Regulation of G Protein-coupled Receptor Kinases by Caveolin
1999; Elsevier BV; Volume: 274; Issue: 13 Linguagem: Inglês
10.1074/jbc.274.13.8858
ISSN1083-351X
AutoresChristopher V. Carman, Michael P. Lisanti, Jeffrey Benovic,
Tópico(s)Ion channel regulation and function
ResumoG 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. 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Here, we investigate the interaction of GRKs with caveolin both in vitro and in intact cells, and we demonstrate a previously unappreciated mode of regulation for these kinases. Hemagglutinin (HA)-specific polyclonal antibody was from Babco. GRK1-, GRK5-, and caveolin-1-specific polyclonal antibodies were from Santa Cruz Biotechnology, and a caveolin-1-specific monoclonal antibody (2297) was from Transduction Laboratories, Inc. AP-2 α-subunit- and clathrin-specific antibodies were generously provided by Dr. J. H. Keen. Transferrin receptor-specific antibody was from Chemicon International. Maltose-binding protein (MBP) vector, pMAL, and amylose resin were from New England Biolabs. Most other reagents were from sources previously described (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 overexpressed in and purified from Sf9 insect cells (33Kim C.M. Dion S.B. Onorato J.J. Benovic J.L. Receptor. 1993; 3: 39-55PubMed Google Scholar, 34Kunapuli P. Onorato J.J. Hosey M.M. Benovic J.L. J. Biol. Chem. 1994; 269: 1099-1105Abstract Full Text PDF PubMed Google Scholar), and purified GRK1 was generously provided by Drs. J. Pitcher and R. J. Lefkowitz. Purified GST, GST-caveolin-1, GST-GRK2, and GST-GRK5 fusion proteins, and urea stripped rod outer segments were prepared as described previously (27Li S. Okamoto T. Chun M. Sargiacomo M. Casanova J.E. Hansen S.E. Nishimoto I. Lisanti M.P. J. Biol. Chem. 1995; 270: 15693-15701Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar, 35Pronin A.N. Satpaev D.K. Slepak V.Z. Benovic J.L. J. Biol. Chem. 1997; 272: 18273-18280Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 36Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar). Sf9 expressed and purified Gβ1γ2 and GRK2 PH domain were generously provided by Dr. S. P. Kennedy. An MBP-caveolin-1 fusion construct containing caveolin residues 1–101 (MBP-caveolin-11–101) was generated by polymerase chain reaction amplification of base pairs 1–303 of the caveolin-1 cDNA. The product was then subcloned into the pMal vector in-frame with the upstream MBP via EcoRI and HindIII restriction sites in the polylinker region. Purified MBP and MBP-caveolin-1 fusion proteins were generated as described previously (37Vojtek B.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1654) Google Scholar). SDS-PAGE was performed using standard methods (38Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205523) Google Scholar). Following electrophoresis, proteins were electroblotted onto nitrocellulose. Immunoblotting was performed using caveolin-1-, GRK1-, GRK2-, GRK5-, AP-2-, clathrin-, transferrin receptor-, Gβ-, and GST-specific primary antibodies, horseradish peroxidase-conjugated secondary antibody (1:2000 dilution), and visualization by ECL following the manufacturer's guidelines. Five μg of purified GST or GST-caveolin-1 fusion proteins containing either the N-terminal residues 1–61 (GST-caveolin1–61) or membrane proximal residues 61–101 (GST-caveolin61–101) immobilized on glutathione-agarose beads were incubated with 2 μg of purified GRK1, GRK2, GRK2 PH domain (GRK2 residues 553–670), GRK5 or Gβ1γ2 in 100 μl of binding buffer (20 mm Tris-HCl, pH 7.5, 5 mm EDTA, 100 mm NaCl and either 0.02% or 1% Triton X-100) at 30 °C for 60 min. The samples were chilled on ice for 5 min, and the beads were then pelleted in a microcentrifuge for 10 s, washed three times with 400 μl of binding buffer, and boiled with SDS sample buffer. Samples were subjected to 10% SDS-PAGE and immunoblotting using GRK- or Gβ-specific antibodies. One μg of purified MBP or MBP-caveolin-11–101 immobilized on amylose resin was incubated with 200 ng of purified GRKs or soluble GST, GST-GRK2 (residues 1–184, 1–147, 1–122, 1–88, 1–63, 70–184, 80–184, 87–184, 94–184, 185–467, or 468–689), or GST-GRK5 (residues 1–200, 1–98, 1–39, 20–49, or 489–590) fusion proteins in 100 μl of binding buffer (20 mm Tris-HCl, pH 7.5, 5 mmEDTA, 100 mm NaCl, and 0.2% Triton X-100) at 30 °C for 60 min. Washing, elution, and analysis were identical to GST-caveolin binding experiments with the exception that GST fusion proteins were immunoblotted with a GST-specific antibody. COS-1, A431 and NIH-3T3 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (or 10% bovine calf serum for NIH-3T3 cells), 100 units/ml penicillin G, and 100 μg/ml streptomycin sulfate at 37 °C in a humidified atmosphere containing 5% CO2. COS-1 cells grown to 75–95% confluence in a 100-mm dish were transfected with 20 μg total of pcDNA3, pcDNA3-GRK2, and pCB7-caveolin-1 DNA using FugeneTM following the manufacturer's instructions. Five 150-mm dishes of A431 or NIH-3T3 cells were grown to confluence, and caveolin-rich fractions were generated by a detergent-free sodium carbonate method, essentially as described previously (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). Briefly, dishes were placed on ice for 5 min, rinsed three times with ice-cold phosphate-buffered saline, and then scraped into ∼1.5 ml of 500 mm sodium carbonate, pH 11.0 buffer containing protease inhibitors (1 mmphenylmethylsulfonyl fluoride, 20 μg/ml benzamidine, and 10 μg/ml aprotinin). Cells were then subjected to 15 strokes with a Dounce homogenizer, three 20-s bursts with a Brinkman Polytron (2500 rpm), and three 30-s bursts with a tip sonicator, all on ice. The extracts were diluted 1:2 with 90% sucrose in MES-buffered saline, overlaid with 6 ml of 35% sucrose/MES-buffered saline and 3 ml of 5% sucrose/MES-buffered saline, and centrifuged at 4 °C for ∼16 h at 39,000 rpm in a Beckman SW41 rotor. After centrifugation, nine 1.3-ml fractions were collected, and aliquots of each were subjected to SDS-PAGE and immunoblotting using either caveolin-1-, GRK2-, AP-2-, clathrin-, or transferrin receptor-specific antibodies. COS-1 cells, co-transfected with pcDNA3-GRK2 and pCB7-caveolin-1, were rinsed with ice-cold phosphate-buffered saline 24 h after transfection and harvested by addition of 1 ml of extraction buffer (10 mm Tris-HCl, pH 7.4, 1 mm EDTA, 5 mm dithiothreitol, 100 mm NaCl, 1.0% Triton X-100, 60 mm octyl glucoside, 1 mm phenylmethylsulfonyl fluoride, 20 μg/ml benzamidine, and 10 μg/ml each of leupeptin, pepstatin A and aprotinin) and rocking at 4 °C for 30 min. The cells were scraped, vortexed 5 times, and homogenized with two 15-s bursts with a Brinkman Polytron (2500 rpm). Lysates were centrifuged at 4 °C for 10 min at maximum speed in a microcentrifuge, and the supernatant was removed. For immunoprecipitation, 100 μl of supernatant was incubated with either a GRK2-, caveolin-1, or HA-specific polyclonal antibody or a transferrin receptor-specific monoclonal antibody for 30 min at 4 °C followed by addition of 50 μl of 50% protein A-agarose pre-equilibrated in extraction buffer and an additional 60 min incubation at 4 °C. Samples were centrifuged for 10 s, pellets were washed three times for 30 min at 4 °C with extraction buffer, and bound proteins were eluted with 50 μl of SDS sample buffer and boiling for 10 min. Samples were subjected to 10% SDS-PAGE and immunoblotting using GRK2-, caveolin-1-, and transferrin receptor-specific antibodies. Rhodopsin phosphorylation reactions contained, in a total volume of 20 μl, 30 nm GRK1, GRK2, or GRK5, 400 nm rhodopsin, 100 μm[γ-32P]ATP (5 cpm/fmol), 20 mm Tris-HCl, pH 7.5, 2 mm EDTA, 7.5 mm MgCl2, and either Me2SO (vehicle) or 0.1–12.5 μmcaveolin peptides (17Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (667) Google Scholar, 30Michel J.B. Feron O. Sase K. Prabhakar P. Michel T. J. Biol. Chem. 1997; 272: 25907-25912Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar) in Me2SO. Reactions were incubated at 37 °C for 5 min, stopped with SDS sample buffer, and subjected to 10% SDS-PAGE. After autoradiography the32P-labeled rhodopsin bands were excised and counted. Peptide phosphorylation reactions were identical except they contained 0.1–10 mm peptide substrate (RRREEEEESAAA) instead of rhodopsin and 50 nm GRK2 in a total volume of 100 μl. Reactions were incubated at 30 °C for either 20 or 60 min, blotted to P81 paper, washed 5 times with 75 mm phosphoric acid, and counted. Since many of the proteins involved in G protein-coupled receptor signaling associate with caveolae and/or caveolin, we analyzed the primary sequence of GRK2 and found that a consensus caveolin binding motif (φXφXXXXφ, φXXXXφXXφ, or φXφXXXXφXXφ; where φ is an aromatic residue (32Couet J. Li S. Okamoto T. Ikezu T. Lisanti M.P. J. Biol. Chem. 1997; 272: 6525-6533Abstract Full Text Full Text PDF PubMed Scopus (797) Google Scholar)) exists within the C-terminal PH domain (576WQRRYFYQF584). To test whether GRK2 can bind to caveolin, purified GRK2 was incubated with GST, GST-caveolin1–61, and GST-caveolin61–101 fusion proteins immobilized on glutathione-agarose. This analysis demonstrated specific binding of GRK2 (∼15% of the total) to GST-caveolin61–101 (Fig.1), a protein that contains the caveolin scaffolding domain (residues 81–101) previously implicated in caveolin interaction with other signaling molecules such as the epidermal growth factor receptor, c-Src, PKC, and endothelial nitric-oxide synthase (12Couet J. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1997; 272: 30429-30438Crossref PubMed Scopus (538) 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, 28Oka N. Yamamoto M. Schwencke C. Kawabe J. Ebina T. Ohno S. Couet J. Lisanti M.P. Ishikawa Y. J. Biol. Chem. 1997; 272: 33416-33421Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 30Michel J.B. Feron O. Sase K. Prabhakar P. Michel T. J. Biol. Chem. 1997; 272: 25907-25912Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Importantly, there was minimal binding to GST alone (data not shown) or GST-caveolin1–61 (Fig. 1). Moreover, GRK2 binding to GST-caveolin61–101 was modestly enhanced at higher ionic strength (data not shown), consistent with the binding being primarily mediated by hydrophobic interactions. Given the localization of the identified consensus caveolin binding sequence to the PH domain, we also analyzed the direct binding of purified GRK2 PH domain and observed significant and specific binding to GST-caveolin61–101 (data not shown). Interestingly, GRK1 and GRK5, which lack a PH domain, also displayed specific binding to GST-caveolin61–101 (although a low level of binding to GST-caveolin1–61 was also observed for GRK5) (Fig. 1). To test further the binding specificity, similar experiments were performed in the presence of 1% Triton X-100 in order to minimize the role of nonspecific hydrophobic interactions. Whereas the overall extent of binding was slightly reduced for all of the GRKs tested, GRK1, GRK2, GRK5, and the GRK2-PH domain, retained specific binding to GST-caveolin61–101 (data not shown). To demonstrate further the specificity of GRK/caveolin interactions, we tested the binding of purified Gβ1γ2 to the GST-caveolin-1 constructs. Despite modification with an extremely hydrophobic geranylgeranyl moiety, Gβ1γ2did not significantly interact with either GST-caveolin1–61 or GST-caveolin61–101 (Fig.1). These results demonstrate that the observed GRK/caveolin binding is not simply due to nonspecific hydrophobic interactions. Overall, these data suggest that caveolin binding may be a common feature of GRKs and that a conserved caveolin binding motif may be present. Analysis of the aromatic residues present in GRKs failed to reveal conserved sequences that strictly fit the previously identified consensus caveolin binding motifs (32Couet J. Li S. Okamoto T. Ikezu T. Lisanti M.P. J. Biol. Chem. 1997; 272: 6525-6533Abstract Full Text Full Text PDF PubMed Scopus (797) Google Scholar). However, three potential caveolin binding motifs were identified in all GRKs as follows:63LXXXXφXXφ71,162φXXφXXXXXφXXφXXφ177, and 375φXLXXXXφ382. In order to map caveolin-binding determinants in GRKs, amylose resins containing either purified MBP or an MBP-caveolin1–101fusion protein were generated and incubated with either full-length GRKs or various soluble GST-GRK2 or GST-GRK5 fusion proteins. Whereas full-length GRK1, GRK2, and GRK5 failed to interact with MBP, they each bound to MBP-caveolin1–101 similar to their demonstrated binding to GST-caveolin61–101 (data not shown). Analysis of GST-GRK2 fusion proteins containing the N terminus (residues 1–184), catalytic domain (residues 185–467), and C terminus (residues 468–689) were then tested. This revealed specific binding of GST-GRK2468–689 (Fig. 2) in agreement with the observed binding of purified GRK2 PH domain to GST-caveolin61–101 mentioned above. Interestingly, GST-GRK21–184 also bound significantly to MBP-caveolin1–101, whereas GST-GRK2184–467 exhibited weak binding to both MBP-caveolin1–101 and MBP, suggesting that this interaction is not specific (Fig. 2). Importantly, neither GST alone nor any of the other GST-GRK fusion proteins tested bound to the MBP-amylose resin under these conditions (data not shown). To map further the caveolin-binding determinants within the N terminus of GRK2, GST-GRK21–184 was progressively truncated, and the resulting fusions were analyzed for caveolin binding. GST-GRK21–147, GST-GRK21–122, and GST-GRK21–88 all retained binding to MBP-caveolin1–101, whereas GST-GRK21–63failed to bind (Fig. 2). Additional GST-GRK2 fusions (GST-GRK270–184, GST-GRK280–184, GST-GRK287–184, and GST-GRK294–184) were also tested, and all failed to bind MBP-caveolin1–101. This suggests that the critical binding determinants lie between residues 63 and 70 in GRK2. Indeed, residues 63–71 were initially identified as a conserved domain with similarity to the consensus sequences for caveolin binding (Fig. 2 C). In order to determine if this N-terminal domain was responsible for the conserved GRK/caveolin interactions, the binding of several GST-GRK5 fusion proteins to MBP-caveolin1–101 was also tested. Initial analysis of an N-terminal construct (GST-GRK51–200) demonstrated significant and specific binding to MBP-caveolin1–101, whereas a C-terminal construct (GST-GRK5489–590) failed to bind (Fig. 2). To map further the N-terminal caveolin-binding site, GST-GRK51–98, GST-GRK51–39, and GST-GRK520–49 were analyzed. Although GST-GRK51–98 retained binding to MBP-caveolin1–101, GST-GRK51–39 and GST-GRK520–49 failed to bind (Fig. 2) suggesting that the critical caveolin binding region in GRK5 lies between residues 49 and 98, a region that overlaps the N-terminal caveolin binding region identified in GRK2. These data suggest that the N-terminal region, including GRK2 residues 63–71 (LXXXXφXXφ) (Fig. 2 C), is important for the conserved GRK/caveolin binding characteristics. The initial study that proposed caveolin binding motifs (φXφXXXXφ, φXXXXφXXφ, or φXφXXXXφXXφ) used the caveolin scaffolding domain to select random peptide ligands from phage display libraries (32Couet J. Li S. Okamoto T. Ikezu T. Lisanti M.P. J. Biol. Chem. 1997; 272: 6525-6533Abstract Full Text Full Text PDF PubMed Scopus (797) Google Scholar). Interestingly, in that study nearly 10% of the selected 15-mer peptides contained only two aromatic residues. Additionally, whereas a generally conserved caveolin consensus motif, including four aromatic residues (φXφXXXXφXXφ) has been identified in Gα subunits, the Gαq subunit has substitutions of valine and leucine for two of these aromatic residues (32Couet J. Li S. Okamoto T. Ikezu T. Lisanti M.P. J. Biol. Chem. 1997; 272: 6525-6533Abstract Full Text Full Text PDF PubMed Scopus (797) Google Scholar). Despite this substitution, Gαq has been shown to co-immunoprecipitate with caveolin (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). Thus, it seems possible that caveolin binding requirements may be broader than initially described. Finally, although the functional significance for the existence of two caveolin binding regions in GRK2 (residues 63–71 and the PH domain) remains to be elucidated, it is of interest that the PH domain-localized motif overlaps with a region that includes phospholipid-binding determinants (39Harlan J.E. Hadjuk P.J. Yoon H.S. Fesik S.W. Nature. 1994; 371: 168-170Crossref PubMed Scopus (666) Google Scholar). Two approaches were used to ascertain whether GRK2 and caveolin associate in intact cells. The first employed a widely used extraction and fractionation method that enables the separation of caveolae from other cellular organelles (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). For these studies, A431 cells were lysed in a detergent-free sodium carbona
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