Regulation of G Protein-coupled Receptor Kinases by Caveolin

G protein-coupled receptor kinases (GRKs) have been principally characterized by their ability to phosphorylate and desensitize G protein-coupled receptors. However, recent studies suggest that GRKs may have more diverse protein/protein interactions in cells. Based on the identification of a consensus caveolin binding motif within the pleckstrin homology domain of GRK2, we tested the direct binding of purified full-length GRK2 to various glutathioneS-transferase-caveolin-1 fusion proteins, and we discovered a specific interaction of GRK2 with the caveolin scaffolding domain. Interestingly, analysis of GRK1 and GRK5, which lack a pleckstrin homology domain, revealed in vitro binding properties similar to those of GRK2. Maltose-binding protein caveolin and glutathione S-transferase-GRK fusion proteins were used to map overlapping regions in the N termini of both GRK2 and GRK5 that appear to mediate conserved GRK/caveolin interactions. In vivo association of GRK2 and caveolin was suggested by co-fractionation of GRK2 with caveolin in A431 and NIH-3T3 cells and was further supported by co-immunoprecipitation of GRK2 and caveolin in COS-1 cells. Functional significance for the GRK/caveolin interaction was demonstrated by the potent inhibition of GRK-mediated phosphorylation of both receptor and peptide substrates by caveolin-1 and -3 scaffolding domain peptides. These data reveal a novel mode for the regulation of GRKs that is likely to play an important role in their cellular function. G protein-coupled receptor kinases (GRKs) have been principally characterized by their ability to phosphorylate and desensitize G protein-coupled receptors. However, recent studies suggest that GRKs may have more diverse protein/protein interactions in cells. Based on the identification of a consensus caveolin binding motif within the pleckstrin homology domain of GRK2, we tested the direct binding of purified full-length GRK2 to various glutathioneS-transferase-caveolin-1 fusion proteins, and we discovered a specific interaction of GRK2 with the caveolin scaffolding domain. Interestingly, analysis of GRK1 and GRK5, which lack a pleckstrin homology domain, revealed in vitro binding properties similar to those of GRK2. Maltose-binding protein caveolin and glutathione S-transferase-GRK fusion proteins were used to map overlapping regions in the N termini of both GRK2 and GRK5 that appear to mediate conserved GRK/caveolin interactions. In vivo association of GRK2 and caveolin was suggested by co-fractionation of GRK2 with caveolin in A431 and NIH-3T3 cells and was further supported by co-immunoprecipitation of GRK2 and caveolin in COS-1 cells. Functional significance for the GRK/caveolin interaction was demonstrated by the potent inhibition of GRK-mediated phosphorylation of both receptor and peptide substrates by caveolin-1 and -3 scaffolding domain peptides. These data reveal a novel mode for the regulation of GRKs that is likely to play an important role in their cellular function. G protein-coupled receptor kinases (GRKs) 1The abbreviations used are:GRK, G protein-coupled receptor kinase; PKC, protein kinase C; PH, pleckstrin homology; MBP, maltose-binding protein; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; HA, hemagglutinin; MES, 4-morpholineethanesulfonic acid.1The abbreviations used are:GRK, G protein-coupled receptor kinase; PKC, protein kinase C; PH, pleckstrin homology; MBP, maltose-binding protein; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; HA, hemagglutinin; MES, 4-morpholineethanesulfonic acid. phosphorylate the agonist-activated form of G protein-coupled receptors that in turn promotes the high affinity binding of arrestins (1Carman C.V. Benovic J.L. Curr. Opin. Neurobiol. 1998; 8: 335-344Crossref PubMed Scopus (230) Google Scholar). This process functions to both uncouple the receptor from the G protein and to promote receptor internalization via clathrin-coated pits. The activity and cellular localization of GRKs appear to be regulated by a variety of molecules including activated receptors, Gβγ subunits, phosphatidylinositol 4,5-bisphosphate, PKC, and calmodulin (1Carman C.V. Benovic J.L. Curr. Opin. Neurobiol. 1998; 8: 335-344Crossref PubMed Scopus (230) Google Scholar). Many of these interactions are thought to be important largely for their ability to regulate interaction of GRKs with the plasma membrane where receptor substrates reside. Recent studies have provided novel information regarding the function and cellular localization of GRKs. For example, it was shown that GRK2 can traffic along with β2-adrenergic receptors to the endosome following receptor activation (2Ruiz-Gomez A. Mayor Jr., F. J. Biol. Chem. 1997; 272: 9601-9604Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Mayor and co-workers (3Murga C. Ruiz-Gomez A. Garcia-Higuera I. Kim C.M. Benovic J.L. Mayor Jr., F. J. Biol. Chem. 1996; 271: 985-994Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) have also demonstrated an association of GRK2 with microsomes that appears to be mediated via an unidentified GRK2-binding protein. In addition, we and others (4Carman C.V. Som T. Kim C.M. Benovic J.L. J. Biol. Chem. 1998; 273: 20308-20316Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 5Pitcher J.A. Hall R.A. Daaka Y. Zhang J. Ferguson S.S.G. 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 (133) Google Scholar, 6Freeman J.L.R. De La Cruz E.M. Pollard T.D. Lefkowitz R.J. Pitcher J.A. J. Biol. Chem. 1998; 273: 20653-20657Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) have recently demonstrated novel interactions between GRKs and the cytoskeleton. Collectively, these studies suggest that the function and regulation of GRKs may involve diverse protein/protein interactions. Caveolae represent distinct cholesterol- and glycosphingolipid-enriched plasma membrane and vesicular structures in cells that function in a variety of cellular processes including endothelial transcytosis and potocytosis (7Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1336) Google Scholar). Caveolin, a 22–24-kDa integral membrane protein composed of cytoplasmic N and C termini and a central intramembrane domain, is thought to be a major structural component of caveolae (7Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1336) Google Scholar). A 20-amino acid juxtamembrane region (the scaffolding domain) within the N-terminal domain has been shown to mediate the association of caveolin with other proteins (8Couet J. Shengwen L. Okamoto T. Scherer P.E. Lisanti M.P. Trends Cardiovasc. Med. 1997; 7: 103-110Crossref PubMed Scopus (111) Google Scholar). Recently, a wide variety of cellular signaling molecules have been shown to associate with caveolae leading to the hypothesis that caveolae may serve as cell-surface microdomains that concentrate and organize cellular signaling pathways (7Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1336) Google Scholar, 8Couet J. Shengwen L. Okamoto T. Scherer P.E. Lisanti M.P. Trends Cardiovasc. Med. 1997; 7: 103-110Crossref PubMed Scopus (111) Google Scholar). Whereas some of the initial data supporting this hypothesis was derived from cell fractionation methods that may be less specific than originally thought (9Stan R.V. Roberts W.G. Predescu D. Ihida K. Saucan L. Ghitescu L. Palade G.E. Mol. Biol. Cell. 1997; 8: 595-605Crossref PubMed Scopus (176) Google Scholar, 10Huang C. Hepler J.R. Chen L.T. Gilman A.G. Anderson R.G.W. Mumby S.M. Mol. Biol. Cell. 1997; 8: 2365-2378Crossref PubMed Scopus (188) Google Scholar, 11Waugh M.G. Lawson D. Tan S.K. Hsuan J.J. J. Biol. Chem. 1998; 273: 17115-17121Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), more recent studies have demonstrated interactions between signaling molecules and caveolin using a variety of methods including immunoprecipitation, immunofluorescence microscopy, immunogold electron microscopy, and in vitrobinding. These studies reveal that many proteins involved in mitogenic signaling cascades, including the epidermal growth factor, platelet-derived growth factor, insulin and Neu (c-ErbB2) receptors, c-Src, Fyn, Erk-2, and Ras, associate with caveolin (12Couet J. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1997; 272: 30429-30438Crossref PubMed Scopus (538) Google Scholar, 13Liu P. Ying Y. Ko Y.-G. Anderson R.G.W. J. Biol. Chem. 1996; 271: 10299-10303Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 14Liu P. Ying Y. Anderson R.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13666-13670Crossref PubMed Scopus (190) Google Scholar, 15Yamamoto M. Toya Y. Schwencke C. Lisanti M.P. Myers Jr., M.G. Ishikawa Y. J. Biol. Chem. 1998; 273: 26962-26968Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 16Engelman J.A. Lee R.J. Karnezis A. Bearss D.J. Webster M. Siegel P. Muller W.J. Windle J.J. Pestell R.G. Lisanti M.P. J. Biol. Chem. 1998; 273: 20448-20455Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 17Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (667) Google Scholar, 18Song K. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (915) Google Scholar, 19Wary K.K. Margiotti A. Zurzolo C. Giancotti F.G. Cell. 1998; 94: 625-634Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar). Similarly, various G protein-coupled signaling components including receptors (β2-adrenergic, m2-muscarinic, B2-bradykinin, cholecystokinin, ETA-endothelin, calcium-sensing and angiotensin II receptors), G proteins (Gsα, Giα, Goα, and Gqα) and various downstream effector molecules (adenylyl cyclase, PKCα, -Y, -ε, and -ζ, and endothelial and neuronal nitric-oxide synthase) have been shown to interact with caveolin suggesting a potential role of caveolae in regulating such pathways (20Dupree P. Parton R.G. Raposo G. Kurzchalia T.V. Simons K. EMBO J. 1993; 12: 1597-1605Crossref PubMed Scopus (399) Google Scholar, 21Feron O. Smith T.W. Michel T. Kelly R.A. J. Biol. Chem. 1997; 272: 17744-17748Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 22de Weerd W.F.C. Leeb-Lundberg L.M.F. J. Biol. Chem. 1997; 272: 17858-17866Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 23Roettger B.F. Rentsch R.U. Pinon D. Holicky E. Hadac E. Larkin J.M. Miller L. J. Cell Biol. 1995; 128: 1029-1041Crossref PubMed Scopus (202) Google Scholar, 24Kifor O. Diaz R. Butters R. Kifor I. Brown E.M. J. Biol. Chem. 1998; 273: 21708-21713Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 25Chun M. Liyanage U.K. Lisanti M. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, K.K. 1998; PubMed Scopus Google Scholar, S. Okamoto T. M. Sargiacomo M. I. Lisanti M.P. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, M. Schwencke C. J. T. S. Couet J. Lisanti M.P. Ishikawa Y. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Schwencke C. Couet J. Lisanti M. Ishikawa Y. 1998; PubMed Google Scholar, O. K. P. Michel T. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, R. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). GRKs also play an important role in regulating G protein-coupled signaling we the of the GRKs for consensus caveolin binding J. Li S. Okamoto T. T. Lisanti M.P. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). GRK2 and -3 were to a pleckstrin homology consensus caveolin binding we the interaction of GRKs with caveolin both in vitro and in and we a mode of regulation for these was from and were from Cruz and a was from and were provided by J. was from Maltose-binding protein and were from other were from (4Carman C.V. Som T. Kim C.M. Benovic J.L. J. Biol. Chem. 1998; 273: 20308-20316Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). GRK2 and GRK5 were in and purified from cells C.M. J.J. Benovic J.L. 1993; Google Scholar, P. J.J. Benovic J.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and purified GRK1 was provided by J. Pitcher and R. J. GST, and fusion proteins, and were as S. Okamoto T. M. Sargiacomo M. I. Lisanti M.P. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, Benovic J.L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, Benovic J.L. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). and purified and GRK2 domain were provided by S. P. fusion caveolin was by of of the caveolin-1 The was the with the via and in the and fusion proteins were as S.M. J.A. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar). was using methods U.K. PubMed Scopus Google Scholar). proteins were was using and and by following the of purified fusion proteins the N-terminal membrane on were with of purified GRK2, GRK2 domain GRK5 in of binding and for The were on for and the were in a for with of binding and with were to and using of purified on was with of purified GRKs GST, fusion proteins in of binding and for and analysis were to binding with the that fusion proteins were with a A431 and NIH-3T3 cells were in with for NIH-3T3 and in a COS-1 cells to in a were with of and using following the of A431 NIH-3T3 cells were to and were by a as K. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (915) Google Scholar). were on for with and of and were to with a with a and with a on The were with in with of and of and for in a were and of were to and using COS-1 with and were with and by of of and of A and and for The cells were and with with a were for in a and the was For immunoprecipitation, of was with a a for by of of protein in and an were for were for with and proteins were with of and for were to and using and phosphorylation in a of GRK2, GRK5, and S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (667) Google Scholar, O. K. P. Michel T. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) in were for with and to were and phosphorylation were peptide of and GRK2 in a of were for to with and many of the proteins involved in G protein-coupled receptor signaling associate with caveolae we the of GRK2 and that a consensus caveolin binding motif where is an J. Li S. Okamoto T. T. Lisanti M.P. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google within the domain GRK2 can to purified GRK2 was with GST, and fusion proteins on This analysis demonstrated specific binding of GRK2 of the to a protein that the caveolin scaffolding domain in caveolin interaction with other signaling molecules such as the epidermal growth c-Src, PKC, and endothelial nitric-oxide (12Couet J. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1997; 272: 30429-30438Crossref PubMed Scopus (538) Google S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (667) Google Scholar, M. Schwencke C. J. T. S. Couet J. Lisanti M.P. Ishikawa Y. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, O. K. P. Michel T. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). was binding to GRK2 binding to was with the binding mediated by interactions. the localization of the consensus caveolin binding to the domain, we also the direct binding of purified GRK2 domain and and specific binding to Interestingly, GRK1 and GRK5, which lack a domain, also specific binding to a of binding to was also for further the binding similar were in the of in to the role of interactions. Whereas the of binding was for of the GRKs GRK2, GRK5, and the domain, specific binding to further the of GRK/caveolin we tested the binding of purified to the with an interact with These that the GRK/caveolin binding is to interactions. these data suggest that caveolin binding may be a of GRKs and that a conserved caveolin binding motif may be of the in GRKs to reveal conserved that the consensus caveolin binding J. Li S. Okamoto T. T. Lisanti M.P. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). However, potential caveolin binding were in GRKs as and In to map in purified an protein were and with full-length GRKs various fusion Whereas full-length GRK2, and GRK5 to interact with MBP, to similar to their demonstrated binding to of fusion proteins the N domain and C were This revealed specific binding of in with the binding of purified GRK2 domain to Interestingly, also to binding to both and MBP, suggesting that this interaction is specific of the other fusion proteins tested to the these map further the within the N of GRK2, was and the were for caveolin and binding to to and were also and to This that the binding between and in GRK2. were as a conserved domain with to the consensus for caveolin binding In to this N-terminal domain was for the conserved GRK/caveolin the binding of fusion proteins to was also analysis of an N-terminal demonstrated and specific binding to a to map further the N-terminal and were binding to and to suggesting that the caveolin binding region in GRK5 between and a region that the N-terminal caveolin binding region in GRK2. These data suggest that the N-terminal including GRK2 is important for the conserved GRK/caveolin binding The initial that caveolin binding used the caveolin scaffolding domain to peptide from J. Li S. Okamoto T. T. Lisanti M.P. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). Interestingly, in that of the a conserved caveolin consensus including has been in subunits, the has of and for of these J. Li S. Okamoto T. T. Lisanti M.P. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). this has been shown to with caveolin Weerd W.F.C. Leeb-Lundberg L.M.F. J. Biol. Chem. 1997; 272: 17858-17866Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). it that caveolin binding may be than the significance for the of caveolin binding regions in GRK2 and the domain) to be it is of that the motif with a region that PubMed Scopus Google Scholar). were used to GRK2 and caveolin associate in cells. The a used and fractionation that the of caveolae from other cellular K. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (915) Google Scholar). For these A431 cells were in a on a and for caveolin and GRK2 by and the the of the cellular of the cellular protein was in and of GRK2 revealed that it was in and a also with caveolin A similar co-fractionation was in NIH-3T3 cells a of molecules to associate with a variety of cellular including the plasma the and the Cell Biol. Scopus Google Scholar, K. J. Cell Biol. 1993; PubMed Scopus Google Scholar) were to be to and these data that this fractionation a specific of cellular including caveolae However, recent studies have further characterized the and proteins in from these similar methods and demonstrated the of (9Stan R.V. Roberts W.G. Predescu D. Ihida K. Saucan L. Ghitescu L. Palade G.E. Mol. Biol. Cell. 1997; 8: 595-605Crossref PubMed Scopus (176) Google Scholar, 10Huang C. Hepler J.R. Chen L.T. Gilman A.G. Anderson R.G.W. Mumby S.M. Mol. Biol. Cell. 1997; 8: 2365-2378Crossref PubMed Scopus (188) Google Scholar, 11Waugh M.G. Lawson D. Tan S.K. Hsuan J.J. J. Biol. Chem. 1998; 273: 17115-17121Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). data are with localization of GRK2 in A431 and NIH-3T3 we the that GRK2 be with of these other In to a association in we used an In these COS-1 cells GRK2 and caveolin-1 were in the of and in to were with and by of protein and proteins were with and to and using and GRK2 and caveolin were by their specific to GRK2 GRK2 were with for a of caveolin was of the Similarly, caveolin with a GRK2 revealed a of GRK2 of the further the of this using a was This receptor to of caveolin GRK2 these data that a of the cellular GRK2 was with caveolin in these cells and the in and and the co-fractionation and co-immunoprecipitation of GRK2 and caveolin suggest that GRK/caveolin interactions are likely to is important to that many studies have to membrane and cell using a variety of In these studies was to be cytoplasmic with a with the membrane (2Ruiz-Gomez A. Mayor Jr., F. J. Biol. Chem. 1997; 272: 9601-9604Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, Benovic J.L. Caron M.G. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, I. J. S. W.J. Lefkowitz R.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the of the cellular GRK2 with the and with caveolin likely a of the GRK2. such as GRK-mediated receptor may be by GRK/caveolin In to the potential significance of the GRK/caveolin we that activity may be regulated by the caveolin scaffolding domain in a similar to the demonstrated inhibition of the epidermal growth receptor (12Couet J. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1997; 272: 30429-30438Crossref PubMed Scopus (538) Google Scholar), S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (667) Google Scholar), M. Schwencke C. J. T. S. Couet J. Lisanti M.P. Ishikawa Y. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar), Y. Schwencke C. Couet J. Lisanti M. Ishikawa Y. 1998; PubMed Google Scholar), and endothelial nitric-oxide O. K. P. Michel T. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). In to this GRK2 phosphorylation of the receptor was in the of scaffolding domain from -3 a In addition, the scaffolding domain are we also a caveolin-1 scaffolding domain peptide in these studies O. K. P. Michel T. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) the acid of this peptide is to the caveolin-1 scaffolding domain it between binding and interactions. In these studies caveolin-1 and scaffolding domain GRK2 activity in a with of and In the scaffolding domain and on GRK2 phosphorylation of of the other GRKs revealed that GRK1 was also by caveolin-1 and -3 scaffolding domain with of and as was GRK5 with of and Similarly, GRK1 and GRK5 activity were by the scaffolding domain peptides. for of the GRKs tested the caveolin-1 peptide was to be less receptor phosphorylation that this peptide is in and in it is that this peptide is with these that the inhibition of GRK-mediated receptor phosphorylation by caveolin scaffolding domain is to GRK/caveolin is that the for caveolin peptide inhibition of phosphorylation is than the for This may be to the of the caveolin binding region in GRK2 within the domain. In the caveolin binding motif in the domain a region suggested to be important for binding PubMed Scopus Google Scholar). it is that caveolin with binding in inhibition of In the inhibition of GRKs be to inhibition of the domain direct inhibition of receptor binding to of the domain. In to between these we tested the of the caveolin scaffolding domain on GRK2 phosphorylation of the peptide In these caveolin demonstrated a similar for inhibition of peptide phosphorylation as with the with the caveolin-1 and -3 scaffolding domain inhibition with of and The caveolin-1 peptide GRK2 with an than than that of the caveolin-1 peptide These data suggest that the properties of the caveolin are specific and likely to be on the domain than by interactions of the domain. In to between and of analysis was by of peptide in the of caveolin-1 scaffolding domain of these data reveal a of a mode of inhibition Similarly, a of the peptide phosphorylation data of and for in the of and caveolin from to in the of and it appears that an mode of inhibition is likely to be the for caveolin inhibition of GRKs. This is with the of to of the domain. The that the and are by caveolin that the conserved N-terminal is likely to be important for this However, caveolin binding to the domain of GRK2 may to binding of to this domain of GRK2 has been shown to mediate on GRK2 activity J.J. Y. Benovic J.L. J. Biol. Chem. 1995; PubMed Scopus Google Scholar). suggest that GRKs may be to some of inhibition in cells that caveolin-1 This potential role may be of in the of of the in of including and receptor signaling M. M. Ishikawa Y. J. Mol. Cell. 1997; Full Text PDF Scopus Google Scholar, W.J. J.J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, P. T. M. J. 1997; 273: Google Scholar). also in a in GRK2 and GRK5 an that may to the in receptor signaling in W.J. J.J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, P. T. M. J. 1997; 273: Google Scholar). Interestingly, a recent demonstrated an in caveolin in from to K. Toya Y. Schwencke C. Ishikawa Y. J. 1997; 273: PubMed Google Scholar). it that in the a in and in caveolin have on activity and the of the In the data a novel interaction between GRKs and caveolin that in potent inhibition of These interactions are suggested to in vivo by the co-fractionation and co-immunoprecipitation of GRK2 and caveolin in cells. the cellular role of this interaction to be is that caveolin to is that localization of GRKs in caveolae may direct a distinct of protein/protein interactions regulating the cellular localization and the of protein kinase A is regulated in such a via interaction with a of scaffolding proteins Google Scholar, J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). this of novel protein interactions for as as further of the role of GRK/caveolin J. Pitcher and R. Lefkowitz for purified kinase; J. for and R. for of the fusion and A. for purified GRK5 and fusion proteins and for