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• W2117501535 abstract "CCR5 is the major coreceptor for macrophage-tropic human immunodeficiency virus type I (HIV-1). For most G-protein-coupled receptors that have been tested so far, the disulfide bonds linking together the extracellular loops (ECL) are required for maintaining the structural integrity necessary for ligand binding and receptor activation. A natural mutation affecting Cys20, which is thought to form a disulfide bond with Cys269, has been described in various human populations, although the consequences of this mutation for CCR5 function are not known. Using site-directed mutagenesis, we mutated the four extracellular cysteines of CCR5 singly or in combination to investigate their role in maintaining the structural conformation of the receptor, its ligand binding and signal transduction properties, and its ability to function as a viral coreceptor. Alanine substitution of any single Cys residue reduced surface expression levels by 40–70%. However, mutation of Cys101 or Cys178, predicted to link ECL1 and ECL2 of the receptor, abolished recognition of CCR5 by a panel of conformation sensitive anti-CCR5 antibodies. The effects of the mutations on receptor expression and conformation were partially temperature-sensitive, with partial restoration of receptor expression and conformation achieved by incubating cells at 32 °C. All cysteine mutants were unable to bind detectable levels of MIP-1β, and did not respond functionally to CCR5 agonists. Surprisingly, all cysteine mutants did support infection by R5 strains of HIV, though at reduced levels. These results indicate that both disulfide bonds of CCR5 are necessary for maintaining the structural integrity of the receptor necessary for ligand binding and signaling. Env binding and the mechanisms of HIV entry appear much less sensitive to alterations of CCR5 conformation. CCR5 is the major coreceptor for macrophage-tropic human immunodeficiency virus type I (HIV-1). For most G-protein-coupled receptors that have been tested so far, the disulfide bonds linking together the extracellular loops (ECL) are required for maintaining the structural integrity necessary for ligand binding and receptor activation. A natural mutation affecting Cys20, which is thought to form a disulfide bond with Cys269, has been described in various human populations, although the consequences of this mutation for CCR5 function are not known. Using site-directed mutagenesis, we mutated the four extracellular cysteines of CCR5 singly or in combination to investigate their role in maintaining the structural conformation of the receptor, its ligand binding and signal transduction properties, and its ability to function as a viral coreceptor. Alanine substitution of any single Cys residue reduced surface expression levels by 40–70%. However, mutation of Cys101 or Cys178, predicted to link ECL1 and ECL2 of the receptor, abolished recognition of CCR5 by a panel of conformation sensitive anti-CCR5 antibodies. The effects of the mutations on receptor expression and conformation were partially temperature-sensitive, with partial restoration of receptor expression and conformation achieved by incubating cells at 32 °C. All cysteine mutants were unable to bind detectable levels of MIP-1β, and did not respond functionally to CCR5 agonists. Surprisingly, all cysteine mutants did support infection by R5 strains of HIV, though at reduced levels. These results indicate that both disulfide bonds of CCR5 are necessary for maintaining the structural integrity of the receptor necessary for ligand binding and signaling. Env binding and the mechanisms of HIV entry appear much less sensitive to alterations of CCR5 conformation. human immunodeficiency virus monoclonal antibody G-protein-coupled receptor phosphate-buffered saline Chinese hamster ovary Dulbecco's modified Eagle's medium fluorescence-activated cell sorting bovine serum albumin mean channel fluorescence wild-type dithiothreitol The entry of human immunodeficiency virus type-1 (HIV-1)1 into its target cells requires the interaction of the envelope glycoprotein (gp120) with CD4 and a coreceptor belonging to the rhodopsin-like G-protein-coupled receptor (GPCR) family. CCR5, a chemokine receptor for MIP-1α, MIP-1β, MCP-2, and RANTES (1Samson M. Labbe O. Mollereau C. Vassart G. Parmentier M. Biochemistry. 1996; 35: 3362-3367Crossref PubMed Scopus (588) Google Scholar) plays a key role in HIV-1 transmission and pathogenesis. It is used as the main coreceptor by macrophage-tropic HIV-1 strains and primary isolates that are responsible for viral transmission and which predominate during the asymptomatic phase of the disease (2Schuitemaker H. Koot M. Kootstra N.A. Dercksen M.W. de Goede R.E. van Steenwijk R.P. Lange J.M. Schattenkerk J.K. Miedema F. Tersmette M. J. Virol. 1992; 66: 1354-1360Crossref PubMed Google Scholar, 3Schuitemaker H. Kootstra N.A. de Goede R.E. de Wolf F. Miedema F. Tersmette M. J. Virol. 1991; 65: 356-363Crossref PubMed Google Scholar). The central role of CCR5 in HIV pathogenesis was demonstrated by the occurrence of a deletion mutant of CCR5 (CCR5Δ32 or Δccr5), frequent in populations of European origin, that confers a strong, although incomplete, protection to homozygotes (4Samson M. Libert F. Doranz B.J. Rucker J. Liesnard C. Farber C.M. Saragosti S. Lapoumeroulie C. Cognaux J. Forceille C. Muyldermans G. Verhofstede C. Burtonboy G. Georges M. Imai T. Rana S. Yi Y. Smyth R.J. Collman R.G. Doms R.W. Vassart G. Parmentier M. Nature. 1996; 382: 722-725Crossref PubMed Scopus (2454) Google Scholar, 5Liu R. Paxton W.A. Choe S. Ceradini D. Martin S.R. Horuk R. MacDonald M.E. Stuhlmann H. Koup R.A. Landau N.R. Cell. 1996; 86: 367-377Abstract Full Text Full Text PDF PubMed Scopus (2567) Google Scholar, 6Michael N.L. Nelson J.A. KewalRamani V.N. Chang G. O'Brien S.J. Mascola J.R. Volsky B. Louder M. White G.C. Littman D.R. Swanstrom R. O'Brien T.R. J. Virol. 1998; 72: 6040-6047Crossref PubMed Google Scholar, 7O'Brien T.R. Winkler C. Dean M. Nelson J.A. Carrington M. Michael N.L. White G.C. Lancet. 1997; 349: 1219Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). CXCR4, the receptor for the CXC chemokine SDF-1 (8Oberlin E. Amara A. Bachelerie F. Bessia C. Virelizier J.L. Arenzana-Seisdedos F. Schwartz O. Heard J.M. Clark-Lewis I. Legler D.F. Loetscher M. Baggiolini M. Moser B. Nature. 1996; 382: 833-835Crossref PubMed Scopus (1482) Google Scholar, 9Bleul C.C. Farzan M. Choe H. Parolin C. Clark-Lewis I. Sodroski J. Springer T.A. Nature. 1996; 382: 829-833Crossref PubMed Scopus (1751) Google Scholar), acts as a coreceptor for T-cell line-adapted strains of HIV-1 and T-cell tropic strains that appear during the late stages of the disease. Other GPCRs including the chemokine receptors CCR8, CCR2, CCR3, and CX3CR1, and the orphan receptors BOB/GPR15, Bonzo/STRL33, GPR1, APJ, and ChemR23 have been shown to function as co-receptors in vitro for subsets of HIV-1, HIV-2, or simian immunodeficiency virus strains, but their role in vivo remains to be clarified (10Rucker J. Edinger A.L. Sharron M. Samson M. Lee B. Berson J.F. Yi Y. Margulies B. Collman R.G. Doranz B.J. Parmentier M. Doms R.W. J. Virol. 1997; 71: 8999-9007Crossref PubMed Google Scholar, 11Samson M. Edinger A.L. Stordeur P. Rucker J. Verhasselt V. Sharron M. Govaerts C. Mollereau C. Vassart G. Doms R.W. Parmentier M. Eur. J. Immunol. 1998; 28: 1689-1700Crossref PubMed Scopus (203) Google Scholar, 12Edinger A.L. Hoffman T.L. Sharron M. Lee B. Yi Y. Choe W. Kolson D.L. Mitrovic B. Zhou Y. Faulds D. Collman R.G. Hesselgesser J. Horuk R. Doms R.W. J. Virol. 1998; 72: 7934-7940Crossref PubMed Google Scholar, 13Choe H. Farzan M. Konkel M. Martin K. Sun Y. Marcon L. Cayabyab M. Berman M. Dorf M.E. Gerard N. Gerard C. Sodroski J. J. Virol. 1998; 72: 6113-6118Crossref PubMed Google Scholar, 14Deng H.K. Unutmaz D. KewalRamani V.N. Littman D.R. Nature. 1997; 388: 296-300Crossref PubMed Scopus (599) Google Scholar, 15Alkhatib G. Liao F. Berger E.A. Farber J.M. Peden K.W. Nature. 1997; 388: 238Crossref PubMed Scopus (122) Google Scholar, 16Farzan M. Choe H. Martin K. Marcon L. Hofmann W. Karlsson G. Sun Y. Barrett P. Marchand N. Sullivan N. Gerard N. Gerard C. Sodroski J. J. Exp. Med. 1997; 186: 405-411Crossref PubMed Scopus (253) Google Scholar, 17Liao F. Alkhatib G. Peden K.W. Sharma G. Berger E.A. Farber J.M. J. Exp. Med. 1997; 185: 2015-2023Crossref PubMed Scopus (343) Google Scholar, 18Reeves J.D. McKnight A. Potempa S. Simmons G. Gray P.W. Power C.A. Wells T. Weiss R.A. Talbot S.J. Virology. 1997; 231: 130-134Crossref PubMed Scopus (132) Google Scholar). Structure-function studies performed by various groups have shown the involvement of all extracellular domains of CCR5 in its HIV coreceptor function, particularly the N-terminal domain and second extracellular loop (19Rucker J. Samson M. Doranz B.J. Libert F. Berson J.F. Yi Y. Smyth R.J. Collman R.G. Broder C.C. Vassart G. Doms R.W. Parmentier M. Cell. 1996; 87: 437-446Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 20Atchison R.E. Gosling J. Monteclaro F.S. Franci C. Digilio L. Charo I.F. Goldsmith M.A. Science. 1996; 274: 1924-1926Crossref PubMed Scopus (279) Google Scholar, 21Bieniasz P.D. Fridell R.A. Aramori I. Ferguson S.S. Caron M.G. Cullen B.R. EMBO J. 1997; 16: 2599-2609Crossref PubMed Scopus (201) Google Scholar, 22Alkhatib G. Berger E.A. Murphy P.M. Pease J.E. J. Biol. Chem. 1997; 272: 20420-20426Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 23Picard L. Simmons G. Power C.A. Meyer A. Weiss R.A. Clapham P.R. J. Virol. 1997; 71: 5003-5011Crossref PubMed Google Scholar). In addition, the second extracellular loop of CCR5 is responsible for the binding selectivity of the receptor, and determines the range of chemokines to which it responds functionally (24Samson M. LaRosa G. Libert F. Paindavoine P. Detheux M. Vassart G. Parmentier M. J. Biol. Chem. 1997; 272: 24934-24941Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Detailed antigenic mapping of CCR5 also indicates that CCR5 is structurally complex, with the reactivity of many antibodies being dependent upon multiple CCR5 domains (25Lee B. Sharron M. Blanpain C. Doranz B.J. Vakili J. Setoh P. Berg E. Liu G. Guy H.R. Durell S.R. Parmentier M. Chang C.N. Gaylord H. Tsang M. Doms R.W. J. Biol. Chem. 1999; 274: 9617-9626Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Disulfide bonds linking extracellular domains of GPCRs are thought to be important for maintaining the conformational integrity of the receptor, and in particular for allowing ligand access to the binding pocket (26Perlman J.H. Wang W. Nussenzveig D.R. Gershengorn M.C. J. Biol. Chem. 1995; 270: 24682-24685Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Within the rhodopsin family of GPCRs, two cysteines are almost invariably present that form a disulfide bond linking the first and second extracellular loops (26Perlman J.H. Wang W. Nussenzveig D.R. Gershengorn M.C. J. Biol. Chem. 1995; 270: 24682-24685Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 27Fraser C.M. J. Biol. Chem. 1989; 264: 9266-9270Abstract Full Text PDF PubMed Google Scholar, 28Dohlman H.G. Caron M.G. DeBlasi A. Frielle T. Lefkowitz R.J. Biochemistry. 1990; 29: 2335-2342Crossref PubMed Scopus (192) Google Scholar, 29Savarese T.M. Wang C.D. Fraser C.M. J. Biol. Chem. 1992; 267: 11439-11448Abstract Full Text PDF PubMed Google Scholar). Mutation of these cysteines in the β2-adrenergic, muscarinic M1, and thyrotropin-releasing hormone receptors results in a dramatic impairment of receptor function (27Fraser C.M. J. Biol. Chem. 1989; 264: 9266-9270Abstract Full Text PDF PubMed Google Scholar, 28Dohlman H.G. Caron M.G. DeBlasi A. Frielle T. Lefkowitz R.J. Biochemistry. 1990; 29: 2335-2342Crossref PubMed Scopus (192) Google Scholar, 30Noda K. Saad Y. Graham R.M. Karnik S.S. J. Biol. Chem. 1994; 269: 6743-6752Abstract Full Text PDF PubMed Google Scholar). A few rhodopsin-like G protein-coupled receptors, such as the CB1 cannabinoid receptor and the mas oncogene do not share this conserved disulfide bond (31Gerard C.M. Mollereau C. Vassart G. Parmentier M. Biochem. J. 1991; 279: 129-134Crossref PubMed Scopus (600) Google Scholar, 32Young D. Waitches G. Birchmeier C. Fasano O. Wigler M. Cell. 1986; 45: 711-719Abstract Full Text PDF PubMed Scopus (326) Google Scholar). In addition to this conserved disulfide bridge, the various chemokine receptors have in common two additional cysteines located in the N-terminal domain and the third extracellular loop. These two cysteines are thought to form a second disulfide bond (33Baggiolini M. Dewald B. Moser B. Annu. Rev. Immunol. 1997; 15: 675-705Crossref PubMed Scopus (1986) Google Scholar), which is expected to provide an additional structural constraint that contributes to the stability and conformation of the receptor. Indeed, all GPCRs that have been demonstrated to interact with chemokines contain these additional Cys residues, suggesting that the structure imposed by the additional bond is essential for the interaction of the receptors with the common core structure of their ligands. A natural variant of CCR5 has been described in which the N-terminal domain Cys20 is replaced by Ser (34Carrington M. Kissner T. Gerrard B. Ivanov S. O'Brien S.J. Dean M. Am. J. Hum. Genet. 1997; 61: 1261-1267Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Although the consequences of this mutation on receptor function was not described, this allele was found in two seropositive non-progressors, suggesting that the mutant receptor could play a role similar to that of the prevalent CCR5 mutant allele CCR5Δ32. In order to address the role of the disulfide bonds of CCR5 for its receptor and coreceptor functions and to investigate the consequences of the Cys20polymorphism, we substituted alanine residues for the four extracellular cysteines, individually or in combination. The various mutants were tested for their ability to bind chemokines, to respond functionally to agonists and to allow infection by macrophage-tropic HIV-1 strains. The mutants were also tested by flow cytometry, using a panel of well defined monoclonal antibodies to determine surface expression and/or modifications of receptor conformation. We show that alanine substitution of any of the four conserved cysteines resulted in a moderate decrease of CCR5 expression level, but abolished completely chemokine binding and functional response to chemokines. The disulfide bond bridging extracellular loops 1 and 2 was found to be necessary for maintaining the complex conformational structure of the extracellular domains. Finally, none of the disulfide bonds was an absolute requirement for the coreceptor function of CCR5. Recombinant MIP-1β was obtained from R&D Systems. It was shown locally to be active at adequate concentrations on wild-type CCR5 expressed in CHO-K1 cells. The lyophilized chemokine was dissolved as a 10 μm solution in sterile phosphate-buffered saline (PBS) and stored at −20 °C in aliquots. It was diluted to the working concentration immediately before use.125I-MIP-1β (specific activity, 2200 Ci/mmol) was obtained from NEN Life Science Products. A plasmid containing the coding region of the CCR5 gene (1Samson M. Labbe O. Mollereau C. Vassart G. Parmentier M. Biochemistry. 1996; 35: 3362-3367Crossref PubMed Scopus (588) Google Scholar) in the pcDNA3 vector (Stratagene) was used as a template for site directed mutagenesis using the QuickChange kit (Stratagene) as described by the manufacturer. After sequencing, the mutated cassettes were transferred into pcDNA3-CCR5. Double and quadruple mutants were generated by further transfer of mutated cassettes. The mutant CCR5 genes were also transferred into a bicistronic vector (pEFIN3) as described previously (24Samson M. LaRosa G. Libert F. Paindavoine P. Detheux M. Vassart G. Parmentier M. J. Biol. Chem. 1997; 272: 24934-24941Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). All final constructs were verified by sequencing. 293T cells were maintained in DMEM (Life Technologies) supplemented with 10% fetal calf serum (HyClone), 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mmglutamine. CHO-K1 cells were cultured using Ham's F-12 medium supplemented with 10% fetal calf serum (Life Technologies, Inc.), 100 units/ml penicillin, and 100 μg/ml streptomycin (Life Technologies, Inc.). The 2D7 and the 3A9 monoclonal antibodies (35Wu L. LaRosa G. Kassam N. Gordon C.J. Heath H. Ruffing N. Chen H. Humblias J. Samson M. Parmentier M. Moore J.P. Mackay C.R. J. Exp. Med. 1997; 186: 1373-1381Crossref PubMed Scopus (331) Google Scholar, 36Wu L. Paxton W.A. Kassam N. Ruffing N. Rottman J.B. Sullivan N. Choe H. Sodroski J. Newman W. Koup R.A. Mackay C.R. J. Exp. Med. 1997; 185: 1681-1691Crossref PubMed Scopus (639) Google Scholar) were kindly provided by Charles Mackay (Leukocyte). The monoclonal antibodies CTC5 and CTC8 were received from Protein Design Labs (25Lee B. Sharron M. Blanpain C. Doranz B.J. Vakili J. Setoh P. Berg E. Liu G. Guy H.R. Durell S.R. Parmentier M. Chang C.N. Gaylord H. Tsang M. Doms R.W. J. Biol. Chem. 1999; 274: 9617-9626Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). The monoclonal antibodies 501, 531, and 549 were donated by R&D Systems (25Lee B. Sharron M. Blanpain C. Doranz B.J. Vakili J. Setoh P. Berg E. Liu G. Guy H.R. Durell S.R. Parmentier M. Chang C.N. Gaylord H. Tsang M. Doms R.W. J. Biol. Chem. 1999; 274: 9617-9626Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). 293T cells were transfected with wild-type CCR5 or the various cysteine mutants in the pcDNA3 vector using the calcium phosphate method (37Lee B. Doranz B.J. Rana S. Yi Y. Mellado M. Frade J.M. Martinez A. O'Brien S.J. Dean M. Collman R.G. Doms R.W. J. Virol. 1998; 72: 7450-7458Crossref PubMed Google Scholar). 18 h following transfection, the cells were suspended in PBS containing 2 mm EDTA and washed with PBS. The cells were incubated in FACS staining buffer (PBS supplemented with 2.5% calf serum, 0.5% BSA, and 0.02% sodium azide), containing 10 μg/ml monoclonal antibody, followed by a phycoerythrin-conjugated horse anti-mouse secondary antibody (Vector Laboratories) at a 1/100 dilution. The concentration used for the monoclonal antibodies was at least 2-fold the saturating concentration. FACS analysis was performed on a FACScan flow cytometer using the CellQuest software (Becton Dickinson). The mean channel fluorescence (MCF) was used to compare the levels of receptor expression at the cell surface. Results were normalized for the MCF obtained for a particular antibody against wild-type (wt) CCR5 (normalized as 100%) after subtraction of the background MCF obtained against pcDNA3 transfected cells (normalized as 0%). A plasmid encoding apoaequorin and Gα16 under control of the SRα promoter (38Takebe Y. Seiki M. Fujisawa J. Hoy P. Yokota K. Arai K. Yoshida M. Arai N. Mol. Cell. Biol. 1988; 8: 466-472Crossref PubMed Google Scholar) was transfected into CHO-K1 cells, using Fusgene 6 (Roche Molecular Biochemicals). Zeocin (250 μg/ml, Invitrogen) selection of transfectants was initiated 2 days after transfection, and 3 weeks later, stably transfected cell lines were cloned by limit dilution. The best responding clone was selected on the basis of the functional responses to ionomycin A (100 nm) and ATP (10 μm). Constructs encoding wild-type CCR5 and alanine substitution mutants were transfected using Fusgene 6 in this apoaequorin expressing cell line. Selection of stably transfected cells was made for 14 days with 400 μg/ml G418 (Life Technologies), and the population of mixed cell clones expressing each of the constructs was used for binding and functional studies. The level of receptor expression was measured by flow cytometry using antibodies directed against the N terminus (3A9) and second extracellular loop (2D7) of CCR5. Transfected CHO-K1 cells stably expressing wild-type or mutant CCR5 were collected from plates with Ca2+/Mg2+-free PBS supplemented with 5 mm EDTA, gently pelleted for 2 min at 1000 ×g and resuspended in binding buffer (50 mmHepes, pH 7.4, 1 mm CaCl2, 5 mmMgCl2, 0.5% BSA). Competition binding assays were performed in Minisorb tubes (Nunc), using 0.24 mm125I-MIP-1β (2200 Ci/mmol, NEN Life Science Products) as tracer, variable concentrations of competitors and 40,000 cells in a final volume of 0.1 ml. Total binding was measured in the absence of competitor and nonspecific binding was measured with a 100-fold excess of unlabeled ligand. Samples were incubated for 90 min at 27 °C, then bound tracer was separated by filtration through GF/B filters presoaked in 1% BSA. Filters were counted in a β-scintillation counter. Binding parameters were determined with PRISM software (Graph-Pad software) using nonlinear regression applied to a one-site competition model. Dithiothreitol (DTT) treatment on wtCCR5 was carried out with 100 nm at 37° for 1 h. Cells were than washed three times, and binding experiments were performed as described above. Functional response to chemokines was analyzed by measuring the luminescence of aequorin as described (39Stables J. Green A. Marshall F. Fraser N. Knight E. Sautel M. Milligan G. Lee M. Rees S. Anal. Biochem. 1997; 252: 115-126Crossref PubMed Scopus (182) Google Scholar). The stably transfected cell lines were collected from plates with Ca2+/Mg2+-free DMEM supplemented with 5 mm EDTA, pelleted for 2 min at 1000 × g, resuspended in DMEM at a density of 5 × 106 cells/ml and incubated for 2 h in the dark in the presence of coelenterazine H (Molecular Probes) at a final concentration of 5 μm. Cells were diluted 7.5-fold before use. Agonists in a volume of 50 μl were added to 50 μl of cell suspension (33,000 cells), and luminescence was measured for 30 s in a Packard luminometer. Plasmids encoding the HIV-1 ADA and BaL Env. were provided by John Moore (Aaron Diamond AIDS Research Center, New York, NY). The NL4–3 luciferase virus backbone (pNL-Luc-E− R−) was provided by Ned Landau (Aaron Diamond AIDS Research Center). Luciferase reporter viruses were prepared as described previously by cotransfecting 293T cells with the indicated Env and the NL4–3 luciferase virus backbone (40Connor R.I. Chen B.K. Choe S. Landau N.R. Virology. 1995; 206: 935-944Crossref PubMed Scopus (1092) Google Scholar). Target cells were prepared by co-transfecting 293T cells with CD4 and a constant amount of appropriate coreceptor cloned in pcDNA3 vector. Incubation was done at 37 or 32 °C as indicated. Four days after infection, cells were lysed with 0.5% Triton X-100 in PBS, and an appropriate aliquot was analyzed for luciferase activity. The four extracellular cysteines of CCR5 believed to be involved in the formation of disulfide bonds were mutated to alanine, individually or in combination. A schematic representation of the CCR5 extracellular domains highlighting the position of the four cysteines and the location of the two putative bonds is presented in Fig. 1. The disulfide bond linking Cys101 and Cys178 is common to most G protein-coupled receptors, while the bond linking Cys20 and Cys269 is specific to chemokine receptors and is not found outside this subfamily. Plasmids encoding the mutant receptors were transfected into 293T cells and cell surface expression evaluated by FACS using two monoclonal antibodies (mAbs) directed against linear epitopes located within the N-terminal domain of CCR5 (CTC5 and CTC8, Figs. 1 and2 a). CTC5 recognizes the very distal part of CCR5 N terminus, including Asp2; CTC8 reacts with a more proximal region involving Tyr10 to Asn13 (25Lee B. Sharron M. Blanpain C. Doranz B.J. Vakili J. Setoh P. Berg E. Liu G. Guy H.R. Durell S.R. Parmentier M. Chang C.N. Gaylord H. Tsang M. Doms R.W. J. Biol. Chem. 1999; 274: 9617-9626Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). The location of these epitopes coupled with the fact that these mAbs recognize fully denatured and reduced CCR5 by Western blot argues that binding of these mAbs to CCR5 is unlikely to be affected by the Cys mutations, either directly or indirectly through altered receptor conformation. Using these two mAbs, we found that surface expression of the CCR5 mutants was consistently reduced by 40–70%, demonstrating that the disulfide bonds are dispensable for surface expression but nevertheless contribute to the generation of a receptor conformation compatible with efficient trafficking to the cell surface. Simultaneous substitution of the two cysteines involved in a putative disulfide bond (e.g. Cys101 and Cys178 or Cys20 and Cys269) did not result in further impairment of surface expression, which is compatible with the hypothesis that disulfide bond disruption is more important than the actual amino acid replacements. Disruption of both disulfide bonds by concomitant replacement of the four cysteines by alanine did not affect expression more dramatically than single bond disruption.Figure 2a, FACS analysis of CCR5 mutants using different anti-CCR5 mAbs. 293T cells were transfected with wild-type CCR5 or mutant receptors, kept for 18 h at 37 °C, and stained with six mAbs directed against different domains of CCR5. CTC5 and CTC8 recognize well defined linear epitopes at the N terminus of the receptor. 2D7 and 531 recognize epitopes of the second extracellular loop, located, respectively, before and after Cys178. mAbs 501 and 549 recognize multidomain epitopes involving at least ECL1 and ECL2. Results have been normalized against the MCF values obtained with each antibody for wild-type CCR5 (set at 100%) after subtraction of background fluorescence (MCF value obtained for pcDNA3 transfected cells). The results shown are representative of two to three independent experiments (depending on the antibodies) that provided similar results (less than 10% variation between experiments). b, influence of temperature on CCR5 mutant conformation. Following transfection, the cells were incubated for 18 h at 32 °C instead of 37 °C. FACS analysis was performed with the same panel of mAbs as that used in Fig. 1. Results were normalized as for Fig. 1. Mutation of Cys101 and Cys178, or their combination, strongly impaired the recognition by multi-domain mAbs 501 and 549 without altering the surface expression of the mutants.View Large Image Figure ViewerDownload (PPT) The reduced levels of surface expression observed after mutation of any single Cys residue suggested that disulfide bond disruption could affect receptor conformation, resulting in partial impairment of receptor trafficking through the endoplasmic reticulum and Golgi complex. To test this hypothesis further, mAbs recognizing other regions of the receptors were used. mAbs 2D7 and 531 recognize conformation-dependent determinants in the second extracellular loop of CCR5 (Fig. 1). The epitope of 2D7 maps to the first part of ECL2 (35Wu L. LaRosa G. Kassam N. Gordon C.J. Heath H. Ruffing N. Chen H. Humblias J. Samson M. Parmentier M. Moore J.P. Mackay C.R. J. Exp. Med. 1997; 186: 1373-1381Crossref PubMed Scopus (331) Google Scholar) and involves Lys172 and Asp173, while that of mAb 531 maps to the second half of ECL2, from Tyr184 to Phe189. In addition, two mAbs (mAb 501 and mAb 549) which interact with multiple CCR5 extracellular domains were also used (Fig. 1). mAb 501 requires both the first and second loops of CCR5, while mAb 549 requires the combination of the N terminus and the first two extracellular loops for efficient recognition of this receptor (25Lee B. Sharron M. Blanpain C. Doranz B.J. Vakili J. Setoh P. Berg E. Liu G. Guy H.R. Durell S.R. Parmentier M. Chang C.N. Gaylord H. Tsang M. Doms R.W. J. Biol. Chem. 1999; 274: 9617-9626Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). None of the four mAbs recognizes CCR5 by Western blot. All four conformation-dependent mAbs recognized the C20A and C269A single and combination mutants as well as the N-terminal mAbs CTC5 and CTC8. Thus, disruption of the disulfide bond involving the N-terminal domain of CCR5 and the third extracellular loop did not affect conformationally sensitive antigenic determinants involving extracellular loops 1 and 2 (Fig. 2 a). By sharp contrast, 2D7 barely detected the C101A and C178A mutants, while mAbs 531, 501, and 549 were totally unable to label these mutants. Interestingly, the mutant combining C101A and C178A was recognized more efficiently by 2D7 and mAb 531 than the individual mutants (Fig. 2 a). These results are consistent with these two cysteine residues forming a disulfide bond, since elimination of one Cys residue involved in a disulfide bond leaves behind an unpaired, reactive Cys residue that has the potential to form aberrant intra- or intermolecular disulfide bonds. By contrast, simultaneous replacement of both cysteines involved in a disulfide bond will not leave an unpaired Cys, and so might result in more efficient protein folding. These data demonstrate that the disulfide bond linking ECL1 and ECL2, but not the bond linking the N terminus to ECL3, is essential for rendering the extracellular domains of CCR5 compatible with their recognition by conformation-sensitive antibodies. Mutations that affect protein folding are sometimes temperature-sensitive. Thus, a mutation that results in misfolding at 37 °C may not do so at 32 °C. To determine if the Cys mutants exhibited a temperature-sensitive phenotype, cells were incubated for 18 h at 32 °C following transfection and CCR5 expression monitored by FACS using the panel of mAbs described above (Fig.2 b). We found that incubation at 32 °C dramatically increased the relative expression level of all the CCR5 mutants as de" @default.
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• W2117501535 date "1999-07-01" @default.
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• W2117501535 title "Extracellular Cysteines of CCR5 Are Required for Chemokine Binding, but Dispensable for HIV-1 Coreceptor Activity" @default.
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• W2117501535 doi "https://doi.org/10.1074/jbc.274.27.18902" @default.
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