Artigo Acesso aberto Revisado por pares

Constitutive Activation of CCR5 and CCR2 Induced by Conformational Changes in the Conserved TXP Motif in Transmembrane Helix 2

2003; Elsevier BV; Volume: 278; Issue: 38 Linguagem: Inglês

10.1074/jbc.m303739200

ISSN

1083-351X

Autores

Diana Alvarez Arias, Jean-Marc Navenot, Wenbo Zhang, James R. Broach, Stephen C. Peiper,

Tópico(s)

Immune Cell Function and Interaction

Resumo

CCR5 is a G protein-coupled receptor for RANTES, MIP-1α, MIP-1β, and MCP-2 that functions as the front line coreceptor for human immunodeficiency virus type 1 infection. To elucidate the mechanism for CCR5 activation, this coreceptor was expressed in yeast coupled to the pheromone response pathway and a constitutively active mutant (CAM) was derived by random mutagenesis. Conversion of Thr-82 in the highly conserved TXP motif in transmembrane helix 2 to Pro, His, Tyr, Arg, or Lys conferred autonomous signaling activity in yeast and mammalian cells. This substitution also imparted constitutive signaling to CCR2 in yeast and mammalian cells, but not CCR1, CCR3, CCR4, CXCR2, or CXCR4. The CCR5-CAM, but not the CCR2-CAM had a reduction in ligand binding affinity. Whereas the amplitude of calcium mobilization induced by RANTES stimulation was lower in the CCR5-CAM than the wild-type (WT) receptor, MCP-1 induced a higher signal in the CCR2-CAM than in CCR2-WT. The chemotactic response of CCR5-CAM(T82P) to RANTES was similar to that of CCR5-WT, but CCR5-CAM(T82K) was dramatically decreased. The chemotactic response of CCR2-WT and CCR2-CAM(T94K) were similar. These findings extend insight into the role of the TXP motif in the mechanism for CCR5 signaling. CCR2, the receptor most closely genetically related to CCR5, shared a similar signaling mechanism, but other receptors containing the TXP motif did not. The expression of CCR5 and CCR2 in yeast and the availability of variants with autonomous signaling represent critical tools for characterizing receptor antagonists and developing approaches to block their role in human diseases. CCR5 is a G protein-coupled receptor for RANTES, MIP-1α, MIP-1β, and MCP-2 that functions as the front line coreceptor for human immunodeficiency virus type 1 infection. To elucidate the mechanism for CCR5 activation, this coreceptor was expressed in yeast coupled to the pheromone response pathway and a constitutively active mutant (CAM) was derived by random mutagenesis. Conversion of Thr-82 in the highly conserved TXP motif in transmembrane helix 2 to Pro, His, Tyr, Arg, or Lys conferred autonomous signaling activity in yeast and mammalian cells. This substitution also imparted constitutive signaling to CCR2 in yeast and mammalian cells, but not CCR1, CCR3, CCR4, CXCR2, or CXCR4. The CCR5-CAM, but not the CCR2-CAM had a reduction in ligand binding affinity. Whereas the amplitude of calcium mobilization induced by RANTES stimulation was lower in the CCR5-CAM than the wild-type (WT) receptor, MCP-1 induced a higher signal in the CCR2-CAM than in CCR2-WT. The chemotactic response of CCR5-CAM(T82P) to RANTES was similar to that of CCR5-WT, but CCR5-CAM(T82K) was dramatically decreased. The chemotactic response of CCR2-WT and CCR2-CAM(T94K) were similar. These findings extend insight into the role of the TXP motif in the mechanism for CCR5 signaling. CCR2, the receptor most closely genetically related to CCR5, shared a similar signaling mechanism, but other receptors containing the TXP motif did not. The expression of CCR5 and CCR2 in yeast and the availability of variants with autonomous signaling represent critical tools for characterizing receptor antagonists and developing approaches to block their role in human diseases. CCR5 is a G protein-coupled receptor (GPCR) 1The abbreviations used are: GPCR, G protein-coupled receptor; AT-1A, angiotensin II receptor 1A; BSA, bovine serum albumin; CAM, constitutively active mutant; CHO, Chinese hamster ovary; HIV-1, human immunodeficiency virus type 1; HPLC, high pressure liquid chromatography; mAb, monoclonal antibody; MCP-1, monocyte chemoattractant protein 1; MCP-2, monocyte chemoattractant protein 2; MEM, minimum essential medium; MIP-1α, MIP-1β; macrophage inflammatory protein 1α/β; ORF, open reading frame; RANTES, regulated and normal T cell expressed and secreted; TM, transmembrane domain; GTPγS, guanosine 5′-3-O-(thio)triphosphate; WT, wild type.1The abbreviations used are: GPCR, G protein-coupled receptor; AT-1A, angiotensin II receptor 1A; BSA, bovine serum albumin; CAM, constitutively active mutant; CHO, Chinese hamster ovary; HIV-1, human immunodeficiency virus type 1; HPLC, high pressure liquid chromatography; mAb, monoclonal antibody; MCP-1, monocyte chemoattractant protein 1; MCP-2, monocyte chemoattractant protein 2; MEM, minimum essential medium; MIP-1α, MIP-1β; macrophage inflammatory protein 1α/β; ORF, open reading frame; RANTES, regulated and normal T cell expressed and secreted; TM, transmembrane domain; GTPγS, guanosine 5′-3-O-(thio)triphosphate; WT, wild type. for RANTES, MIP-1α, MIP-1β, and MCP-2 (1Samson M. Labbe O. Mollereau C. Vassart G. Parmentier M. Biochemistry. 1996; 35: 3362-3367Crossref PubMed Scopus (583) Google Scholar), members of the C-C chemokine family, that is expressed by CD4+ T-lymphocytes (2Bonecchi R. Bianchi G. Bordignon P.P. D'Ambrosio D. Lang R. Borsatti A. Sozzani S. Allavena P. Gray P.A. Mantovani A. Sinigaglia F. J. Exp. Med. 1998; 187: 129-134Crossref PubMed Scopus (1820) Google Scholar, 3Lee B. Sharron M. Montaner L.J. Weissman D. Doms R.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5215-5220Crossref PubMed Scopus (479) Google Scholar, 4Sallusto F. Lenig D. Mackay C.R. Lanzavecchia A. J. Exp. Med. 1998; 187: 875-883Crossref PubMed Scopus (1362) Google Scholar). CCR5 has been shown to function as the front line coreceptor for commonly transmitted strains of human immunodeficiency virus (HIV-1) type 1 (5Choe H. Farzan M. Sun Y. Sullivan N. Rollins B. Ponath P.D. Wu L. Mackay C.R. LaRosa G. Newman W. Gerard N. Gerard C. Sodroski J. Cell. 1996; 85: 1135-1148Abstract Full Text Full Text PDF PubMed Scopus (2078) Google Scholar, 6Wu L. Gerard N.P. Wyatt R. Choe H. Parolin C. Ruffing N. Borsetti A. Cardoso A.A. Desjardin E. Newman W. Gerard C. Sodroski J. Nature. 1996; 384: 179-183Crossref PubMed Scopus (1077) Google Scholar, 7Trkola A. Dragic T. Arthos J. Binley J.M. Olson W.C. Allaway G.P. Cheng-Mayer C. Robinson J. Maddon P.J. Moore J.P. Nature. 1996; 384: 184-187Crossref PubMed Scopus (952) Google Scholar, 8Alkhatib G. Combadiere C. Broder C.C. Feng Y. Kennedy P.E. Murphy P.M. Berger E.A. Science. 1996; 272: 1955-1958Crossref PubMed Scopus (2432) Google Scholar). The discovery that individuals homozygous for a 32 base pair deletion in the gene encoding CCR5, who lack a functional receptor, are highly resistant to HIV-1 infection, has revealed the clinical significance of this target for blocking viral entry (9Liu 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 (2527) Google Scholar, 10Rana S. Besson G. Cook D.G. Rucker J. Smyth R.J. Yi Y. Turner J.D. Guo H.H. Du J.G. Peiper S.C. Lavi E. Samson M. Libert F. Liesnard C. Vassart G. Doms R.W. Parmentier M. Collman R.G. J. Virol. 1997; 71: 3219-3227Crossref PubMed Google Scholar, 11Samson 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 (2416) Google Scholar). This insight has fueled intensive characterization of the structural basis of CCR5 interaction with the gp120 subunit of HIV-1 envelope glycoproteins and with physiologic ligands, which block coreceptor utilization by this pathologic ligand (12Doranz B.J. Lu Z.H. Rucker J. Zhang T.Y. Sharron M. Cen Y.H. Wang Z.X. Guo H.H. Du J.G. Accavitti M.A. Doms R.W. Peiper S.C. J. Virol. 1997; 71: 6305-6314Crossref PubMed Google Scholar, 13Rucker 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, 14Blanpain C. Doranz B.J. Vakili J. Rucker J. Govaerts C. Baik S.S. Lorthioir O. Migeotte I. Libert F. Baleux F. Vassart G. Doms R.W. Parmentier M. J. Biol. Chem. 1999; 274: 34719-34727Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 15Dragic T. Trkola A. Lin S.W. Nagashima K.A. Kajumo F. Zhao L. Olson W.C. Wu L. Mackay C.R. Allaway G.P. Sakmar T.P. Moore J.P. Maddon P.J. J. Virol. 1998; 72: 279-285Crossref PubMed Google Scholar, 16Farzan M. Choe H. Vaca L. Martin K. Sun Y. Desjardins E. Ruffing N. Wu L. Wyatt R. Gerard N. Gerard C. Sodroski J. J. Virol. 1998; 72: 1160-1164Crossref PubMed Google Scholar, 17Cormier E.G. Persuh M. Thompson D.A. Lin S.W. Sakmar T.P. Olson W.C. Dragic T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5762-5767Crossref PubMed Scopus (175) Google Scholar, 18Farzan M. Vasilieva N. Schnitzler C.E. Chung S. Robinson J. Gerard N.P. Gerard C. Choe H. Sodroski J. J. Biol. Chem. 2000; 275: 33516-33521Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 19Siciliano S.J. Kuhmann S.E. Weng Y. Madani N. Springer M.S. Lineberger J.E. Danzeisen R. Miller M.D. Kavanaugh M.P. DeMartino J.A. Kabat D. J. Biol. Chem. 1999; 274: 1905-1913Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 20Wang Z. Lee B. Murray J.L. Bonneau F. Sun Y. Schweickart V. Zhang T. Peiper S.C. J. Biol. Chem. 1999; 274: 28413-28419Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 21Navenot J.M. Wang Z.X. Trent J.O. Murray J.L. Hu Q.X. DeLeeuw L. Moore P.S. Chang Y. Peiper S.C. J. Mol. Biol. 2001; 313: 1181-1193Crossref PubMed Scopus (46) Google Scholar). GPCRs have seven hydrophobic helices that function as transmembrane spanning domains. In addition to this hydrophobic core, the receptor contains three intracellular loops, three extracellular loops, an N-terminal extracellular domain, and a C-terminal cytoplasmic tail. Amino acid residues in the N-terminal extracellular domain and the second extracellular loop have been shown to be critical for CCR5 utilization as a coreceptor for M-tropic gp120 subunits (14Blanpain C. Doranz B.J. Vakili J. Rucker J. Govaerts C. Baik S.S. Lorthioir O. Migeotte I. Libert F. Baleux F. Vassart G. Doms R.W. Parmentier M. J. Biol. Chem. 1999; 274: 34719-34727Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 15Dragic T. Trkola A. Lin S.W. Nagashima K.A. Kajumo F. Zhao L. Olson W.C. Wu L. Mackay C.R. Allaway G.P. Sakmar T.P. Moore J.P. Maddon P.J. J. Virol. 1998; 72: 279-285Crossref PubMed Google Scholar, 16Farzan M. Choe H. Vaca L. Martin K. Sun Y. Desjardins E. Ruffing N. Wu L. Wyatt R. Gerard N. Gerard C. Sodroski J. J. Virol. 1998; 72: 1160-1164Crossref PubMed Google Scholar, 20Wang Z. Lee B. Murray J.L. Bonneau F. Sun Y. Schweickart V. Zhang T. Peiper S.C. J. Biol. Chem. 1999; 274: 28413-28419Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 21Navenot J.M. Wang Z.X. Trent J.O. Murray J.L. Hu Q.X. DeLeeuw L. Moore P.S. Chang Y. Peiper S.C. J. Mol. Biol. 2001; 313: 1181-1193Crossref PubMed Scopus (46) Google Scholar). The utilization of coreceptor domains also has been shown to be influenced by the conformation of transmembrane-spanning domains and residues predicted to occur at the helical-cytosolic interface (19Siciliano S.J. Kuhmann S.E. Weng Y. Madani N. Springer M.S. Lineberger J.E. Danzeisen R. Miller M.D. Kavanaugh M.P. DeMartino J.A. Kabat D. J. Biol. Chem. 1999; 274: 1905-1913Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 20Wang Z. Lee B. Murray J.L. Bonneau F. Sun Y. Schweickart V. Zhang T. Peiper S.C. J. Biol. Chem. 1999; 274: 28413-28419Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). In addition to membrane fusion and viral entry, the interaction of CD4-activated gp120 with CCR5 results in receptor activation, which may increase viral replication (22Weissman D. Rabin R.L. Arthos J. Rubbert A. Dybul M. Swofford R. Venkatesan S. Farber J.M. Fauci A.S. Nature. 1997; 389: 981-985Crossref PubMed Scopus (310) Google Scholar, 23Cicala C. Arthos J. Ruiz M. Vaccarezza M. Rubbert A. Riva A. Wildt K. Cohen O. Fauci A.S. J. Immunol. 1999; 163: 420-426PubMed Google Scholar, 24Arthos J. Rubbert A. Rabin R.L. Cicala C. Machado E. Wildt K. Hanbach M. Steenbeke T.D. Swofford R. Farber J.M. Fauci A.S. J. Virol. 2000; 74: 6418-6424Crossref PubMed Scopus (90) Google Scholar). Some envelope glycoproteins demonstrate weak CCR5 signaling and have low level replication, which can be augmented by activating CCR5 with physiologic ligands (24Arthos J. Rubbert A. Rabin R.L. Cicala C. Machado E. Wildt K. Hanbach M. Steenbeke T.D. Swofford R. Farber J.M. Fauci A.S. J. Virol. 2000; 74: 6418-6424Crossref PubMed Scopus (90) Google Scholar). The mechanism for GPCR signaling has been shown to involve re-orientation of hydrophobic helices and consequent alteration in the conformation of the cytoplasmic domains (25Hubbell W.L. Cafiso D.S. Altenbach C. Nat. Struct. Biol. 2000; 7: 735-739Crossref PubMed Scopus (717) Google Scholar, 26Farrens D.L. Altenbach C. Yang K. Hubbell W.L. Khorana H.G. Science. 1996; 274: 768-770Crossref PubMed Scopus (1101) Google Scholar). The structural basis for the dynamic equilibrium between inactive and active conformations is best understood for rhodopsin. Although some insight into the mechanism for ligand binding has been developed, understanding of the mechanism for CCR5 activation is limited. Mutation of the Pro residue in a highly conserved sequence TXP in transmembrane domain (TM2) 2 of CCR5 has been shown to render the receptor refractory to conformational activation by ligand binding (27Govaerts C. Blanpain C. Deupi X. Ballet S. Ballesteros J.A. Wodak S.J. Vassart G. Pardo L. Parmentier M. J. Biol. Chem. 2001; 276: 13217-13225Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). In order to gain insight we have functionally expressed human CCR5 in Saccharomyces cerevisiae coupled to the pheromone response pathway by a hybrid Gα subunit. CCR5 variants with autonomous signaling activity were selected using this system. A randomly generated constitutively active mutant (CAM) contained a conversion of Thr-82 to Pro in the TXP sequence in TM2. This CCR5 mutant also demonstrated constitutive signaling in mammalian cells. Analogous mutations conferred autonomous signaling to CCR2, but not CCR1, CCR3, or CCR4. The conformational shift in CCR5 induced by activating mutations in Thr-82 resulted in decreased binding affinity to its ligands, but the analogous substitution in CCR2 did not alter the binding of MCP-1. Exposure of the CCR5 and CCR2 CAMs to their respective ligands activated cytosolic mobilization of calcium ions. These findings provide insight into the mechanism for CCR5 and CCR2 activation and furnish valuable reagents for developing a new generation of antagonists. Strains, Cell Lines, and Plasmids—For expression in yeast open reading frame (ORF) of human CCR5 or other chemokine receptors, was cloned into the yeast expression vector Cp4258 and the Saccharomyces cerevisiae strain CY12946 was transformed as described previously (28Zhang W.B. Navenot J.M. Haribabu B. Tamamura H. Hiramatu K. Omagari A. Pei G. Manfredi J.P. Fujii N. Broach J.R. Peiper S.C. J. Biol. Chem. 2002; 277: 24515-24521Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). For mammalian expression, CHO cells were transfected with pcDNA3.1-CCR5 or CCR2 constructs and single cell clones were derived by immunofluorescent sorting (MoFlo, Cytomation, Fort Collins, CO) with monoclonal antibodies (mAbs) 227R (ICOS, Bothell, WA) or anti-human CCR2 (R&D Systems, Minneapolis, MN), respectively. Recombinant RANTES Production—The ORF encoding the mature protein human RANTES was amplified by PCR and subcloned into the pPICZαA shuttle vector (Invitrogen, Carlsbad, CA) using unique restriction sites, EcoRI and XbaI. Pichia Pastoris were transformed with the linearized vector by electroporation. Colonies with high level expression were selected on 1000 μg/ml Zeocin YPDS plates. The protein was targeted for secretion into the culture medium and purified to homogeneity by cation exchange HPLC using an UNO™ S1 column (Bio-Rad), followed by reversed phase HPLC using a MICROSORB-MV™ C4 column (VARIAN, Walnut Creek, CA). Construction and Screening of CCR5 Mutant Library—The CCR5 ORF was subjected to random mutagenesis by PCR in the presence of manganese resulting in a mutation rate of 0.1–0.3%. This mutant library was transformed into the histidine auxotrophic CY12946 yeast strain and functionally expressed upstream of the pheromone response pathway as previously described (28Zhang W.B. Navenot J.M. Haribabu B. Tamamura H. Hiramatu K. Omagari A. Pei G. Manfredi J.P. Fujii N. Broach J.R. Peiper S.C. J. Biol. Chem. 2002; 277: 24515-24521Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Autonomous CCR5 activation of FUS1-HIS3 reporter gene allows selection of these colonies on histidine deficient medium. Plasmid DNA from positive colonies was extracted, sequenced, and retransformed into yeast for further characterization. Site-directed mutagenesis was performed using a QuikChange kit (Invitrogen). Yeast Receptor Activation Assay—Ligand-induced or autonomous receptor activation in yeast strains carrying a FUS1-HIS3 reporter gene was evaluated by growth on histidine-deficient medium. Receptor activation in yeast strains carrying a FUS1-lacZ gene was performed using a fluorescein di-β-d-galactopyranoside (Molecular Probes, Eugene, OR) as described previously (28Zhang W.B. Navenot J.M. Haribabu B. Tamamura H. Hiramatu K. Omagari A. Pei G. Manfredi J.P. Fujii N. Broach J.R. Peiper S.C. J. Biol. Chem. 2002; 277: 24515-24521Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). The experimental data were normalized using β-galactosidase activity of nonstimulated wild-type (WT) CCR5. Ligand Binding and Displacement—CHO cells stably expressing CCR5 or CCR2 variants were incubated with 0.1 nm125I-MIP-1α or 125I-MCP-1 (Amersham Biosciences, Piscataway, NJ) in the presence of incremental concentrations of RANTES (Leinco Tech., St. Louis, MO), MIP-1α, MIP-1β, and MCP-2 (PeproTech, Rocky Hill, NJ) for CCR5, or MCP-1 (PeproTech) for CCR2 as described previously (21Navenot J.M. Wang Z.X. Trent J.O. Murray J.L. Hu Q.X. DeLeeuw L. Moore P.S. Chang Y. Peiper S.C. J. Mol. Biol. 2001; 313: 1181-1193Crossref PubMed Scopus (46) Google Scholar). All binding experiments were conducted on ice to prevent ligand-induced receptor internalization. The affinity was calculated using Prism (GraphPad Software, San Diego, CA) and is expressed as the EC50 ± S.D. averaged from the results obtained in three independent experiments and based on duplicate samples of each concentration. [35S]GTPγS Binding Assay—The [35S]GTPγS binding assay was carried out essentially as described (28Zhang W.B. Navenot J.M. Haribabu B. Tamamura H. Hiramatu K. Omagari A. Pei G. Manfredi J.P. Fujii N. Broach J.R. Peiper S.C. J. Biol. Chem. 2002; 277: 24515-24521Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 29Mueller A. Mahmoud N.G. Goedecke M.C. McKeating J.A. Strange P.G. Br J. Pharmacol. 2002; 135: 1033-1043Crossref PubMed Scopus (47) Google Scholar). Briefly, 7 μg of cell membranes were preincubated with 40 μm GDP (Sigma) in the absence (basal binding) or presence of an agonmist for 30 min at 30 °C before addition of [35S]GTPγS (Amersham Biosciences) to a final concentration of 0.25 nm. Nonspecific binding was determined in the presence of 10 μm GTPγS (Sigma). After incubating for another 30 min, reactions were terminated by rapid filtration through GF/C filters (Whatman Inc., Clifton, NJ). Bound radioactivity was measured by scintillation counting. Cells were treated with 100 ng/ml pertussis toxin (Calbiochem, La Jolla, CA) for 14–16 h to uncouple the Gi/oα from the receptors. The percentage of specific binding was calculated as 100 × (sample cpm – nonspecific cpm) ÷ (basal cpm – nonspecific cpm). The values are the means ± S.E. of triplicate samples, and the results are representative of four independent experiments. Calcium Mobilization—CHO cells were loaded with 2 μg/ml fura-2 acetoxymethyl ester (Molecular Probes). Agonist-dependent increases in cytoplasmic calcium were determined as described (21Navenot J.M. Wang Z.X. Trent J.O. Murray J.L. Hu Q.X. DeLeeuw L. Moore P.S. Chang Y. Peiper S.C. J. Mol. Biol. 2001; 313: 1181-1193Crossref PubMed Scopus (46) Google Scholar). The results are representative of two independent experiments. Chemotaxis Assay—Cells were resuspended in MEMα medium containing 0.5% bovine serum albumin. The cell density was adjusted to 2 × 106 cells/ml and 100 μl was added to the top chamber of 24-well transwell apparatus (6.5-mm diameter, 8.0-μm pore size; Corning Inc., Corning, NY). Agonist was added to the lower chamber. The plates were incubated for 4 h at 37 °C. Cells that passed through membranes into the lower chamber were collected and counted by flow cytometry. The chemotactic index was determined as a ratio of cells in lower chamber in the presence versus in the absence of agonist. The results are representative of three independent experiments. Derivation of CCR5 CAMs in Yeast—CCR5 was expressed in yeast coupled to the pheromone response pathway by a hybrid Gα subunit, as previously described for CXCR4 (28Zhang W.B. Navenot J.M. Haribabu B. Tamamura H. Hiramatu K. Omagari A. Pei G. Manfredi J.P. Fujii N. Broach J.R. Peiper S.C. J. Biol. Chem. 2002; 277: 24515-24521Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Histidine auxotrophic yeast strains expressing human CCR5 and a pheromone-responsive FUS1-HIS3 reporter gene grew in the absence of histidine when exposed to RANTES (Fig. 1A), demonstrating that the receptor was functional and linked to the pheromone response pathway. High concentrations of RANTES were required to activate CCR5 signaling in yeast, but chemokines that do not bind CCR5 had no effect at any concentration (data not shown). Control cells (Fig. 1A) and yeast strains expressing CXCR4 (data not shown) were not stimulated to grow in the absence of histidine when exposed to RANTES, establishing the specificity of the system. This was confirmed by the ability of RANTES to activate human CCR5 in parallel experiments in yeast strains containing a FUS1-lacZ reporter gene, resulting in expression of β-galactosidase activity (Fig. 2A, see inset).Fig. 2Characterization of CCR5 signaling variants in yeast and mammalian cells. A, analysis of FUS1-lacZ reporter gene in yeast indicated that T82P and T82K mutations conferred constitutive activity while T82G had an inhibitory effect on RANTES-induced CCR5 signaling (-▪- CCR5; -▴- T82P; -·- T82K; -·- T82G; -×- Cp4258). Inset shows the response of CCR5-WT to incremental doses of RANTES, but the absence of effect on the control strain transformed with vector alone. B, expression level of CCR5 mutants in CHO cells was determined by flow cytometry with 227R mAb. Staining with irrelevant IgG1 antibody was used as a negative control. C, [35S]GTPγS binding of CCR5 mutants in CHO cells showed increased association of Gα subunits with T82P and T82K mutants, but not the T82G mutant, in the absence of agonist. The results are representative of four independent experiments.View Large Image Figure ViewerDownload (PPT) In order to derive CCR5-CAMs, the CCR5 ORF was randomly mutated by PCR in the presence of manganese at a mutational rate of 0.1–0.3% and these cDNA pools were screened for the ability to confer histidine-independent growth in the histidine auxotrophic yeast strain containing the FUS1-HIS3 reporter gene. Screening of over 105 recombinant events for growth in the absence of histidine and CCR5 ligands yielded three clones with an A to C substitution resulting in the T82P mutation. No other activating mutation was identified among 18 autonomously growing clones that were sequenced. Yeast cells expressing CCR5(T82P) grew in histidine free medium in the absence of RANTES and this was not significantly altered by the addition of this ligand, as shown in Fig. 1B. The proliferation of cells expressing the CCR5-CAM without ligand stimulation was greater than that of those expressing CCR5-WT grown in the presence of 1 μm RANTES. Cells expressing CCR5-WT did not grow in the absence of RANTES. The specificity of WT receptor and CCR5-CAM coupling to pheromone response pathway was confirmed by inhibition of yeast proliferation with 3-amino-1, 2,4-triazole, a competitive inhibitor of histidine production (data not shown). Amino acid substitutions that confer CCR5 autonomous signaling were determined by saturation mutagenesis of Thr-82. As shown in Fig. 1C, substitution of Thr-82 with Lys or Arg resulted in high levels of basal β-galactosidase expression in yeast cells containing the FUS1-lacZ reporter gene. Conversion of Thr-82 to His, Tyr, or Pro, but not Gly, Ala, Val, Leu, Met, Trp, Asp, or Glu, resulted in CAM activity, albeit at lower levels. Exposure of CAMs to RANTES did not result in additional activation of receptor signaling. Activation of β-galactosidase expression in yeast cells expressing CCR5(T82K) or CCR5(T82P) was not significantly altered by exposure to RANTES at concentrations up to 10 μm (Fig. 2A). CCR5 CAMs Selected in Yeast Are Active in Mammalian Cells—The authenticity of the activated conformation of the CCR5-CAMs selected in yeast was determined by analysis of signaling in mammalian cells. CHO transfectants expressing CCR5-WT or the T82P, T82K, or T82G mutants were prepared by lipofection and cell sorting. Staining of the transfectants with 227R, a mAb to the N-terminal extracellular domain of CCR5, revealed that all were expressed on the surface of CHO cells at similar levels, as shown in Fig. 2B. The autonomous signaling activity of the CCR5-CAMs was determined in [35S]GTPγS binding experiments (Fig. 2C). This response was inhibited by pertussis toxin (data not shown). Membrane fractions from CHO transfectants expressing CCR5(T82P) or CCR5(T82K) have basal levels of [35S]GTPγS binding that were ∼2–3-fold higher than that of transfectants expressing the WT receptor or CCR5(T82G). Exposure to incremental concentrations of RANTES demonstrated that CCR5(T82P) was more sensitive to ligand activation than the WT receptor and the other CCR5 mutants in some experiments (Fig. 2C). The Role of TXP in Autonomous Signaling is Common to CCR5 and CCR2—Since the TXP sequence in TM2 is highly conserved in receptors for C-C and C-X-C chemokines, the analogous Thr residue in other C-C receptors was substituted with Pro or Lys to determine whether it conferred autonomous signaling. As shown in Fig. 3A, introduction of Thr → Pro or Thr → Lys conversions in CCR1, CCR3, and CCR4 had no effect on the expression of β-galactosidase activity in yeast cells containing a FUS1-lacZ reporter gene. In contrast, yeast cells containing CCR2(T94K), but not CCR2(T94P) demonstrated elevated basal levels of β-galactosidase activity. CCR2(T94K) was expressed in CHO transfectants to establish that the conformation induced by this mutation also conferred autonomous coupling to G proteins in mammalian cells. Transfectants expressing CCR2-WT or the T94K mutation were prepared by lipofection and cell sorting. The respective tranfectants expressed CCR2-WT and CCR2(T94K) at similar levels on the cell surface, as shown in Fig. 3B. The authenticity of the signaling phenotype of the CCR2-CAM was determined in [35S]GTPγS binding experiments. This response was inhibited by pertussis toxin (data not shown). Membrane fractions from transfectants expressing CCR2(T94K) had a 30–40% higher basal level of [35S]GTPγS binding than those from transfectants expressing CCR2-WT. Levels of [35S]GTPγS binding induced by stimulation of CCR2(T94K) transfectants with incremental concentrations of MCP-1 were ∼2-fold higher than that observed in transfectants expressing CCR2-WT, including the maximum level of activation. Conformational Changes in Extracellular Domains of CCR5, but not CCR2, CAMs—Ligand binding experiments were performed to determine whether the altered orientation of transmembrane helices induced by the activating mutations influences the conformation of extracellular domains. To minimize the effect of receptor internalization induced by the ligands, the experiments were conducted at 0 °C. The binding of 125I-MIP-1α by CCR5 mutants was characterized in homologous and heterologous displacement experiments. The EC50 of MIP-1α binding to CCR5(T82P) (0.9 nm) was identical to that of the WT receptor in homologous displacement experiments (Fig. 4A). In contrast, parallel experiments demonstrated that the binding of CCR5(T82K) to 125I-MIP-1α (17.2 nm) was dramatically decreased. The EC50 of MIP-1α binding to CCR5(T82G) (3.2 nm) was intermediate between that observed with CCR5-WT/CCR5(T82P) and CCR5(T82K). Heterologous displacement of 125I-MIP-1α binding to these CCR5 variants by RANTES is shown in Fig. 4B. CCR5(T82P) and CCR5(T82G) demonstrated a decrease in the affinity of RANTES displacement in comparison to the WT receptor (EC50 = 2.6 nm and 2.1 nm versus 0.4 nm, respectively). The loss of affinity of CCR5(T82K) for RANTES was even more pronounced (51.6 nm) than observed for MIP-1α. The results for the displacement of 125I-MIP-1α by MIP-1β illustrate a similar trend with MIP-1β having the highest affinity for CCR5-WT (3.9 nm) and the lowest for CCR5(T82K) (20.7 nm), even though in that case the difference was only about 5-fold (Fig. 4C). When MCP-2 was used to displace 125I-MIP-1α, its binding to CCR5(T82G) was similar to CCR5-WT (6.3 nm versus 4.0 nm). In contrast CCR5(T82P) showed about 3-fold decrease of affinity for this ligand. The T82K mutation resulted in an almost complete loss of binding of MCP-2 (ab

Referência(s)