Analysis of Ligand-stimulated CC Chemokine Receptor 5 (CCR5) Phosphorylation in Intact Cells Using Phosphosite-specific Antibodies
2003; Elsevier BV; Volume: 278; Issue: 4 Linguagem: Inglês
10.1074/jbc.m209844200
ISSN1083-351X
AutoresBeatrix Pollok‐Kopp, Katrin Schwarze, Viola Katharina Baradari, Martin Oppermann,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoHuman CC chemokine receptor 5 (CCR5), a member of the superfamily of G protein-coupled receptors, regulates the activation and directed migration of leukocytes and serves as the main coreceptor for the entry of R5 tropic strains of human immunodeficiency viruses. We have previously shown that RANTES/CCL5 binding to CCR5 induces GPCR kinase (GRK)- and protein kinase C (PKC)-mediated phosphorylation of four distinct C-terminal serine residues. To study these phosphorylation events in vivo, we have generated monoclonal antibodies, which specifically react only with either phosphorylated or nonphosphorylated CCR5. These phosphosite-specific antibodies reveal that following ligand stimulation of the receptor serine 337 is exclusively phosphorylated by a PKC-mediated mechanism, while GRKs phosphorylate serine 349. GRK-mediated receptor phosphorylation proceeds in a regular time-dependent manner (t 12 ∼2 min) with an apparent EC50 of 5 nm. In contrast, PKC phosphorylates serine 337 at 50-fold lower concentrations and in a very rapid, albeit transient manner. Protein phosphatases that are active at neutral pH and are inhibited by okadaic acid rapidly dephosphorylate phosphoserine 337, but less efficiently phosphoserine 349, in intact cells and in an in vitro assay. Immunofluorescence microscopy demonstrates that phosphorylated receptors accumulate in a perinuclear compartment, which resembles recycling endosomes. This study is the first to analyze in detail the spatial and temporal dynamics of GRK- versus PKC-mediated phosphorylation of a G protein-coupled receptor and its subsequent dephosphorylation on the level of individual phosphorylation sites. Human CC chemokine receptor 5 (CCR5), a member of the superfamily of G protein-coupled receptors, regulates the activation and directed migration of leukocytes and serves as the main coreceptor for the entry of R5 tropic strains of human immunodeficiency viruses. We have previously shown that RANTES/CCL5 binding to CCR5 induces GPCR kinase (GRK)- and protein kinase C (PKC)-mediated phosphorylation of four distinct C-terminal serine residues. To study these phosphorylation events in vivo, we have generated monoclonal antibodies, which specifically react only with either phosphorylated or nonphosphorylated CCR5. These phosphosite-specific antibodies reveal that following ligand stimulation of the receptor serine 337 is exclusively phosphorylated by a PKC-mediated mechanism, while GRKs phosphorylate serine 349. GRK-mediated receptor phosphorylation proceeds in a regular time-dependent manner (t 12 ∼2 min) with an apparent EC50 of 5 nm. In contrast, PKC phosphorylates serine 337 at 50-fold lower concentrations and in a very rapid, albeit transient manner. Protein phosphatases that are active at neutral pH and are inhibited by okadaic acid rapidly dephosphorylate phosphoserine 337, but less efficiently phosphoserine 349, in intact cells and in an in vitro assay. Immunofluorescence microscopy demonstrates that phosphorylated receptors accumulate in a perinuclear compartment, which resembles recycling endosomes. This study is the first to analyze in detail the spatial and temporal dynamics of GRK- versus PKC-mediated phosphorylation of a G protein-coupled receptor and its subsequent dephosphorylation on the level of individual phosphorylation sites. heterotrimeric GTP-binding protein-coupled receptor arbitrary unit CC chemokine receptor 5 enzyme-linked immunosorbent assay GPCR kinase human embryonic kidney 293 human immunodeficiency virus monoclonal antibody phosphorylated CCR5 protein kinase C phorbol 12-myristate 13-acetate released on activation normal T cell expressed and secreted (also known as CCL5) rat basophilic leukemia cells succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate phosphate-buffered saline fluorescein isothiocyanate bovine serum albumin high performance liquid chromatography 1,4-piperazinediethanesulfonic acid G protein-coupled receptors (GPCR)1 comprise the largest known family of signal-transducing proteins and respond to a large variety of external stimuli (1Bockaert J. Pin J.P. EMBO J. 1999; 18: 1723-1729Crossref PubMed Scopus (1233) Google Scholar, 2Pierce K.L. Premont R.T. Lefkowitz R.J. Nat. Rev. Mol. Cell. Biol. 2002; 3: 639-650Crossref PubMed Scopus (2125) Google Scholar). The receptors relay the information encoded by the ligand through the activation of heterotrimeric guanine nucleotide-binding proteins and intracellular effector molecules. Many GPCR undergo a process of rapid desensitization, which involves ligand-induced phosphorylation of serine and threonine residues located in the third intracellular loop or C-terminal domain by two different families of protein kinases. (i) GPCR kinases (GRKs) specifically phosphorylate only the agonist-occupied GPCR and thus mediate agonist-specific or homologous receptor phosphorylation (3Böhm S.K. Grady E.F. Bunnett N.W. Biochem. J. 1997; 322: 1-18Crossref PubMed Scopus (466) Google Scholar, 4Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1072) Google Scholar). (ii) In contrast, the second messenger-activated kinases, such as cyclic AMP-dependent protein kinase and protein kinase C (PKC), potentially phosphorylate both the ligand-bound GPCR and multiple other receptors in a heterologous manner. Receptor phosphorylation enhances the affinity of the agonist-occupied receptor for interaction with arrestin which interdicts signal transduction between the receptor and G proteins by steric mechanisms. The nonvisual arrestins, β-arrestin-1 and β-arrestin-2, also promote clathrin-mediated endocytosis of phosphorylated receptors and have been implicated in cross-talk with other signaling pathways. Once internalized, GPCR are targeted to recycling or degradative pathways (5Ferguson S.S. Pharmacol. Rev. 2001; 53: 1-24PubMed Google Scholar). Some GPCR are dephosphorylated by membrane-associated G protein-coupled receptor phosphatases and recycled rapidly to the plasma membrane where they can again respond to agonists while other receptors appear to be retained within the cell. Although reversible receptor phosphorylation is a well recognized mechanism that plays important roles in multiple aspects of GPCR signaling, with few exceptions the exact sites of second messenger kinase- and GRK-mediated receptor phosphorylation have not been identified. Most insights into receptor phosphorylation derive fromin vitro assays with purified proteins in reconstituted systems or from mutagenesis studies with elimination of the presumed consensus sites for receptor phosphorylation under experimental conditions of protein overexpression. Results obtained by these various methods can sometimes prove misleading, as illustrated by studies with the β2-adrenergic receptor (6Hausdorff W.P. Campbell P.T. Ostrowski J., Yu, S.S. Caron M.G. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2979-2983Crossref PubMed Scopus (139) Google Scholar, 7Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 13796-13803Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 8Seibold A. January B.G. Friedman J. Hipkin R.W. Clark R.B. J. Biol. Chem. 1998; 273: 7637-7642Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The observed discrepancies in the outcome of these studies are probably explained by unspecific effects of receptor mutagenesis and by the poor substrate specificity of receptor kinases in reconstituted systems. Furthermore, very little is known about the different kinetics and intracellular localization of agonist-induced GPCR phosphorylation, which is mediated by second messenger-dependent kinases versusGRKs at normal levels of protein expression. In the present study we have taken a new approach to the analysis of receptor phosphorylation in intact cells. By using phosphosite-specific monoclonal antibodies we determined with high temporal and spatial resolution the ligand-induced phosphorylation and dephosphorylation of CC chemokine receptor CCR5 at two separate phosphorylation sites. This receptor is well suited as a model protein for this kind of analysis since its function as cofactor for the entry of R5 tropic strains of human immunodeficiency viruses (HIV) has stimulated detailed investigations into the mechanisms that regulate CCR5 signaling and cell surface expression in recent years. In particular, this receptor was shown to be a potential substrate for all major GRK isoforms (9Aramori I. Zhang J. Ferguson S.S. Bieniasz P.D. Cullen B.R. Caron M.G. EMBO J. 1997; 16: 4606-4616Crossref PubMed Scopus (225) Google Scholar,10Oppermann M. Mack M. Proudfoot A.E. Olbrich H. J. Biol. Chem. 1999; 274: 8875-8885Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), and inhibitor studies revealed that PKC as well as GRK2 and/or GRK3 are responsible for the chemokine-induced receptor phosphorylation in rat basophilic leukemia (RBL) cells, which stably express CCR5 (10Oppermann M. Mack M. Proudfoot A.E. Olbrich H. J. Biol. Chem. 1999; 274: 8875-8885Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Two-dimensional phosphoamino acid analysis in combination with site-directed mutagenesis identified four serine residues that are located in the CCR5 C terminus as the only potential phosphorylation sites on this receptor (10Oppermann M. Mack M. Proudfoot A.E. Olbrich H. J. Biol. Chem. 1999; 274: 8875-8885Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Intact C-terminal phosphorylation sites were found to be necessary for β-arrestin binding as well as efficient receptor desensitization and internalization, but not for CCR5-mediated chemotaxis (11Kraft K. Olbrich H. Majoul I. Mack M. Proudfoot A. Oppermann M. J. Biol. Chem. 2001; 276: 34408-34418Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 12Hüttenrauch F. Nitzki A. Lin F.T. Höning S. Oppermann M. J. Biol. Chem. 2002; 277: 30769-30777Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). HIV-1 entry does not require receptor phosphorylation (13Gosling J. Monteclaro F.S. Atchison R.E. Arai H. Tsou C.L. Goldsmith M.A. Charo I.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5061-5066Crossref PubMed Scopus (160) Google Scholar), yet a fully phosphorylation-deficient CCR5 mutant is largely resistant to the inhibitory effect of CC chemokines on in vitro HIV infection (14Brandt S.M. Mariani R. Holland A.U. Hope T.J. Landau N.R. J. Biol. Chem. 2002; 277: 17291-17299Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). It was previously unknown whether phosphorylation of different serine residues can be attributed to particular kinases or whether they compete for the same phosphoacceptor sites. The current report demonstrates that agonist-induced CCR5 phosphorylation at two distinct sites proceeds with different kinetics and characteristic intracellular distribution in a dynamic manner that involves different protein kinases and phosphatases. Tissue culture reagents were from Biochrom; RBL-2H3, HEK-293, and X63-Ag8.653 cells were from the American Type Culture Collection; LipofectAMINE was from Invitrogen; geniticin, detergents, potato acid phosphatase, activators and inhibitors of protein kinase C, proteases, and phosphatase inhibitors were fromCalbiochem; synthetic phospho-/peptides were from Jerini; enhanced chemiluminescence (ECL) Western blotting reagents and protein G-Sepharose were from Amersham Biosciences; the CCR5 antagonist TAK-779 was kindly provided by Takeda; anti-phosphotyrosine antibody PY20 was from Becton Dickinson Transduction Laboratories; horseradish-conjugated and FITC-conjugated anti-mouse antibodies were from Dako; streptavidin peroxidase was from Jackson ImmunoResearch; all other reagents were from Sigma-Aldrich. Rat basophilic leukemia cells, which stably express wild-type CCR5 (RBL-CCR5 (10Oppermann M. Mack M. Proudfoot A.E. Olbrich H. J. Biol. Chem. 1999; 274: 8875-8885Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar)) or CCR5 mutants with alanine exchange of three serine residues at amino acid positions 336, 337, and 342 (RBL-CCR5-AAAS, Ref. 12Hüttenrauch F. Nitzki A. Lin F.T. Höning S. Oppermann M. J. Biol. Chem. 2002; 277: 30769-30777Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) or of all four C-terminal serine phosphorylation sites (RBL-CCR5(P-), Ref. 11Kraft K. Olbrich H. Majoul I. Mack M. Proudfoot A. Oppermann M. J. Biol. Chem. 2001; 276: 34408-34418Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) were maintained in 80:20-10 medium (80 parts RPMI 1640, 20 parts medium 199, supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 units/ml), 100 μg/ml streptomycin, and 600 μg/ml geniticin) in a 5% CO2 incubator at 37 °C. Human embryonic kidney (HEK-293) cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells were seeded at a density of 3 × 105 cells per 600-mm dish and transfected using LipofectAMINE with pEF-BOS expression vectors (1.5 μg/dish), which encode various CCR5 Ser/Ala mutants (10Oppermann M. Mack M. Proudfoot A.E. Olbrich H. J. Biol. Chem. 1999; 274: 8875-8885Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Transfection efficiencies as determined by flow cytometry with an anti-CCR5 mAb (Q10/19) ranged between 43 and 68%. A phosphopeptide (CEAPERA(pS)(pS)VYTR(pS)TGEQI(pS)VGL) corresponding to the 22 C-terminal amino acid residues of human CCR5 with phosphoserine (highlighted in boldface type) incorporated at the four known phosphorylation sites as well as a nonphosphorylated version of the same peptide were synthesized by standard solid phase methods. The peptides were purified to >70% purity by reversed phase HPLC and displayed the correct mass spectrum. The peptides were conjugated via the N-terminal cysteine residues to bovine serum albumin using succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and BALB/c mice were immunized at four monthly intervals with peptide-BSA conjugates (75 μg per animal and injection). Monoclonal antibodies were generated according to standard techniques by the fusion of spleen cells with X63-Ag8.653 myeloma cells and were identified initially by differential ELISA reactivities toward phosphorylated and nonphosphorylated receptor peptides. Polyclonal phosphopeptide-specific antisera directed against a putative tyrosine phosphorylation site within a highly conserved Asp-Arg-Tyr sequence at the end of the third transmembrane domain of CCR5 (15Rodriguez-Frade J.M. Vila-Coro A.J. Martin A. Nieto M. Sanchez-Madrid F. Proudfoot A.E. Wells T.N. Martinez A. Mellado M. J. Cell Biol. 1999; 144: 755-765Crossref PubMed Scopus (110) Google Scholar) were generated by immunizing New Zealand White rabbits with the following synthetic phosphopeptide which corresponds to amino acids Ile-119 to Val-131 coupled to bovine serum albumin: C(ε-aminocaproic acid)IILLTIDR(pY)LAVV (phosphotyrosine highlighted in boldface type). IgG was purified using protein A chromatography, and antibodies reactive with the corresponding nonphosphorylated receptor sequence were removed by adsorption to a nonphosphopeptide affinity column. Two murine mAb (R22/7 and T21/8; both IgG1/κ) which bind with high affinity to a CCR5 N-terminal epitope were generated following the immunization of mice with intact RBL-CCR5 cells (10Oppermann M. Mack M. Proudfoot A.E. Olbrich H. J. Biol. Chem. 1999; 274: 8875-8885Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). RBL-2H3 cells which express wild-type or phosphorylation-deficient CCR5 (7 × 105 cells per 60-mm dish) were stimulated with varying concentrations of RANTES for 10 min at 37 °C, washed once with PBS, and solubilized in detergent buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 5 mm EDTA, 1% Triton X-100, 0.05% SDS with phosphatase and protease inhibitors as described, Ref. 16Oppermann M. Freedman N.J. Alexander R.W. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 13266-13272Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar) on ice. Experiments that focused on the detection of tyrosine phosphorylation were performed in the presence of 10 μm sodium orthovanadate. Receptors were immunoprecipitated by the incubation (2 h/4 °C) of cellular lysates with 20 μg of anti-CCR5 R22/7 and protein G-Sepharose. Receptors were eluted by incubation at 37 °C for 30 min in SDS sample buffer containing 2% SDS and 5% 2-mercaptoethanol and subjected to 10% SDS-polyacrylamide gel electrophoresis. Immunoblots were performed using monoclonal anti-pS337 V14/2 (5 μg/ml), anti-pTyr PY20 (0.1 μg/ml), or polyclonal anti-pY127 (10 μg/ml) antibodies in Tris-buffered saline containing 0.1% Tween 20/5% nonfat dry milk. Enhanced chemiluminescence detection of antigens was achieved with horseradish peroxidase-conjugated secondary antibodies. Afterward membranes were stripped and reprobed for total cellular receptors with anti-CCR5 mAb R22/7 (10 μg/ml). The agonist-induced phosphorylation of CCR5 was determined in RBL-CCR5 cells grown to confluency in 24-well dishes, which were stimulated for various incubation times with the indicated concentrations of RANTES or PMA in 80:20–10 medium. The cells were transferred to ice and scraped in 200 μl of lysis buffer containing protease and phosphatase inhibitors. Cell debris was removed by centrifugation at 3,000 × g for 10 min and supernatants (100 μl) were applied either directly or after 2-fold dilution in lysis buffer into wells of microtiter plates, which were adsorbed with anti-CCR5 mAb T21/8 (5 μg/ml in 50 mmcarbonate, pH 10.6) as the capture antibody. The same cellular lysates were probed, in parallel, with several biotinylated mAb (anti-pS349 E11/19; anti-pS337 V14/2; anti-S337 R-C10; 1 μg/ml in PBS, 0.05% Tween 20), which react with distinct phosphorylation states and sites on the CCR5 C terminus. After a 2-h incubation period (non-)phosphorylated CCR5 were detected by adding a 4000-fold dilution of streptavidin peroxidase (Jackson ImmunoResearch) in PBS/Tween for 1 h and 2.2-azino-di-(3-ethyl-benzthiazoline sulfonate) as substrate. Incubation periods were terminated by 3 wash cycles with PBS/Tween, respectively. The assays were calibrated with a standard protein, which was obtained by the conjugation of bovine serum albumin with synthetic N-terminal and non-/phosphorylated C-terminal CCR5 peptides at 1:5:5 molar ratios using SMCC as a cross-linking reagent. Results were expressed in arbitrary units (1 AU equals 1 ng of BSA-peptide per ml). RBL-CCR5 cells were incubated with 30 nm RANTES (10 min) and 200 nmPMA (5 min) to induce maximal receptor phosphorylation. Cells were scraped in lysis buffer and filled directly into wells of ELISA plates, which were precoated with anti-CCR5 mAb T21/8. Receptors bound to the solid phase were incubated for up to 1 h at 37 °C with potato acid phosphatase (0.6 units/ml in 40 mm PIPES, 1 mm dithiothreitol, pH 6.0 with protease inhibitors). Alternatively, RBL-2H3 cells were scraped in 50 mmTris-HCl, pH 7.0, 0.1 mm EDTA, 50 mm2-mercaptoethanol with protease inhibitors and homogenized by sonication (four 10-s bursts at 100 watts). Nuclei were removed by centrifugation (10 min; 300 × g) and the supernatant was incubated for 1 h at 37 °C with receptors bound to the ELISA plate in the presence or absence of 200 nm okadaic acid. RBL-CCR5 cells were grown overnight on glass coverslips in 24-well plates. Cells were washed once in warm 80:20-10 medium and then treated with 25 nm RANTES in medium for varying time intervals at 37 °C. Thereafter, the coverslips were placed on ice, washed with cold medium, and fixed with an ice-cold solution of 3% paraformaldehyde, pH 7.4 in PBS for 20 min. Free aldehyde groups were quenched with 50 mmNH4Cl in PBS for 30 min. After permeabilization with cold PBS containing 0.05% saponin and 0.2% gelatin for 15 min, cells were washed with the same buffer and stained with anti-CCR5 (T21/8), anti-pS337 (V14/2), or anti-pS349 (E11/19) antibodies (5 μg/ml in PBS/saponin) for 1 h on ice. After washing with PBS/saponin/gelatin, secondary antibody staining was carried out with a FITC-conjugated goat anti-mouse IgG (1:100 dilution) for 1 h. After further washes in PBS, coverslips were mounted in Mowiol containing 0.1% p-phenylenediamine. The samples were analyzed by confocal laser-scanning microscopy utilizing a Leica TCS SP2 system, images were assembled in Corel Draw. Synthetic (phospho-)peptides that encompass the four C-terminal serine phosphorylation sites of CCR5 were used to generate monoclonal antibodies which differentially bind either to phosphorylated or nonphosphorylated versions of the immunogenic peptides. Two hybridomas (E11/19, IgG1/κ; V14/2, IgG1/κ) were identified, which selectively react only with ligand-activated CCR5 in a phosphorylation-dependent manner as shown by several independent immunological techniques. Another mAb (R-C10, IgG1/κ) was derived from a separate fusion after immunization with a nonphosphorylated C-terminal receptor peptide which exclusively recognizes non-activated CCR5. The immunoblot shown in Fig. 1 demonstrates that the pCCR5-specific mAb V14/2 reacts with CCR5, which migrates as a broad 40-kDa protein in SDS-PAGE analysis typical of a glycosylated G protein-coupled receptor, but only in its ligand-activated, i.e. phosphorylated form. Under the experimental conditions used in this study we did not observe higher molecular weight forms of CCR5. Despite RANTES activation this mAb did not recognize a phosphorylation-deficient CCR5 mutant, which was generated by alanine replacement of all four C-terminal serine phosphorylation sites. Similar results were obtained with the mAb E11/19, whereas the mAb R-C10 displayed reverse reactivity and lost its ability to react with CCR5 upon ligand activation in a dose-dependent manner. It has been proposed that chemokine binding to their receptors exposes the tyrosine residue within the highly conserved Asp-Arg-Tyr motif in the second intracellular receptor loop which is then rapidly phosphorylated by Janus kinases (JAKs) (15Rodriguez-Frade J.M. Vila-Coro A.J. Martin A. Nieto M. Sanchez-Madrid F. Proudfoot A.E. Wells T.N. Martinez A. Mellado M. J. Cell Biol. 1999; 144: 755-765Crossref PubMed Scopus (110) Google Scholar, 17Rodriguez-Frade J.M. Mellado M. Martinez C. Trends Immunol. 2001; 22: 612-617Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). We tested this hypothesis by generating phosphopeptide-specific antibodies, which specifically recognize phospho-Tyr-127 and probed the ligand-activated CCR5 with this and anti-phosphotyrosine antibodies, in parallel, by immunoblotting and enzyme-linked immunosorbent assay. We did not observe phosphorylation of either this specific or any other tyrosine residue of this receptor after incubation of RBL-CCR5 cells with saturating concentrations of RANTES (30 nm) within a time frame of 30 s up to 20 min (Fig. 2). To exactly map the epitopes that are recognized by the various phospho-CCR5-specific mAb we determined ELISA reactivities of RBL cellular lysates containing maximally phosphorylated CCR5 Ser/Ala mutants (Fig. 3). Substitution of serine 337 by alanine either alone or in combination with any other mutation resulted in the complete loss of V14/2 binding to the receptor, whereas the mutation of serine 349 eliminated E11/19 reactivity. This indicates that V14/2 and E11/19 recognize phosphoserines at positions 337 and 349, respectively, and independent of the phosphorylation state of any of the other three serine residues. Using the same cellular lysates in the absence of receptor activation we found that the mAb R-C10 exclusively reacts with nonphosphorylated serine 337 (not shown). Epitope mapping with monophosphorylated CCR5 peptides independently confirmed these results. Exposure to CC-chemokines produces a rapid increase in CCR5 phosphorylation, which is mediated by protein kinases belonging to the PKC and GRK families. We tested the hypothesis that both protein kinases phosphorylate distinct CCR5 C-terminal serine residues. To this end ELISA procedures were established that are based on a capture antibody with specificity for a CCR5 N-terminal epitope and different (non)phospho-CCR5-specific detecting antibodies. These assays enabled us to determine, in parallel, the phosphorylation status of serine residues at positions 337 and 349 after stimulation of CCR5-expressing cells with various agonists. Stimulation with PMA caused maximal phosphorylation at serine 337 and pretreatment with bisindolylmaleimide I, a selective and potent inhibitor of classical and novel PKC isoforms, completely prevented phorbol ester- and ligand-induced receptor phosphorylation (Fig. 4). In contrast, in the same experiment neither the PMA-induced activation nor the inhibition of PKC by bisindolylmaleimide in cells that were exposed to receptor-saturating concentrations (30 nm) of RANTES had any noticeable effect on the phosphorylation state of serine 349. Several other protein kinase inhibitors, including the protein kinase A inhibitor H-89 (5 μm), the calmodulin kinase inhibitor KN-93 (10 μm), the phosphatidylinositol 3-kinase inhibitor wortmannin (200 nm), or the broad spectrum protein kinase inhibitor staurosporine (500 nm) also did not inhibit the chemokine-stimulated phosphorylation of Ser-349. Pretreatment of cells with pertussis toxin for 16 h at a concentration (100 ng/ml) known to disrupt G protein-mediated signaling in RBL-CCR5 cells (11Kraft K. Olbrich H. Majoul I. Mack M. Proudfoot A. Oppermann M. J. Biol. Chem. 2001; 276: 34408-34418Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) abrogated RANTES-induced phosphorylation at serine 337 but, again, had no effect on phosphorylation of serine 349. Previous studies that employed receptor mutants (18Hausdorff W.P. Bouvier M. O'Dowd B.F. Irons G.P. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1989; 264: 12657-12665Abstract Full Text PDF PubMed Google Scholar) or various kinase inhibitors (19Roth N.S. Campbell P.T. Caron M.G. Lefkowitz R.J. Lohse M.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6201-6204Crossref PubMed Scopus (118) Google Scholar, 20Ali H. Richardson R.M. Tomhave E.D. Didsbury J.R. Snyderman R. J. Biol. Chem. 1993; 268: 24247-24254Abstract Full Text PDF PubMed Google Scholar) suggested that second messenger-dependent kinases and GRKs differently contribute to GPCR phosphorylation upon exposure to different agonist concentrations and at different time intervals. Phosphosite-specific mAb now allowed us to monitor the kinetics and dose-dependence of the RANTES-induced phosphorylation of non-mutated CCR5 by different protein kinases under close to physiological conditions. When RBL-CCR5 cells were stimulated with increasing concentrations of agonist for various time intervals half-maximal phosphorylation of Ser-349 was observed at 5 nm RANTES and ∼4-fold higher concentrations were required to achieve maximal phosphorylation (Fig. 5 B). The concentration dependence of RANTES-induced phosphorylation paralleled ligand binding to the receptor (11Kraft K. Olbrich H. Majoul I. Mack M. Proudfoot A. Oppermann M. J. Biol. Chem. 2001; 276: 34408-34418Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) and thus conformed to the characteristics of a GRK-mediated mechanism. In contrast, phosphorylation of Ser-337 by the second messenger-activated kinase (PKC) was detected at 50-fold lower concentrations when cells were exposed to agonist for 1 min (Fig. 5 A). This indicates significant signal amplification downstream of the receptor under these experimental conditions. Unexpectedly, the dose dependence of this effect shifted to higher concentrations when cells were exposed to RANTES for longer incubation times. A detailed kinetic analysis revealed that stimulation with low RANTES concentrations results in the phosphorylation of Ser-337 in a very rapid, albeit transient manner (Fig. 5 C). Maximal CCR5 phosphorylation at this site was obtained by stimulation for 40 s, and thereafter the receptor was rapidly dephosphorylated. In the presence of receptor-saturating concentrations of ligand dephosphorylation of phospho-Ser-337 was significantly retarded. When lysates of RBL-CCR5 cells that had been stimulated with 0.5 nm RANTES for various times were probed with a mAb (R-C10), which specifically recognizes only nonphosphorylated Ser-337 the results represented a mirror image of the values obtained with anti-phospho-Ser-337 mAb V14/2 (Fig. 6 A). This experiment shows that stimulation of only a small fraction of the cellular complement of CCR5 with low concentrations of RANTES is capable of causing the transient phosphorylation of ∼50% of all receptors in a heterologous manner. A strikingly different time course of Ser-337 phosphorylation was observed after incubation of cells with 50 nm PMA. Phosphorylation was irreversible within the time frame of this experiment and did not peak until after 5 min (Fig. 6 B). Phosphorylation of substrates by serine/threonine protein kinases often requires phosphorylation of a nearby priming site by another protein kinase (21Fiol C.J. Mahrenholz A.M. Wang Y. Roeske R.W. Roach P.J. J. Biol. Chem. 1987; 262: 14042-14048Abstract Full Text PDF PubMed Google Scholar, 22Roach P.J. J. Biol. Chem. 1991; 266: 14139-14142Abstract Full Text PDF PubMed Google Scholar) and this mechanism was also proposed to underlie the sequential phosphorylation of phosphoacceptor sites in certain G protein-coupled receptors (23Ohguro H. Palczewski K. Ericsson L.H. Walsh K.A. Johnson R.S. Biochemistry. 1993; 32: 5718-5724Crossref PubMed Scopus (152) Google Scholar, 24Giannini E. Brouchon L. Boulay F. J. Biol. Chem. 1995; 270: 19166-19172Abstract Full Text Full Text PDF Pu
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