from that previously reported for the cAMP-dependent protein kinase. Analysis of GRK2 phosphorylation of a variety of deletion mutants of phosducin and PhLP indicates that the critical region for GRK2 phosphorylation is localized in the C-terminal domain of both phosducin and PhLP (between residues 204 and 245 and 195 and 218, respectively). This region is important for the interaction of these proteins with Gβγ subunits. Phosphorylation of phosducin by GRK2 markedly reduces its Gβγ binding ability, suggesting that GRK2 may modulate the activity of the phosducin protein family by disrupting this interaction. The identification of phosducin and PhLP as new substrates for GRK2 further expands the cellular roles of this kinase and suggests new mechanisms for modulating GPCR signal transduction. G protein-coupled receptor kinase 2 (GRK2) is able to phosphorylate a variety of agonist-occupied G protein-coupled receptors (GPCR) and plays an important role in GPCR modulation. However, recent studies suggest additional cellular functions for GRK2. Phosducin and phosducin-like protein (PhLP) are cytosolic proteins that bind Gβγ subunits and act as regulators of G-protein signaling. In this report, we identify phosducin and PhLP as novel GRK2 substrates. The phosphorylation of purified phosducin and PhLP by recombinant GRK2 proceeds rapidly and stoichiometrically (0.82 ± 0.1 and 0.83 ± 0.09 mol of Pi/mol of protein, respectively). The phosphorylation reactions exhibit apparent Km values in the range of 40–100 nm, strongly suggesting that both proteins could be endogenous targets for GRK2 activity. Our data show that the site of phosducin phosphorylation by GRK2 is different and independent from that previously reported for the cAMP-dependent protein kinase. Analysis of GRK2 phosphorylation of a variety of deletion mutants of phosducin and PhLP indicates that the critical region for GRK2 phosphorylation is localized in the C-terminal domain of both phosducin and PhLP (between residues 204 and 245 and 195 and 218, respectively). This region is important for the interaction of these proteins with Gβγ subunits. Phosphorylation of phosducin by GRK2 markedly reduces its Gβγ binding ability, suggesting that GRK2 may modulate the activity of the phosducin protein family by disrupting this interaction. The identification of phosducin and PhLP as new substrates for GRK2 further expands the cellular roles of this kinase and suggests new mechanisms for modulating GPCR signal transduction. G protein-coupled receptor G protein-coupled receptor kinase glutathione S-transferase hexahistidine-tagged phosducin phosducin-like protein cAMP-dependent protein kinase protein kinase A inhibitor, Tricine, N-tris(hydroxymethyl)methylglycine polyacrylamide gel electrophoresis Agonist occupancy of G protein-coupled receptors (GPCR)1 triggers interaction with different types of cellular proteins. Interaction with heterotrimeric G proteins promotes GTP/GDP exchange and dissociation into Gα and Gβγ subunits, both of which modulate different membrane-bound effector proteins. This signal transduction process is regulated at different levels. Agonist-activated GPCRs are phosphorylated by a family of specific G protein-coupled receptor kinases (GRKs). This is followed by binding of regulatory proteins termed arrestins to the phosphorylated receptor leading to uncoupling from G proteins, a process known as desensitization (reviewed in Refs.1Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1059) Google Scholar, 2Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 238-319Crossref Scopus (855) Google Scholar, 3Lohse M.J. Krasel C. Winstel R. Mayor Jr., F. Kidney Int. 1996; 49: 1047-1052Abstract Full Text PDF PubMed Scopus (56) Google Scholar). GRK2 is a ubiquitous member of the GRK family, which is able to phosphorylate a variety of GPCR (4Chuang T.T. Iacovelli L. Sallese M. DeBlasi A. Trends Pharmacol. Sci. 1996; 17: 416-421Abstract Full Text PDF PubMed Scopus (223) Google Scholar, 5Aragay A.M. Ruiz-Gómez A. Penela P. Sarnago S. Elorza A. Jimenez-Sainz M.C. Mayor Jr., F. FEBS Lett. 1998; 430: 37-40Crossref PubMed Scopus (63) Google Scholar). Recent data indicate that GRK2 and β-arrestins play additional roles in GPCR regulation and signaling. These proteins also initiate receptor internalization by facilitating the interaction of the receptor complex with the endocytic machinery. This is followed by receptor dephosphorylation and recycling (reviewed in Refs. 6Mayor Jr., F. Penela P. Ruiz-Gómez A. Trends Cardiovasc. Med. 1998; 8: 235-241Crossref Scopus (28) Google Scholar, 7Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar). Moreover, recent data suggest that GRK2 and β-arrestin may directly participate in GPCR-mediated activation of the mitogen-activated protein kinase (MAPK) cascade by allowing the recruitment of signaling proteins such as Src-tyrosine kinase (8Luttrell L.M. Daaka Y. Della Rocca G.J. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31648-31656Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 9Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S.G. Caron Lefkowitz R.J. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, L.M. Ferguson S.S.G. Daaka Y. S. Della Rocca G.J. Luttrell Caron Lefkowitz R.J. PubMed Scopus Google further in GPCR cellular and to be different mechanisms J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1059) Google Scholar, 5Aragay A.M. Ruiz-Gómez A. Penela P. Sarnago S. Elorza A. Jimenez-Sainz M.C. Mayor Jr., F. FEBS Lett. 1998; 430: 37-40Crossref PubMed Scopus (63) Google Scholar, Benovic J.L. 1998; 8: PubMed Scopus Google with the that this kinase plays different important roles in cellular signaling. of GRK2 to the its interaction with receptor and binding of the C-terminal domain of GRK2 to both Gβγ subunits and J.A. Lefkowitz R.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Benovic J.L. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). This binding process in an activity of GRK2 its substrates Benovic J.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of GRK2 is also by protein kinase phosphorylation T.T. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, R. S. Krasel C. M.J. Sci. S. A. 1996; PubMed Scopus Google Scholar, S. Elorza A. Mayor Jr., F. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google by of its interaction with Benovic J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google L. Sallese M. S. A. J. PubMed Scopus Google Scholar). In an as protein in also GRK2 and activity Penela P. C. P. Benovic J.L. Mayor Jr., F. J. Biol. Chem. Full Text PDF PubMed Google Scholar, C. Ruiz-Gómez A. Benovic J.L. Mayor Jr., F. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The of different cellular of GRK2 suggests the of additional substrates and cellular functions for this regulatory mechanisms of GPCR signaling of G-protein Interaction of regulators of family with Gα subunits its activity and of the signal J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). the phosducin and phosducin-like proteins (PhLP) are able to bind to Gβγ subunits, with signaling and with with PubMed Scopus Google Scholar, S. M. S. M.J. PubMed Scopus Google Scholar, R. A. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, S. M. M. M.J. J. 1997; PubMed Scopus Google Scholar, S. M. M. Sci. S. A. PubMed Scopus Google Scholar, S. M.J. Sci. S. A. 1996; PubMed Scopus (63) Google Scholar, C. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). Phosducin is a that in the PubMed Scopus Google and to be S. M. S. M.J. PubMed Scopus Google Scholar, S. M.J. Sci. S. A. 1996; PubMed Scopus Google Scholar). The PhLP also a of S. M. M. Sci. S. A. PubMed Scopus Google Scholar, S. M.J. Sci. S. A. 1996; PubMed Scopus (63) Google Scholar). proteins reported to for Gβγ binding with targets such as in of GPCR signaling S. M. S. M.J. PubMed Scopus Google Scholar, S. M.J. Sci. S. A. 1996; PubMed Scopus (63) Google Scholar, C. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, J. Sci. S. A. PubMed Scopus Google Scholar, S. P. S. M.J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. M.J. Lefkowitz R.J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). In the of its to with Gβγ is phosphorylation by the cAMP-dependent protein kinase a for its J. Biol. Chem. Full Text PDF PubMed Google Scholar, G.J. J. Biol. Chem. Full Text PDF PubMed Google regulation of PhLP phosphorylate PhLP Google this is known these regulatory mechanisms at the receptor and G-protein are cellular to both GRK2 and are able to with Gβγ subunits, is to suggest that may In to GRK2 activation and S. M.J. Sci. S. A. 1996; PubMed Scopus (63) Google Scholar, M. P. M.J. FEBS Lett. PubMed Scopus Google Scholar). In this report, we show that phosducin and PhLP are substrates for GRK2 and that such phosphorylation in of the Gβγ binding with recent data that identify as a for GRK2 J.A. Daaka Y. J. Ferguson S.S.G. S. S. Caron Lefkowitz R.J. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, Benovic J.L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google the of substrates for this kinase and suggest a role for GRK2 in the of GPCR signaling at different this report, we phosducin and PhLP as new substrates for GRK2. In with recent that show that is phosphorylated by this kinase J.A. Daaka Y. J. Ferguson S.S.G. S. S. Caron Lefkowitz R.J. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, Benovic J.L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google data further indicate the of substrates for GRK2. The that the activity of this kinase is to agonist-occupied GPCR suggests that GRK2 may also act as an effector of GPCR signaling and additional cellular of the that phosphorylation of phosducin proteins may The phosphorylation reactions rapidly and The apparent Km for phosducin and for that reported for substrates of GRK2 in the range of J.L. Caron Lefkowitz R.J. J. Biol. Chem. Full Text PDF PubMed Google and with the Km for phosphorylation of Refs. J.A. Daaka Y. J. Ferguson S.S.G. S. S. Caron Lefkowitz R.J. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, Benovic J.L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google and a such as the receptor J.L. Mayor Jr., F. C. Lefkowitz R.J. Caron J. Biol. Chem. Full Text PDF PubMed Google Scholar). The Km data for phosducin and PhLP and the of are also the range of the reported cellular of phosducin S. M.J. Sci. S. A. 1996; PubMed Scopus Google and GRK2 Benovic J.L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). indicate that phosducin and PhLP are substrates for GRK2. the interaction of GRK2 with agonist-occupied receptors and Gβγ subunits promotes an activation of the kinase Benovic J.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google phosducin phosphorylation by GRK2 is in the of suggesting that is the receptor that promotes phosducin be to the mechanisms of this phosphorylation process in and its by GPCR-mediated signaling indicate that the critical for GRK2 phosphorylation are the C-terminal domain of both phosducin and PhLP (between residues and 245 and and 218, suggesting a regulatory in which these the phosphorylation for the of the kinase. These and residues in the of with that GRK2 substrates residues to the phosphorylation site Caron Lefkowitz R.J. Benovic J.L. PubMed Scopus Google Scholar). These C-terminal are in the interaction of phosducin and PhLP with its the Gβγ subunits of heterotrimeric G proteins. The of phosducin with the subunits of R. A. 1996; Full Text Full Text PDF PubMed Scopus Google suggests that phosducin with its C-terminal domain to the of the its is the of the Gβγ with In a the of phosducin to be for Gβγ binding and is able to functions S. M. M. M.J. J. 1997; PubMed Scopus Google Scholar). Moreover, in this region markedly activity of phosducin S. M. M. M.J. J. 1997; PubMed Scopus Google Scholar). a of interaction R. A. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, C. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google and types of studies also indicate that the C-terminal of PhLP plays a role in Gβγ binding C. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, S. C. M.J. FEBS Lett. 1997; PubMed Scopus Google interaction with Gβγ subunits, phosducin and PhLP to with a variety of GPCR signaling by different G proteins S. M. S. M.J. PubMed Scopus Google Scholar, S. M.J. Sci. S. A. 1996; PubMed Scopus (63) Google Scholar, S. M.J. Sci. S. A. 1996; PubMed Scopus Google Scholar, J. Sci. S. A. PubMed Scopus Google Scholar, S. C. M.J. FEBS Lett. 1997; PubMed Scopus Google Scholar). The reported of phosducin by phosphorylation a regulatory in the of receptor signaling leading to in cellular phosphorylation may an additional for the of phosducin interaction with Gβγ subunits regulation of further from that by in important The phosphorylation of phosducin by and GRK2 is independent and at different GRK2 phosducin the of is to GRK2 also These data suggest independent of phosducin functions by the different types of Agonist occupancy of G protein-coupled receptors (GPCR)1 triggers interaction with different types of cellular proteins. Interaction with heterotrimeric G proteins promotes GTP/GDP exchange and dissociation into Gα and Gβγ subunits, both of which modulate different membrane-bound effector proteins. This signal transduction process is regulated at different levels. Agonist-activated GPCRs are phosphorylated by a family of specific G protein-coupled receptor kinases (GRKs). This is followed by binding of regulatory proteins termed arrestins to the phosphorylated receptor leading to uncoupling from G proteins, a process known as desensitization (reviewed in Refs.1Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1059) Google Scholar, 2Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 238-319Crossref Scopus (855) Google Scholar, 3Lohse M.J. Krasel C. Winstel R. Mayor Jr., F. Kidney Int. 1996; 49: 1047-1052Abstract Full Text PDF PubMed Scopus (56) Google Scholar). GRK2 is a ubiquitous member of the GRK family, which is able to phosphorylate a variety of GPCR (4Chuang T.T. Iacovelli L. Sallese M. DeBlasi A. Trends Pharmacol. Sci. 1996; 17: 416-421Abstract Full Text PDF PubMed Scopus (223) Google Scholar, 5Aragay A.M. Ruiz-Gómez A. Penela P. Sarnago S. Elorza A. Jimenez-Sainz M.C. Mayor Jr., F. FEBS Lett. 1998; 430: 37-40Crossref PubMed Scopus (63) Google Scholar). Recent data indicate that GRK2 and β-arrestins play additional roles in GPCR regulation and signaling. These proteins also initiate receptor internalization by facilitating the interaction of the receptor complex with the endocytic machinery. This is followed by receptor dephosphorylation and recycling (reviewed in Refs. 6Mayor Jr., F. Penela P. Ruiz-Gómez A. Trends Cardiovasc. Med. 1998; 8: 235-241Crossref Scopus (28) Google Scholar, 7Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar). Moreover, recent data suggest that GRK2 and β-arrestin may directly participate in GPCR-mediated activation of the mitogen-activated protein kinase (MAPK) cascade by allowing the recruitment of signaling proteins such as Src-tyrosine kinase (8Luttrell L.M. Daaka Y. Della Rocca G.J. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31648-31656Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 9Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S.G. Caron Lefkowitz R.J. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, L.M. Ferguson S.S.G. Daaka Y. S. Della Rocca G.J. Luttrell Caron Lefkowitz R.J. PubMed Scopus Google further in GPCR signaling. GRK2 cellular and to be different mechanisms J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1059) Google Scholar, 5Aragay A.M. Ruiz-Gómez A. Penela P. Sarnago S. Elorza A. Jimenez-Sainz M.C. Mayor Jr., F. FEBS Lett. 1998; 430: 37-40Crossref PubMed Scopus (63) Google Scholar, Benovic J.L. 1998; 8: PubMed Scopus Google with the that this kinase plays different important roles in cellular signaling. of GRK2 to the its interaction with receptor and binding of the C-terminal domain of GRK2 to both Gβγ subunits and J.A. Lefkowitz R.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Benovic J.L. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). This binding process in an activity of GRK2 its substrates Benovic J.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of GRK2 is also by protein kinase phosphorylation T.T. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, R. S. Krasel C. M.J. Sci. S. A. 1996; PubMed Scopus Google Scholar, S. Elorza A. Mayor Jr., F. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google by of its interaction with Benovic J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google L. Sallese M. S. A. J. PubMed Scopus Google Scholar). In an as protein in also GRK2 and activity Penela P. C. P. Benovic J.L. Mayor Jr., F. J. Biol. Chem. Full Text PDF PubMed Google Scholar, C. Ruiz-Gómez A. Benovic J.L. Mayor Jr., F. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The of different cellular of GRK2 suggests the of additional substrates and cellular functions for this kinase. regulatory mechanisms of GPCR signaling of G-protein Interaction of regulators of family with Gα subunits its activity and of the signal J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). the phosducin and phosducin-like proteins (PhLP) are able to bind to Gβγ subunits, with signaling and with with PubMed Scopus Google Scholar, S. M. S. M.J. PubMed Scopus Google Scholar, R. A. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, S. M. M. M.J. J. 1997; PubMed Scopus Google Scholar, S. M. M. Sci. S. A. PubMed Scopus Google Scholar, S. M.J. Sci. S. A. 1996; PubMed Scopus (63) Google Scholar, C. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). Phosducin is a that in the PubMed Scopus Google and to be S. M. S. M.J. PubMed Scopus Google Scholar, S. M.J. Sci. S. A. 1996; PubMed Scopus Google Scholar). The PhLP also a of S. M. M. Sci. S. A. PubMed Scopus Google Scholar, S. M.J. Sci. S. A. 1996; PubMed Scopus (63) Google Scholar). proteins reported to for Gβγ binding with targets such as in of GPCR signaling S. M. S. M.J. PubMed Scopus Google Scholar, S. M.J. Sci. S. A. 1996; PubMed Scopus (63) Google Scholar, C. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, J. Sci. S. A. PubMed Scopus Google Scholar, S. P. S. M.J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. M.J. Lefkowitz R.J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). In the of its to with Gβγ is phosphorylation by the cAMP-dependent protein kinase a for its J. Biol. Chem. Full Text PDF PubMed Google Scholar, G.J. J. Biol. Chem. Full Text PDF PubMed Google regulation of PhLP phosphorylate PhLP Google Scholar). this is known these regulatory mechanisms at the receptor and G-protein are cellular to both GRK2 and are able to with Gβγ subunits, is to suggest that may In to GRK2 activation and S. M.J. Sci. S. A. 1996; PubMed Scopus (63) Google Scholar, M. P. M.J. FEBS Lett. PubMed Scopus Google Scholar). In this report, we show that phosducin and PhLP are substrates for GRK2 and that such phosphorylation in of the Gβγ binding with recent data that identify as a for GRK2 J.A. Daaka Y. J. Ferguson S.S.G. S. S. Caron Lefkowitz R.J. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, Benovic J.L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google the of substrates for this kinase and suggest a role for GRK2 in the of GPCR signaling at different levels. this report, we phosducin and PhLP as new substrates for GRK2. In with recent that show that is phosphorylated by this kinase J.A. Daaka Y. J. Ferguson S.S.G. S. S. Caron Lefkowitz R.J. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, Benovic J.L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google data further indicate the of substrates for GRK2. The that the activity of this kinase is to agonist-occupied GPCR suggests that GRK2 may also act as an effector of GPCR signaling and additional cellular of the that phosphorylation of phosducin proteins may The phosphorylation reactions rapidly and The apparent Km for phosducin and for that reported for substrates of GRK2 in the range of J.L. Caron Lefkowitz R.J. J. Biol. Chem. Full Text PDF PubMed Google and with the Km for phosphorylation of Refs. J.A. Daaka Y. J. Ferguson S.S.G. S. S. Caron Lefkowitz R.J. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, Benovic J.L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google and a such as the receptor J.L. Mayor Jr., F. C. Lefkowitz R.J. Caron J. Biol. Chem. Full Text PDF PubMed Google Scholar). The Km data for phosducin and PhLP and the of are also the range of the reported cellular of phosducin S. M.J. Sci. S. A. 1996; PubMed Scopus Google and GRK2 Benovic J.L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). indicate that phosducin and PhLP are substrates for GRK2. the interaction of GRK2 with agonist-occupied receptors and Gβγ subunits promotes an activation of the kinase Benovic J.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google phosducin phosphorylation by GRK2 is in the of suggesting that is the receptor that promotes phosducin be to the mechanisms of this phosphorylation process in and its by GPCR-mediated signaling indicate that the critical for GRK2 phosphorylation are the C-terminal domain of both phosducin and PhLP (between residues and 245 and and 218, suggesting a regulatory in which these the phosphorylation for the of the kinase. These and residues in the of with that GRK2 substrates residues to the phosphorylation site Caron Lefkowitz R.J. Benovic J.L. PubMed Scopus Google Scholar). These C-terminal are in the interaction of phosducin and PhLP with its the Gβγ subunits of heterotrimeric G proteins. The of phosducin with the subunits of R. A. 1996; Full Text Full Text PDF PubMed Scopus Google suggests that phosducin with its C-terminal domain to the of the its is the of the Gβγ with In a the of phosducin to be for Gβγ binding and is able to functions S. M. M. M.J. J. 1997; PubMed Scopus Google Scholar). Moreover, in this region markedly activity of phosducin S. M. M. M.J. J. 1997; PubMed Scopus Google Scholar). a of interaction R. A. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, C. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google and types of studies also indicate that the C-terminal of PhLP plays a role in Gβγ binding C. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, S. C. M.J. FEBS Lett. 1997; PubMed Scopus Google interaction with Gβγ subunits, phosducin and PhLP to with a variety of GPCR signaling by different G proteins S. M. S. M.J. PubMed Scopus Google Scholar, S. M.J. Sci. S. A. 1996; PubMed Scopus (63) Google Scholar, S. M.J. Sci. S. A. 1996; PubMed Scopus Google Scholar, J. Sci. S. A. PubMed Scopus Google Scholar, S. C. M.J. FEBS Lett. 1997; PubMed Scopus Google Scholar). The reported of phosducin by phosphorylation a regulatory in the of receptor signaling leading to in cellular phosphorylation may an additional for the of phosducin interaction with Gβγ subunits regulation of further from that by in important The phosphorylation of phosducin by and GRK2 is independent and at different GRK2 phosducin the of is to GRK2 also These data suggest independent of phosducin functions by the different types of In this report, we phosducin and PhLP as new substrates for GRK2. In with recent that show that is phosphorylated by this kinase J.A. Daaka Y. J. Ferguson S.S.G. S. S. Caron Lefkowitz R.J. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, Benovic J.L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google data further indicate the of substrates for GRK2. The that the activity of this kinase is to agonist-occupied GPCR suggests that GRK2 may also act as an effector of GPCR signaling and additional cellular of the that phosphorylation of phosducin proteins may The phosphorylation reactions rapidly and The apparent Km for phosducin and for that reported for substrates of GRK2 in the range of J.L. Caron Lefkowitz R.J. J. Biol. Chem. Full Text PDF PubMed Google and with the Km for phosphorylation of Refs. J.A. Daaka Y. J. Ferguson S.S.G. S. S. Caron Lefkowitz R.J. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, Benovic J.L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google and a such as the receptor J.L. Mayor Jr., F. C. Lefkowitz R.J. Caron J. Biol. Chem. Full Text PDF PubMed Google Scholar). The Km data for phosducin and PhLP and the of are also the range of the reported cellular of phosducin S. M.J. Sci. S. A. 1996; PubMed Scopus Google and GRK2 Benovic J.L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). indicate that phosducin and PhLP are substrates for GRK2. the interaction of GRK2 with agonist-occupied receptors and Gβγ subunits promotes an activation of the kinase Benovic J.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google phosducin phosphorylation by GRK2 is in the of suggesting that is the receptor that promotes phosducin be to the mechanisms of this phosphorylation process in and its by GPCR-mediated signaling Our indicate that the critical for GRK2 phosphorylation are the C-terminal domain of both phosducin and PhLP (between residues and 245 and and 218, suggesting a regulatory in which these the phosphorylation for the of the kinase. These and residues in the of with that GRK2 substrates residues to the phosphorylation site Caron Lefkowitz R.J. Benovic J.L. PubMed Scopus Google Scholar). These C-terminal are in the interaction of phosducin and PhLP with its the Gβγ subunits of heterotrimeric G proteins. The of phosducin with the subunits of R. A. 1996; Full Text Full Text PDF PubMed Scopus Google suggests that phosducin with its C-terminal domain to the of the its is the of the Gβγ with In a the of phosducin to be for Gβγ binding and is able to functions S. M. M. M.J. J. 1997; PubMed Scopus Google Scholar). Moreover, in this region markedly activity of phosducin S. M. M. M.J. J. 1997; PubMed Scopus Google Scholar). a of interaction R. A. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, C. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google and types of studies also indicate that the C-terminal of PhLP plays a role in Gβγ binding C. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, S. C. M.J. FEBS Lett. 1997; PubMed Scopus Google Scholar). interaction with Gβγ subunits, phosducin and PhLP to with a variety of GPCR signaling by different G proteins S. M. S. M.J. PubMed Scopus Google Scholar, S. M.J. Sci. S. A. 1996; PubMed Scopus (63) Google Scholar, S. M.J. Sci. S. A. 1996; PubMed Scopus Google Scholar, J. Sci. S. A. PubMed Scopus Google Scholar, S. C. M.J. FEBS Lett. 1997; PubMed Scopus Google Scholar). The reported of phosducin by phosphorylation a regulatory in the of receptor signaling leading to in cellular phosphorylation may an additional for the of phosducin interaction with Gβγ subunits regulation of further from that by in important The phosphorylation of phosducin by and GRK2 is independent and at different GRK2 phosducin the of is to GRK2 also These data suggest independent of phosducin functions by the different types of The for critical of the M. and A. for and F. Mayor for