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• W2021100900 abstract "Agonist binding to the CC chemokine receptor 5 (CCR5) induces the phosphorylation of four distinct serine residues that are located in the CCR5 C terminus. We established a series of clonal RBL-2H3 cell lines expressing CCR5 with alanine mutations of Ser336, Ser337, Ser342, and Ser349 in various combinations and explored the significance of phosphorylation sites for the ability of the receptor to interact with β-arrestins and to undergo desensitization and internalization upon ligand binding. Receptor mutants that lack any two phosphorylation sites retained their ability to recruit endogenous β-arrestins to the cell membrane and were normally sequestered, whereas alanine mutation of any three C-terminal serine residues abolished both β-arrestin binding and rapid agonist-induced internalization. In contrast, RANTES (regulated on activation normal T cell expressed and secreted) stimulation of a S336A/S349A mutant triggered a sustained calcium response and enhanced granular enzyme release. This mutational analysis implies that CCR5 internalization largely depends on a β-arrestin-mediated mechanism that requires the presence of any two phosphorylation sites, whereas receptor desensitization is independently regulated by the phosphorylation of distinct serine residues. Surface plasmon resonance analysis further demonstrated that purified β-arrestin 1 binds to phosphorylated and nonphosphorylated C-tail peptides with similar affinities, suggesting that β-arrestins use additional receptor sites to discriminate between nonactivated and activated receptors. Surface plasmon resonance analysis revealed β-arrestin 1 binding to the second intracellular loop of CCR5, which required an intact Asp-Arg-Tyr triplet. These results suggest that a conserved sequence motif within the second intracellular loop of CCR5 that is known to be involved in G protein activation plays a significant role in β-arrestin binding to CCR5. Agonist binding to the CC chemokine receptor 5 (CCR5) induces the phosphorylation of four distinct serine residues that are located in the CCR5 C terminus. We established a series of clonal RBL-2H3 cell lines expressing CCR5 with alanine mutations of Ser336, Ser337, Ser342, and Ser349 in various combinations and explored the significance of phosphorylation sites for the ability of the receptor to interact with β-arrestins and to undergo desensitization and internalization upon ligand binding. Receptor mutants that lack any two phosphorylation sites retained their ability to recruit endogenous β-arrestins to the cell membrane and were normally sequestered, whereas alanine mutation of any three C-terminal serine residues abolished both β-arrestin binding and rapid agonist-induced internalization. In contrast, RANTES (regulated on activation normal T cell expressed and secreted) stimulation of a S336A/S349A mutant triggered a sustained calcium response and enhanced granular enzyme release. This mutational analysis implies that CCR5 internalization largely depends on a β-arrestin-mediated mechanism that requires the presence of any two phosphorylation sites, whereas receptor desensitization is independently regulated by the phosphorylation of distinct serine residues. Surface plasmon resonance analysis further demonstrated that purified β-arrestin 1 binds to phosphorylated and nonphosphorylated C-tail peptides with similar affinities, suggesting that β-arrestins use additional receptor sites to discriminate between nonactivated and activated receptors. Surface plasmon resonance analysis revealed β-arrestin 1 binding to the second intracellular loop of CCR5, which required an intact Asp-Arg-Tyr triplet. These results suggest that a conserved sequence motif within the second intracellular loop of CCR5 that is known to be involved in G protein activation plays a significant role in β-arrestin binding to CCR5. CC chemokine receptor 5 G protein-coupled receptor GPCR kinase human embryonic kidney released on activation normal T cell expressed and secreted (also known as CCL5) surface plasmon resonance human immunodeficiency virus, type 1 1,4-piperazinediethanesulfonic acid high pressure liquid chromatography intracellular loop carboxyl terminus The CC chemokine receptor CCR51 is a member of the large family of heptahelical receptors that transduce extracellular signals into the cell by activating heterotrimeric G proteins. CCR5 is expressed, among other cells, on memory T lymphocytes, macrophages, and dendritic cells (1Murphy P.M. Baggiolini M. Charo I.F. Hebert C.A. Horuk R. Matsushima K. Miller L.H. Oppenheim J.J. Power C.A. Pharmacol. Rev. 2000; 52: 145-176PubMed Google Scholar). Physiological ligands that bind to this receptor with high affinity include the CC chemokines RANTES, MIP-1α, MIP-1β, and MCP-2 and a proteolytically processed variant of HCC-1. CCR5 is also the principal co-receptor for macrophage-tropic (or R5) strains of HIV-1 (2Berger E.A. Murphy P.M. Farber J.M. Annu. Rev. Immunol. 1999; 17: 657-700Crossref PubMed Scopus (1887) Google Scholar). Individuals homozygous for a nonfunctionalCCR5Δ32 allele express a truncated receptor that fails to reach the cell surface and are thus highly resistant to HIV infection. Chemokines and small molecule receptor antagonists inhibit HIV-1 infection in vitro by blocking the binding of the viral envelope glycoprotein gp120 to CCR5. Another mechanism that underlies the pronounced antiviral effect of certain chemokines and their derivatives relates to their ability to effectively down-modulate CCR5 expression by inducing receptor endocytosis and to thereby decrease co-receptor availability on the cell surface (3Mack M. Luckow B. Nelson P.J. Cihak J. Simmons G. Clapham P.R. Signoret N. Marsh M. Stangassinger M. Borlat F. Wells T.N. Schlöndorff D. Proudfoot A.E. J. Exp. Med. 1998; 187: 1215-1224Crossref PubMed Scopus (379) Google Scholar). According to a current concept of G protein-coupled receptor (GPCR) regulation, largely extrapolated from detailed studies with the prototypic β2-adrenergic receptor, agonist activation of receptors leads to their phosphorylation by both second messenger-dependent protein kinases and GPCR kinases (GRKs) (4Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1070) Google Scholar, 5Bünemann M. Hosey M.M. J. Physiol. 1999; 517: 5-23Crossref PubMed Scopus (165) Google Scholar). This, in turn, promotes binding of members of the arrestin family. Arrestins bind to the ligand-activated/phosphorylated receptor and thereby sterically interfere with further binding of heterotrimeric G proteins. With many GPCRs, β-arrestins are essential for receptor internalization via clathrin-dependent mechanisms by their ability to directly couple the phosphorylated receptor to clathrin heavy chain and to recruit accessory proteins that participate in the formation of clathrin-coated pits (6Ferguson S.S. Pharmacol. Rev. 2001; 53: 1-24PubMed Google Scholar). β-Arrestins were shown to interact with the β2-adaptin subunit of the heterotetrameric AP-2 adaptor complex (7Laporte S.A. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Crossref PubMed Scopus (528) Google Scholar) and to act as co-factors in the activation of small G protein ADP-ribosylation factor 6 (8Claing A. Chen W. Miller W.E. Vitale N. Moss J. Premont R.T. Lefkowitz R.J. J. Biol. Chem. 2001; 276: 42509-42513Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), two processes that fulfill essential roles in the internalization of certain GPCR. Recent evidence suggests that arrestins play a broader role as signaling scaffolds, linking them to the activation of Src family tyrosine kinases and certain mitogen-activated protein kinase modules, in addition to their role in the regulation of receptor trafficking (9Perry S.J. Lefkowitz R.J. Trends Cell Biol. 2002; 12: 130-138Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Together, these findings point to a central role for arrestins both in the termination and in the initiation of certain aspects of GPCR signaling. To date, four members of the arrestin family have been identified. Only β-arrestin 1 (arrestin 2) and β-arrestin 2 (arrestin 3) are ubiquitously expressed throughout the body and are predominantly localized in lymphoid and neuronal tissues (10Attramadal H. Arriza J.L. Aoki C. Dawson T.M. Codina J. Kwatra M.M. Snyder S.H. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 17882-17890Abstract Full Text PDF PubMed Google Scholar). The structural determinants that underlie the stable association of ligand-activated GPCR and arrestins have been studied in detail in the rhodopsin/visual arrestin system (11Gurevich V.V. Dion S.B. Onorato J.J. Ptasienski J. Kim C.M. Sterne-Marr R. Hosey M.M. Benovic J.L. J. Biol. Chem. 1995; 270: 720-731Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 12Gurevich V.V. J. Biol. Chem. 1998; 273: 15501-15506Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 13McDowell J.H. Robinson P.R. Miller R.L. Brannock M.T. Arendt A. Smith W.C. Hargrave P.A. Invest. Ophthalmol. Vis. Sci. 2001; 42: 1439-1443PubMed Google Scholar, 14Vishnivetskiy S.A. Paz C.L. Schubert C. Hirsch J.A. Sigler P.B. Gurevich V.V. J. Biol. Chem. 1999; 274: 11451-11454Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 15Smith W.C. McDowell J.H. Dugger D.R. Miller R. Arendt A. Popp M.P. Hargrave P.A. Biochemistry. 1999; 38: 2752-2761Crossref PubMed Scopus (21) Google Scholar). Much less is known about structural features required for the interaction of these proteins in nonvisual receptor systems. The recent determination of the structures of visual arrestin (16Hirsch J.A. Schubert C. Gurevich V.V. Sigler P.B. Cell. 1999; 97: 257-269Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar) and β-arrestin 1 (17Han M. Gurevich V.V. Vishnivetskiy S.A. Sigler P.B. Schubert C. Structure. 2001; 9: 869-880Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 18Milano S.K. Pace H.C. Kim Y.M. Brenner C. Benovic J.L. Biochemistry. 2002; 41: 3321-3328Crossref PubMed Scopus (172) Google Scholar) at atomic resolution has allowed us to propose a molecular mechanism by which arrestins interact with ligand-activated GPCR. According to this model arrestin in its inactive state is composed of two N- and C-terminal domains and is maintained in this basal conformation by multiple intramolecular hydrophobic interactions among highly conserved residues. Upon binding, the receptor-attached phosphates disrupt these intramolecular forces and thereby induce a structural reorganization of the arrestin molecule. This conformational change is accompanied by the exposure of currently unknown secondary binding sites that allow arrestin to bind with high affinity to the ligand-activated receptor. So far, the structural determinants on ligand-activated receptors that allow arrestins to interact with hundreds of different GPCR are not well characterized. Previously, we have shown that CC chemokines induce the rapid phosphorylation of CCR5 on four C-terminal serine residues, a process that is mediated by the combined action of GRKs and protein kinase C (19Oppermann 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). In a subsequent study the ligand-induced association of β-arrestins with CCR5 was monitored in real time by fluorescence resonance energy transfer (20Kraft 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). Replacement of all four phosphorylated serine residues by alanine abrogated the ability of ligand-activated receptors to interact with β-arrestin, and this was accompanied by a defect in CCR5 desensitization and internalization. Here, we have further investigated the structural determinants that underlie β-arrestin binding to the chemokine-activated CCR5. To this end, we tested several CCR5 Ser/Ala replacement mutants that were stably expressed in RBL cells for their ability to recruit β-arrestins upon chemokine stimulation. An additional β-arrestin 1-binding site within a conserved region in the second cytoplasmic loop of CCR5 was identified by surface plasmon resonance (SPR) analysis. The functional significance of these findings was determined by investigating internalization and desensitization characteristics of the various receptor mutants. Tissue culture media and cell culture supplies were from Biochrom; RBL-2H3 and HEK-293 cells were from the American Type Culture Collection; ECL Western blotting reagents were fromAmersham Biosciences; nitrocellulose membranes were purchased from Schleicher & Schüll; geniticin, detergents, and protease inhibitors were from Calbiochem; the peptides were synthesized by Jerini; anti-β-arrestin 1 monoclonal antibody (clone 10) was from Becton Dickinson Transduction Laboratories; horseradish peroxidase-labeled secondary antibodies and phycoerythrin-labeled goat polyclonal anti-mouse F(ab′)2 were from Dako;125I-RANTES was from PerkinElmer Life Sciences; and all other reagents, unless otherwise indicated, were purchased from Sigma. The pEF-BOS expression vectors that encode human CCR5 with various combinations of C-terminal serine to alanine substitutions have been described before (19Oppermann 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). Rat basophilic leukemia cell 2H3 subline (RBL-2H3) cells were transfected by electroporation (19Oppermann 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 selection of transfected cells was accomplished by adding 600 μg/ml geniticin in the cell culture medium. The RANTES-induced translocation of endogenous β-arrestins present in RBL cells from the cytosol to the membrane fraction was analyzed by subcellular fractionation. RBL-CCR5 cells (1 × 107/135-mm dish) were incubated in the presence of varying concentrations of RANTES for up to 10 min at 37 °C. The cells were placed on ice and scraped into 3 ml of buffer A (10 mm PIPES, 100 mm KCl, 3 mm NaCl, 3.5 mm MgCl2, pH 7.0) containing 50 μg/ml phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A, 10 μg/ml leupeptin, and 5 μg/ml aprotinin. The cells were homogenized by Dounce homogenization (10 strokes) and sonication (four 20-s bursts at 100 W). The nuclei were removed by centrifugation at 1000 × g for 20 min. The supernatant was loaded on a discontinuous gradient of 50, 35, and 20% sucrose in buffer A and centrifuged at 160,000 × g and 4 °C for 2 h. The supernatant (cytosol) was removed, and the 20/35% sucrose interphase (membrane) was collected, diluted in 3 ml of buffer A, and recentrifuged at 160,000 × g and 4 °C for 15 min. The pellet was resuspended in 40 μl of detergent buffer (20Kraft 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). The protein content in 10-μl aliquots of the cytosolic and the membrane fraction was determined by the Bio-Rad DC protein assay kit, and 10 (cytosol) or 25 μg (membrane) of each protein sample was loaded onto 10% SDS-PAGE gels. The proteins were transferred to nitrocellulose membranes, and nonspecific binding sites were blocked by incubation for 1 h with 4% nonfat dry milk in TBS, 0.1% Tween 20, pH 7.4. β-Arrestin 1 and β-arrestin 2 were detected using monoclonal anti-β-arrestin 1 antibodies (1:500) and horseradish peroxidase-labeled secondary antibodies (1:2000). The effect of RANTES stimulation on membrane-associated β-arrestin levels was quantitated by densitometric analysis (ImageMaster TotalLab software; AmershamBiosciences) of enhanced chemiluminescence films. To express β-arrestin 1 in mammalian cells, HEK-293 cells were transfected with pCMV/βarr1-His6 (21Lin F.T. Krueger K.M. Kendall H.E. Daaka Y. Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31051-31057Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar) using the calcium phosphate precipitation technique. Sixty hours after transfection, the cells were scraped into binding buffer (5 mm imidazole, 0.5 m NaCl, 20 mmTris-HCl, pH 7.9) containing protease inhibitors and 0.2% Nonidet P-40. β-Arrestin 1 was purified to apparent homogeneity by nickel affinity chromatography as described (21Lin F.T. Krueger K.M. Kendall H.E. Daaka Y. Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31051-31057Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). The peptides that correspond to cytoplasmic domains of CCR5 and derivatives thereof (see Table II) were synthesized by standard solid phase methods. Preparative purification of the peptides was achieved by reversed phase HPLC. The peptides displayed the correct mass spectrum and were >90% pure by analytical HPLC.Table IIEquilibrium rate constants (Kd) for the interaction between β-arrestin 1 and CCR5 synthetic peptidesPeptidePosition within CCR5Amino acid sequence2-aCysteine residues and biotin-βAla-βAla groups that were used for the conjugation to carrier proteins are underlined, and phosphoserine (pS) and alanine substitutions are highlighted in bold type. Single-letter amino acid codes are used.Equilibrium rate constants2-bβ-Arrestin 1 binding to synthetic CCR5 peptides was determined as outlined in the legend to Fig. 6. From the derived sensorgrams the kinetic rate constants for association (ka) and dissociation (kd) were determined using BIAevaluation software, version 2.1. The derived equilibrium constants (KD) were then calculated using the equation kd/ka. ND, not detectable.μmIL-1Cys58–Ile67CKRLKSMTDINDIL-2Ile124–Val142CIDRYLAVVHAVFALKARTV 6.0IL-3Lys219–Arg235Biotin-βAla-βAla-KTLLRCRNEKKRHRAVRNDCT-1Gly301–Phe320CGEKFRNYLLVFFQKHIAKRF12.5CT-2Glu330–Leu352CEAPERASSVYTRSTGEQEISVGL 5.5phosphoCT-2Glu330–Leu352CEAPERA(pS)(pS)VYTR(pS)TGEQEI(pS)VGL 3.6IL-2sIle124–Leu137CIDRYLAVVHAVFAL12.0IL-2-DRY/AAAIle124–Val142CIAAALAVVHAVFALKARTVNDIL-2-ARYIle124–Val142CIARYLAVVHAVFALKARTV12.5IL-2-DAYIle124–Val142CIDAYLAVVHAVFALKARTV38.0IL-2-DRAIle124–Val142CIDRALAVVHAVFALKARTV30.0IL-2-VV/AAIle124–Val142CIDRYLAAAHAVFALKARTV13.22-a Cysteine residues and biotin-βAla-βAla groups that were used for the conjugation to carrier proteins are underlined, and phosphoserine (pS) and alanine substitutions are highlighted in bold type. Single-letter amino acid codes are used.2-b β-Arrestin 1 binding to synthetic CCR5 peptides was determined as outlined in the legend to Fig. 6. From the derived sensorgrams the kinetic rate constants for association (ka) and dissociation (kd) were determined using BIAevaluation software, version 2.1. The derived equilibrium constants (KD) were then calculated using the equation kd/ka. ND, not detectable. Open table in a new tab The association of β-arrestin 1 with the CCR5 cytoplasmic loop and C-tail derived peptides was analyzed in real time by SPR using a BIAcore 3000 biosensor (BIAcore AB). The peptides were immobilized to CM5 biosensor chips via the thiol group of an N-terminal cysteine residue according to the manufacturer's instructions. The peptide corresponding to the CCR5 cytoplasmic IL-3 loop was synthesized with a biotin moiety attached to the N-terminal amino acid and immobilized to a streptavidin sensor surface (SA5 chip) according to standard procedures. Recombinant β-arrestin 1 was used at concentrations ranging from 50 to 600 nm in running buffer (20 mm HEPES, pH 7.3, 50 mm NaCl, 10 mm KCl, 2 mm MgCl2, 0.2 mm dithiothreitol). All of the measurements were recorded at a flow rate of 20 μl/min. Association (2 min) was followed by dissociation (2 min), during which running buffer was perfused. The sensor surface was regenerated after each experimental cycle by a pulse injection (15 s) of 10 mm NaOH, 0.5% SDS. The kinetic parameters of the interaction between β-arrestin 1 and receptor peptides and the equilibrium constant were calculated using the BIAevaluation software provided by the manufacturer (more details can be found in Ref. 22Jonsson U. Fagerstam L. Ivarsson B. Johnsson B. Karlsson R. Lundh K. Lofas S. Persson B. Roos H. Ronnberg I. Sjolander S. Stenberg E. Stahlberg R. Urbaniczky C. Ostlin H. Malmqvist M. BioTechniques. 1991; 11: 620-627PubMed Google Scholar). The internalization of 125I-RANTES by RBL-CCR5 cells was expressed as a percentage of internalized (acid-resistant) of the total radioactivity at different time points, as described before (20Kraft 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, 23Signoret N. Pelchen-Matthews A. Mack M. Proudfoot A.E. Marsh M. J. Cell Biol. 2000; 151: 1281-1294Crossref PubMed Scopus (161) Google Scholar). For flow cytometric analysis, 2 × 105 RBL-CCR5 cells were washed twice in wash buffer (1% fetal calf serum in phosphate-buffered saline containing 0.05% azide) and then incubated, in sequence, with anti-CCR5 monoclonal antibody T21/8 (10 μg/ml; 45 min/4 °C) and phycoerythrin-labeled goat polyclonal anti-mouse F(ab′)2 (1:100; 45 min/4 °C). The cells were analyzed with an EPICS XL flow cytometer (Coulter). The RANTES-inducedN-acetyl-β-d-glucosaminidase release from CCR5 expressing RBL-2H3 cells was determined as described (24Montz H. Fuhrmann A. Schulze M. Götze O. Cell. Immunol. 1990; 127: 337-351Crossref PubMed Scopus (22) Google Scholar). The values were expressed as percentages of total enzyme present in cells after lysis with 0.1% Triton X-100, and the data were analyzed using nonlinear regression applied to a sigmoidal dose response model with the Ligand Binding module of Sigma-Plot software (SPSS). Agonist-dependent intracellular calcium mobilization was measured in transfected RBL-2H3 cells as described (19Oppermann 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). Calcium decays from 80% of the peak height to basal levels were fitted to an exponential (a + b·e−t/τ), where the time constant τ reflects the ability of CCR5 variants to evoke a more or less sustained calcium response (25Christophe T. Rabiet M.J. Tardif M. Milcent M.D. Boulay F. J. Biol. Chem. 2000; 275: 1656-1664Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Previously, our laboratory analyzed a series of CCR5 serine to alanine mutants that were transiently expressed in COS-7 cells for their abilities to undergo ligand-induced phosphorylation (19Oppermann 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). In this study, several of these receptor constructs were stably transfected into RBL-2H3 cells, and we examined whether the constructs bind β-arrestins and undergo receptor internalization and desensitization upon RANTES stimulation. RBL-2H3 cells are of myeloid origin and have been shown to express high endogenous levels of GRK2/3 and both β-arrestins (arrestin 2 and arrestin 3) (19Oppermann 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, 26Barlic J. Khandaker M.H. Mahon E. Andrews J. DeVries M.E. Mitchell G.B. Rahimpour R. Tan C.M. Ferguson S.S. Kelvin D.J. J. Biol. Chem. 1999; 274: 16287-16294Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 27Santini F. Penn R.B. Gagnon A.W. Benovic J.L. Keen J.H. J. Cell Sci. 2000; 113: 2463-2470Crossref PubMed Google Scholar). Therefore, these cells are well suited for the analysis of GRK/arrestin-driven mechanisms of receptor regulation. CCR5 expression levels of the various cell lines were determined by flow cytometry using a monoclonal antibody (T21/8) with specificity for a CCR5 N-terminal epitope, and signaling characteristics were evaluated by testing the abilities to release glucosaminidase after RANTES stimulation (Table I). In whole cell phosphorylation experiments with these RBL-CCR5 cell lines (data not shown), we confirmed the effect of serine to alanine substitution in various combinations on receptor phosphorylation that we had previously observed when receptor constructs were transiently expressed in COS-7 cells (19Oppermann 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).Table ICell surface expression and functional characteristics of CCR5 wild type and mutant receptors expressed in RBL cellsReceptorMutation1-aThe positions of C-terminal serine phosphorylation sites that were replaced by alanine in various combinations are indicated.Receptor expression (MCF)1-bThe mean channel of fluorescence (MCF) in nontransfected RBL-2H3 cells was 0.5.Glucosaminidase releaseEC50Percentage of maximumnmCCR5-WTWild type52.95.218.3CCR5-Δ1AS349A17.67.210.2CCR5-Δ2AS336A/S337A47.74.529.3CCR5-Δ2BS336A/S342A53.425.747.8CCR5-Δ2CS336A/S349A50.320.946.2CCR5-Δ3AS337A/S342A/S349A6.36.049.4CCR5-Δ3BS336A/S342A/S349A72.414.641.6CCR5-Δ3CS336A/S337A/S342A24.816.459.3CCR5-Δ4S336A/S337A/S342A/S349A40.013.455.01-a The positions of C-terminal serine phosphorylation sites that were replaced by alanine in various combinations are indicated.1-b The mean channel of fluorescence (MCF) in nontransfected RBL-2H3 cells was 0.5. Open table in a new tab We have shown by fluorescence resonance energy transfer technology that wild type CCR5, but not a phosphorylation-deficient CCR5 mutant with alanine substitution of all four C-terminal serine residues rapidly interacts with β-arrestin upon RANTES stimulation (20Kraft 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). We now established a simple translocation assay based on subcellular fractionation of RBL cellular lysates that measures ligand-induced recruitment of β-arrestins from the cytosol to the cell membrane. This assay is based on an anti-β-arrestin 1 monoclonal antibody, which significantly cross-reacts with β-arrestin 2 (Fig. 1A). As shown in Fig.1B, treatment of RBL-CCR5 cells with 10 nmRANTES, but not with phorbol 12-myristate 13-acetate, leads to a more than 3-fold increase of two membrane-associated proteins that were identified as β-arrestin 1 and β-arrestin 2, respectively, by virtue of being immunoreactive with the anti-β-arrestin antibody and migration at the same apparent molecular masses of 54 and 48 kDa as the β-arrestin 1/2 cDNA products from HEK-293 cells. Because this effect was absent in untransfected RBL cells, it directly reflects the ability of ligand-activated CCR5 to recruit β-arrestins to the cell membrane. Translocation of β-arrestins was induced by as little as 1 nm and reached a maximum at 10 nm RANTES (EC50 = 2.4 nm; Fig. 1C). β-Arrestins rapidly associate with the membrane fraction after stimulation with saturating concentrations of RANTES; maximum levels were observed after stimulation for 3–10 min (Fig. 1D). To determine the minimal requirements of receptor phosphorylation for CCR5/β-arrestin association, we investigated RANTES-induced β-arrestin translocation to the cell membrane in RBL cells that express various CCR5 Ser/Ala mutants (Fig.2). All CCR5 mutants with alanine replacements of one or two C-terminal serine residues were capable of inducing translocation of β-arrestin 1 or β-arrestin 2 in a significant (p < 0.05) manner and to a similar degree (2.6–4.6-fold increase of membrane-associated β-arrestin upon RANTES stimulation) to that of wild type CCR5. In contrast, alanine mutation of any three C-terminal serine residues essentially eliminated the ability of the receptor to recruit β-arrestins. Overall, no significant differences were observed in the binding of the two β-arrestin isoforms to the various receptor mutants. We conclude from these experiments that β-arrestin 1 or β-arrestin 2 binding to ligand-activated CCR5 requires the presence of at least two intact C-terminal receptor phosphorylation sites, but the exact position of these sites is not critical. Next, we asked how the abilities of various RBL-CCR5 mutants to induce β-arrestin membrane translocation correlate with their capacities to internalize activated receptors into the intracellular membrane compartment of RBL cells. We had previously reported that125I-RANTES is rapidly taken up by RBL-CCR5 cells and that endocytosis is significantly impaired in RBL cells that express a phosphorylation-deficient CCR5 mutant with alanine mutation of all four serine residues (20Kraft 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). We now show (Fig.3) that internalization of receptor mutants with alanine replacements of any single or two serine residues proceeds in a time-dependent manner that closely resembles wild type receptor internalization. In contrast, substitution of three C-terminal serine residues by alanine in various combinations significantly impaired the early rapid phase of ligand uptake to a similar degree as was observed with the completely phosphorylation-deficient CCR5-Δ4 mutant. Again, the exact position of the various mutated C-terminal phosphorylation sites did not appear to be critical. In a previous study we had shown that phosphorylation-deficient CCR5 mutants that do not b" @default.
• W2021100900 created "2016-06-24" @default.
• W2021100900 creator A5001728125 @default.
• W2021100900 creator A5003085792 @default.
• W2021100900 creator A5011485372 @default.
• W2021100900 creator A5027813230 @default.
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• W2021100900 date "2002-08-01" @default.
• W2021100900 modified "2023-10-16" @default.
• W2021100900 title "β-Arrestin Binding to CC Chemokine Receptor 5 Requires Multiple C-terminal Receptor Phosphorylation Sites and Involves a Conserved Asp-Arg-Tyr Sequence Motif" @default.
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