Journal of Biological Chemistry · 2010 · 75 citations · 26 references
MedicineCell BiologyPharmacologyCell SignalingMembrane TraffickingMolecular PharmacologyChemokine SignalingG Protein-coupled Receptor SignalingLigand BindingReceptor DesensitizationReceptor InternalizationReceptor RegulationCell Surface Receptor TraffickingChemokine Receptor AntagonismChemokine Receptor TraffickingChemokine-mediated TraffickingDesensitization In Chemokine ReceptorsG Protein-coupled Chemokine ReceptorsReceptor Trafficking DynamicsTrans-golgi Transport
CC chemokine receptor 5 (CCR5), the major HIV coreceptor, is a G protein-coupled receptor (GPCR) involved in cell activation and migration in response to chemokines. Blockade of CCR5 is an effective anti-HIV strategy, and potent anti-HIV chemokine analogs such as PSC-RANTES have been developed. These inhibitors act by interfering with receptor trafficking, thereby inducing prolonged intracellular sequestration of CCR5. Like many GPCRs, CCR5 is desensitized following agonist activation. The initial steps in this process are well understood, but later stages, including where CCR5 is sequestered during desensitization, and how anti-HIV chemokine analogs intervene to achieve prolonged sequestration, have yet to be elucidated in detail. In this study we demonstrate that CCR5 cycles to and from the cell surface via the endosome recycling compartment and the trans-Golgi network during desensitization, accumulating in the trans-Golgi network following internalization by both PSC-RANTES and CCL5, the native ligand from which it was derived. In addition, we show that unlike CCR5 sequestered by CCL5, CCR5 sequestered by PSC-RANTES cannot be induced to return to the cell surface by addition of the small molecule CCR5 inhibitor, TAK-779, and that association of PSC-RANTES with CCR5 is more durable than that of native CCL5 during desensitization. Our findings reconcile the previously conflicting descriptions of the location of sequestered CCR5 during desensitization, as well as providing more general insights into potential trafficking routes for endocytosed GPCRs and further elucidation of the unusual inhibitory mechanism of chemokine analogs with potent anti-HIV activity. CC chemokine receptor 5 (CCR5), the major HIV coreceptor, is a G protein-coupled receptor (GPCR) involved in cell activation and migration in response to chemokines. Blockade of CCR5 is an effective anti-HIV strategy, and potent anti-HIV chemokine analogs such as PSC-RANTES have been developed. These inhibitors act by interfering with receptor trafficking, thereby inducing prolonged intracellular sequestration of CCR5. Like many GPCRs, CCR5 is desensitized following agonist activation. The initial steps in this process are well understood, but later stages, including where CCR5 is sequestered during desensitization, and how anti-HIV chemokine analogs intervene to achieve prolonged sequestration, have yet to be elucidated in detail. In this study we demonstrate that CCR5 cycles to and from the cell surface via the endosome recycling compartment and the trans-Golgi network during desensitization, accumulating in the trans-Golgi network following internalization by both PSC-RANTES and CCL5, the native ligand from which it was derived. In addition, we show that unlike CCR5 sequestered by CCL5, CCR5 sequestered by PSC-RANTES cannot be induced to return to the cell surface by addition of the small molecule CCR5 inhibitor, TAK-779, and that association of PSC-RANTES with CCR5 is more durable than that of native CCL5 during desensitization. Our findings reconcile the previously conflicting descriptions of the location of sequestered CCR5 during desensitization, as well as providing more general insights into potential trafficking routes for endocytosed GPCRs and further elucidation of the unusual inhibitory mechanism of chemokine analogs with potent anti-HIV activity. IntroductionReceptor down-modulation, i.e. reduction of cell surface receptor concentration, is a component of the well characterized desensitization/resensitization process that is seen across the G protein-coupled receptor (GPCR) 2The abbreviations used are: GPCRG protein-coupled receptorCCR5CC chemokine receptor 5CCLCC chemokine ligandCHOChinese hamster ovaryERCendosome recycling compartmentTftransferrinTGNtrans-Golgi networkTRITCtetramethylrhodamine isothiocyanate. superfamily (1.Hanyaloglu A.C. von Zastrow M. Annu. Rev. Pharmacol. Toxicol. 2008; 48: 537-568Crossref PubMed Scopus (467) Google Scholar, 2.Marchese A. Paing M.M. Temple B.R. Trejo J. Annu. Rev. Pharmacol. Toxicol. 2008; 48: 601-629Crossref PubMed Scopus (350) Google Scholar). Following endocytosis, GPCRs are either routed for degradation or recycled back to the cell surface in a resensitized form (2.Marchese A. Paing M.M. Temple B.R. Trejo J. Annu. Rev. Pharmacol. Toxicol. 2008; 48: 601-629Crossref PubMed Scopus (350) Google Scholar). Many details of this sorting process are currently unknown, however, and its study represents an emerging field in GPCR biology (1.Hanyaloglu A.C. von Zastrow M. Annu. Rev. Pharmacol. Toxicol. 2008; 48: 537-568Crossref PubMed Scopus (467) Google Scholar).The chemokine receptor CCR5 is a GPCR expressed predominantly on leukocyte subsets, with a principal physiological role in the recruitment of effector cells to sites of inflammation (3.Lederman M.M. Ann. Intern. Med. 1995; 122: 218-222Crossref PubMed Scopus (18) Google Scholar). CCR5 also acts as an HIV coreceptor and plays a key role both in HIV transmission from person to person and in disease progression once infection has occurred (3.Lederman M.M. Ann. Intern. Med. 1995; 122: 218-222Crossref PubMed Scopus (18) Google Scholar). As a consequence, CCR5 is a key target for anti-HIV strategies directed toward both prevention and therapy (4.Kuhmann S.E. Hartley O. Annu. Rev. Pharmacol. Toxicol. 2008; 48: 425-461Crossref PubMed Scopus (60) Google Scholar).The physiological ligands of CCR5, CCL5, CCL3, and CCL4, have intrinsic anti-HIV activity (5.Cocchi F. DeVico A.L. Garzino-Demo A. Arya S.K. Gallo R.C. Lusso P. Science. 1995; 270: 1811-1815Crossref PubMed Scopus (2621) Google Scholar), resulting from both steric blockade of coreceptor interaction with the HIV envelope glycoprotein complex (6.Trkola A. Paxton W.A. Monard S.P. Hoxie J.A. Siani M.A. Thompson D.A. Wu L. Mackay C.R. Horuk R. Moore J.P. J. Virol. 1998; 72: 396-404Crossref PubMed Google Scholar) and induction of CCR5 down-modulation (7.Alkhatib G. Locati M. Kennedy P.E. Murphy P.M. Berger E.A. Virology. 1997; 234: 340-348Crossref PubMed Scopus (198) Google Scholar). N-terminally modified analogs of CCR5 ligands, including AOP-RANTES (8.Simmons G. Clapham P.R. Picard L. Offord R.E. Rosenkilde M.M. Schwartz T.W. Buser R. Wells T.N. Proudfoot A.E. Science. 1997; 276: 276-279Crossref PubMed Scopus (591) Google Scholar) and PSC-RANTES (9.Hartley O. Gaertner H. Wilken J. Thompson D. Fish R. Ramos A. Pastore C. Dufour B. Cerini F. Melotti A. Heveker N. Picard L. Alizon M. Mosier D. Kent S. Offord R. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 16460-16465Crossref PubMed Scopus (136) Google Scholar), exhibit much higher anti-HIV activity than the physiological ligands. Although it is clear that the enhanced potency of these analogs is due to induction of stronger and more prolonged CCR5 down-modulation (9.Hartley O. Gaertner H. Wilken J. Thompson D. Fish R. Ramos A. Pastore C. Dufour B. Cerini F. Melotti A. Heveker N. Picard L. Alizon M. Mosier D. Kent S. Offord R. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 16460-16465Crossref PubMed Scopus (136) Google Scholar, 10.Mack 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 (375) Google Scholar), the details of this unusual inhibitory mechanism have yet to be fully elucidated.The early steps of CCR5 desensitization, which have been quite extensively characterized (for review, see Ref. 11.Oppermann M. Cell. Signal. 2004; 16: 1201-1210Crossref PubMed Scopus (246) Google Scholar), involve processes common to many GPCRs: C-terminal phosphorylation, association with β-arrestin proteins, and clathrin-dependent endocytosis. The subsequent steps are less clearly understood, however. First, although it has been established that CCR5 accumulates in a perinuclear region during desensitization, several different locations for the site of accumulation have been proposed: the early endosome (12.Amara A. Gall S.L. Schwartz O. Salamero J. Montes M. Loetscher P. Baggiolini M. Virelizier J.L. Arenzana-Seisdedos F. J. Exp. Med. 1997; 186: 139-146Crossref PubMed Scopus (518) Google Scholar), the endosomal recycling compartment (ERC) (10.Mack 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 (375) Google Scholar, 13.Signoret N. Pelchen-Matthews A. Mack M. Proudfoot A.E. Marsh M. J. Cell Biol. 2000; 151: 1281-1294Crossref PubMed Scopus (160) Google Scholar, 14.Pollok-Kopp B. Schwarze K. Baradari V.K. Oppermann M. J. Biol. Chem. 2003; 278: 2190-2198Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 15.Signoret N. Christophe T. Oppermann M. Marsh M. Traffic. 2004; 5: 529-543Crossref PubMed Scopus (21) Google Scholar), and the Golgi apparatus (16.Longden J. Cooke E.L. Hill S.J. Br. J. Pharmacol. 2008; 153: 1513-1527Crossref PubMed Scopus (16) Google Scholar, 17.Kiss D.L. Longden J. Fechner G.A. Avery V.M. Cell. Mol. Biol. Lett. 2009; 14: 537-547Crossref PubMed Scopus (13) Google Scholar). Second, it has been demonstrated that during desensitization the pool of sequestered CCR5 receptors remains accessible to the cell surface by continually cycling to and from the plasma membrane (13.Signoret N. Pelchen-Matthews A. Mack M. Proudfoot A.E. Marsh M. J. Cell Biol. 2000; 151: 1281-1294Crossref PubMed Scopus (160) Google Scholar, 15.Signoret N. Christophe T. Oppermann M. Marsh M. Traffic. 2004; 5: 529-543Crossref PubMed Scopus (21) Google Scholar), but little is currently known about how the receptor resensitization process is triggered. Finally, although some progress has been made (10.Mack 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 (375) Google Scholar, 13.Signoret N. Pelchen-Matthews A. Mack M. Proudfoot A.E. Marsh M. J. Cell Biol. 2000; 151: 1281-1294Crossref PubMed Scopus (160) Google Scholar, 18.Pastore C. Picchio G.R. Galimi F. Fish R. Hartley O. Offord R.E. Mosier D.E. Antimicrob. Agents Chemother. 2003; 47: 509-517Crossref PubMed Scopus (73) Google Scholar), the mechanism by which anti-HIV chemokine analogs interfere with the desensitization process to achieve prolonged receptor down-modulation has yet to be elucidated in detail.In this study we used both native chemokine ligands and anti-HIV chemokine analogs to study CCR5 trafficking in response to ligands. Through a series of experiments using Chinese hamster ovary (CHO)-CCR5 cell lines and primary T lymphocytes, we present data that help explain the differences seen in previous work on the location of sequestered CCR5 during desensitization, as well as refining the existing mechanistic model to explain physiological CCR5 trafficking during desensitization and how it is disrupted by anti-HIV chemokine analogs.DISCUSSIONStudies of CCR5 trafficking have led to the establishment of a mechanistic model (11.Oppermann M. Cell. Signal. 2004; 16: 1201-1210Crossref PubMed Scopus (246) Google Scholar, 13.Signoret N. Pelchen-Matthews A. Mack M. Proudfoot A.E. Marsh M. J. Cell Biol. 2000; 151: 1281-1294Crossref PubMed Scopus (160) Google Scholar, 24.Signoret N. Hewlett L. Wavre S. Pelchen-Matthews A. Oppermann M. Marsh M. Mol. Biol. Cell. 2005; 16: 902-917Crossref PubMed Scopus (78) Google Scholar) in which agonist-activated CCR5 is internalized through clathrin-coated pits and delivered to a perinuclear compartment from where it is subsequently recycled to the plasma membrane in a resensitized form. Although there is general agreement about the steps involved in receptor internalization, details of the sequestration and resensitization processes and how they relate to the inhibitory mechanisms of potent anti-HIV chemokine analogs have yet to be elucidated.Reconciling Differences in Current Models for CCR5 TraffickingPast work has led to conflicting proposals for the site of sequestration during desensitization (10.Mack 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 (375) Google Scholar, 12.Amara A. Gall S.L. Schwartz O. Salamero J. Montes M. Loetscher P. Baggiolini M. Virelizier J.L. Arenzana-Seisdedos F. J. Exp. Med. 1997; 186: 139-146Crossref PubMed Scopus (518) Google Scholar, 13.Signoret N. Pelchen-Matthews A. Mack M. Proudfoot A.E. Marsh M. J. Cell Biol. 2000; 151: 1281-1294Crossref PubMed Scopus (160) Google Scholar, 14.Pollok-Kopp B. Schwarze K. Baradari V.K. Oppermann M. J. Biol. Chem. 2003; 278: 2190-2198Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 16.Longden J. Cooke E.L. Hill S.J. Br. J. Pharmacol. 2008; 153: 1513-1527Crossref PubMed Scopus (16) Google Scholar, 17.Kiss D.L. Longden J. Fechner G.A. Avery V.M. Cell. Mol. Biol. Lett. 2009; 14: 537-547Crossref PubMed Scopus (13) Google Scholar). In this study we conducted a thorough investigation of CCR5 desensitization using both physiological chemokines and anti-HIV chemokine analogs as ligands. Like the previous of CCR5 trafficking, work on with CCR5 with for different differences where of to due to of in the perinuclear of from of to of the of the cell than in of to the intracellular of CCR5 as trafficking than a this to that explain and reconcile the different in previous of the site of CCR5 sequestration during desensitization. Our experiments show that CCR5 through the during internalization a following ligand explain the sequestered CCR5 and (10.Mack 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 (375) Google Scholar, 13.Signoret N. Pelchen-Matthews A. Mack M. Proudfoot A.E. Marsh M. J. Cell Biol. 2000; 151: 1281-1294Crossref PubMed Scopus (160) Google Scholar, 14.Pollok-Kopp B. Schwarze K. Baradari V.K. Oppermann M. J. Biol. Chem. 2003; 278: 2190-2198Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). CCR5 subsequently of the to the The of the to the Golgi in the perinuclear region of the cell for the of internalized CCR5 with of the Golgi region (16.Longden J. Cooke E.L. Hill S.J. Br. J. Pharmacol. 2008; 153: 1513-1527Crossref PubMed Scopus (16) Google Scholar, 17.Kiss D.L. Longden J. Fechner G.A. Avery V.M. Cell. Mol. Biol. Lett. 2009; 14: 537-547Crossref PubMed Scopus (13) Google Scholar). to the Golgi region by with Golgi following the CCR5 and that we is of the seen by A. S. P. R. J. 2005; PubMed Scopus Google Scholar) in Golgi in cells with to with and a of the from the Cell to the via the study is the to the as the sequestration site for CCR5 during desensitization. a location for an internalized GPCR be unusual but be internalized receptors have been to to the to recycling back to the plasma membrane in B. C. K. C. P. Traffic. PubMed Scopus Google Scholar, B. G. A. C. P. A. P. J. 2008; PubMed Scopus Google Scholar). is accumulating to the of routes the and the and several have been to be from the cell surface via early to the L. Cell. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). different have been the via the by J. Cell Biol. 1998; PubMed Scopus Google Scholar) and F. T. D. A. C. B. L. J. Cell Biol. PubMed Scopus Google Scholar), and the to by the receptor J. Cell Biol. 2008; PubMed Scopus Google Scholar). Our that the via the than is the by are currently into to following internalization (2.Marchese A. Paing M.M. Temple B.R. Trejo J. Annu. Rev. Pharmacol. Toxicol. 2008; 48: 601-629Crossref PubMed Scopus (350) Google Scholar). of the are to with or ligand to of the from ligands following and are recycled back to the cell surface in a resensitized form via the Our with trafficking of the receptor B. C. K. C. P. Traffic. PubMed Scopus Google Scholar, B. G. A. C. P. A. P. J. 2008; PubMed Scopus Google Scholar), for the of a in which GPCRs for recycling a trafficking to sequestration the through a have that CCR5 sequestered by both native ligands and analogs is and accessible to the cell surface (13.Signoret N. Pelchen-Matthews A. Mack M. Proudfoot A.E. Marsh M. J. Cell Biol. 2000; 151: 1281-1294Crossref PubMed Scopus (160) Google Scholar, 15.Signoret N. Christophe T. Oppermann M. Marsh M. Traffic. 2004; 5: 529-543Crossref PubMed Scopus (21) Google Scholar), and work that this is also the for CCR5 sequestered by PSC-RANTES on the existing model for CCR5 trafficking during desensitization (13.Signoret N. Pelchen-Matthews A. Mack M. Proudfoot A.E. Marsh M. J. Cell Biol. 2000; 151: 1281-1294Crossref PubMed Scopus (160) Google Scholar, 15.Signoret N. Christophe T. Oppermann M. Marsh M. Traffic. 2004; 5: 529-543Crossref PubMed Scopus (21) Google Scholar) and we that CCR5 a trafficking that the receptor from the cell surface to the via N. Hewlett L. Wavre S. Pelchen-Matthews A. Oppermann M. Marsh M. Mol. Biol. Cell. 2005; 16: 902-917Crossref PubMed Scopus (78) Google Scholar), from the to the and from the back to the cell surface cells are the of the is receptor endocytosis, and receptors the cell CCR5, the of clathrin-dependent is and the in the from the to the cell resulting in receptor accumulation in the of TAK-779, an CCR5 inhibitor, of CCR5 to the cell receptors to the cell surface and inducing to an that receptor to the in the for the of anti-HIV chemokine analogs such as AOP-RANTES (8.Simmons G. Clapham P.R. Picard L. Offord R.E. Rosenkilde M.M. Schwartz T.W. Buser R. Wells T.N. Proudfoot A.E. Science. 1997; 276: 276-279Crossref PubMed Scopus (591) Google Scholar) and PSC-RANTES (9.Hartley O. Gaertner H. Wilken J. Thompson D. Fish R. Ramos A. Pastore C. Dufour B. Cerini F. Melotti A. Heveker N. Picard L. Alizon M. Mosier D. Kent S. Offord R. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 16460-16465Crossref PubMed Scopus (136) Google Scholar) potent anti-HIV activity to to more and more prolonged intracellular CCR5 sequestration than the native ligands from which they are (9.Hartley O. Gaertner H. Wilken J. Thompson D. Fish R. Ramos A. Pastore C. Dufour B. Cerini F. Melotti A. Heveker N. Picard L. Alizon M. Mosier D. Kent S. Offord R. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 16460-16465Crossref PubMed Scopus (136) Google Scholar, 10.Mack 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 (375) Google Scholar, 18.Pastore C. Picchio G.R. Galimi F. Fish R. Hartley O. Offord R.E. Mosier D.E. Antimicrob. Agents Chemother. 2003; 47: 509-517Crossref PubMed Scopus (73) Google Scholar). The in this study insights into this unusual inhibitory First, in which cells with either PSC-RANTES or CCL5 in agreement with previously work (13.Signoret N. Pelchen-Matthews A. Mack M. Proudfoot A.E. Marsh M. J. Cell Biol. 2000; 151: 1281-1294Crossref PubMed Scopus (160) Google Scholar), the enhanced CCR5 sequestration by analogs they more receptor internalization than the native ligands. Second, the CCR5 experiments that we on cells with either chemokine analogs or physiological chemokines that enhanced sequestration is a of CCR5 sequestered by analogs directed to a compartment different from that used for CCR5 sequestered by native ligands and and that enhanced sequestration analogs have the to achieve of CCR5 during the desensitization process for the prolonged association PSC-RANTES and CCR5 is that the more to CCR5 than the native although differences in CCR5 native CCL5 and PSC-RANTES have been seen in previous (9.Hartley O. Gaertner H. Wilken J. Thompson D. Fish R. Ramos A. Pastore C. Dufour B. Cerini F. Melotti A. Heveker N. Picard L. Alizon M. Mosier D. Kent S. Offord R. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 16460-16465Crossref PubMed Scopus (136) Google Scholar, H. O. Borlat Cerini F. Dufour B. G. Melotti A. Fish Offord R. M. Hartley O. 2008; PubMed Scopus Google Scholar). are that PSC-RANTES be more than native CCL5 to processes to be for N. Christophe T. Oppermann M. Marsh M. Traffic. 2004; 5: 529-543Crossref PubMed Scopus (21) Google Scholar) or that as a H. O. Borlat Cerini F. Dufour B. G. Melotti A. Fish Offord R. M. Hartley O. 2008; PubMed Scopus Google Scholar), PSC-RANTES is of CCR5 into a that thereby investigation of this help explain the and details of the inhibitory mechanism of analogs and in the of strategies to further anti-HIV that act via blockade of CCR5. IntroductionReceptor down-modulation, i.e. reduction of cell surface receptor concentration, is a component of the well characterized desensitization/resensitization process that is seen across the G protein-coupled receptor (GPCR) 2The abbreviations used are: GPCRG protein-coupled receptorCCR5CC chemokine receptor 5CCLCC chemokine ligandCHOChinese hamster ovaryERCendosome recycling compartmentTftransferrinTGNtrans-Golgi networkTRITCtetramethylrhodamine isothiocyanate. superfamily (1.Hanyaloglu A.C. von Zastrow M. Annu. Rev. Pharmacol. Toxicol. 2008; 48: 537-568Crossref PubMed Scopus (467) Google Scholar, 2.Marchese A. Paing M.M. Temple B.R. Trejo J. Annu. Rev. Pharmacol. Toxicol. 2008; 48: 601-629Crossref PubMed Scopus (350) Google Scholar). Following endocytosis, GPCRs are either routed for degradation or recycled back to the cell surface in a resensitized form (2.Marchese A. Paing M.M. Temple B.R. Trejo J. Annu. Rev. Pharmacol. Toxicol. 2008; 48: 601-629Crossref PubMed Scopus (350) Google Scholar). Many details of this sorting process are currently unknown, however, and its study represents an emerging field in GPCR biology (1.Hanyaloglu A.C. von Zastrow M. Annu. Rev. Pharmacol. Toxicol. 2008; 48: 537-568Crossref PubMed Scopus (467) Google Scholar).The chemokine receptor CCR5 is a GPCR expressed predominantly on leukocyte subsets, with a principal physiological role in the recruitment of effector cells to sites of inflammation (3.Lederman M.M. Ann. Intern. Med. 1995; 122: 218-222Crossref PubMed Scopus (18) Google Scholar). CCR5 also acts as an HIV coreceptor and plays a key role both in HIV transmission from person to person and in disease progression once infection has occurred (3.Lederman M.M. Ann. Intern. Med. 1995; 122: 218-222Crossref PubMed Scopus (18) Google Scholar). As a consequence, CCR5 is a key target for anti-HIV strategies directed toward both prevention and therapy (4.Kuhmann S.E. Hartley O. Annu. Rev. Pharmacol. Toxicol. 2008; 48: 425-461Crossref PubMed Scopus (60) Google Scholar).The physiological ligands of CCR5, CCL5, CCL3, and CCL4, have intrinsic anti-HIV activity (5.Cocchi F. DeVico A.L. Garzino-Demo A. Arya S.K. Gallo R.C. Lusso P. Science. 1995; 270: 1811-1815Crossref PubMed Scopus (2621) Google Scholar), resulting from both steric blockade of coreceptor interaction with the HIV envelope glycoprotein complex (6.Trkola A. Paxton W.A. Monard S.P. Hoxie J.A. Siani M.A. Thompson D.A. Wu L. Mackay C.R. Horuk R. Moore J.P. J. Virol. 1998; 72: 396-404Crossref PubMed Google Scholar) and induction of CCR5 down-modulation (7.Alkhatib G. Locati M. Kennedy P.E. Murphy P.M. Berger E.A. Virology. 1997; 234: 340-348Crossref PubMed Scopus (198) Google Scholar). N-terminally modified analogs of CCR5 ligands, including AOP-RANTES (8.Simmons G. Clapham P.R. Picard L. Offord R.E. Rosenkilde M.M. Schwartz T.W. Buser R. Wells T.N. Proudfoot A.E. Science. 1997; 276: 276-279Crossref PubMed Scopus (591) Google Scholar) and PSC-RANTES (9.Hartley O. Gaertner H. Wilken J. Thompson D. Fish R. Ramos A. Pastore C. Dufour B. Cerini F. Melotti A. Heveker N. Picard L. Alizon M. Mosier D. Kent S. Offord R. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 16460-16465Crossref PubMed Scopus (136) Google Scholar), exhibit much higher anti-HIV activity than the physiological ligands. Although it is clear that the enhanced potency of these analogs is due to induction of stronger and more prolonged CCR5 down-modulation (9.Hartley O. Gaertner H. Wilken J. Thompson D. Fish R. Ramos A. Pastore C. Dufour B. Cerini F. Melotti A. Heveker N. Picard L. Alizon M. Mosier D. Kent S. Offord R. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 16460-16465Crossref PubMed Scopus (136) Google Scholar, 10.Mack 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 (375) Google Scholar), the details of this unusual inhibitory mechanism have yet to be fully elucidated.The early steps of CCR5 desensitization, which have been quite extensively characterized (for review, see Ref. 11.Oppermann M. Cell. Signal. 2004; 16: 1201-1210Crossref PubMed Scopus (246) Google Scholar), involve processes common to many GPCRs: C-terminal phosphorylation, association with β-arrestin proteins, and clathrin-dependent endocytosis. The subsequent steps are less clearly understood, however. First, although it has been established that CCR5 accumulates in a perinuclear region during desensitization, several different locations for the site of accumulation have been proposed: the early endosome (12.Amara A. Gall S.L. Schwartz O. Salamero J. Montes M. Loetscher P. Baggiolini M. Virelizier J.L. Arenzana-Seisdedos F. J. Exp. Med. 1997; 186: 139-146Crossref PubMed Scopus (518) Google Scholar), the endosomal recycling compartment (ERC) (10.Mack 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 (375) Google Scholar, 13.Signoret N. Pelchen-Matthews A. Mack M. Proudfoot A.E. Marsh M. J. Cell Biol. 2000; 151: 1281-1294Crossref PubMed Scopus (160) Google Scholar, 14.Pollok-Kopp B. Schwarze K. Baradari V.K. Oppermann M. J. Biol. Chem. 2003; 278: 2190-2198Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 15.Signoret N. Christophe T. Oppermann M. Marsh M. Traffic. 2004; 5: 529-543Crossref PubMed Scopus (21) Google Scholar), and the Golgi apparatus (16.Longden J. Cooke E.L. Hill S.J. Br. J. Pharmacol. 2008; 153: 1513-1527Crossref PubMed Scopus (16) Google Scholar, 17.Kiss D.L. Longden J. Fechner G.A. Avery V.M. Cell. Mol. Biol. Lett. 2009; 14: 537-547Crossref PubMed Scopus (13) Google Scholar). Second, it has been demonstrated that during desensitization the pool of sequestered CCR5 receptors remains accessible to the cell surface by continually cycling to and from the plasma membrane (13.Signoret N. Pelchen-Matthews A. Mack M. Proudfoot A.E. Marsh M. J. Cell Biol. 2000; 151: 1281-1294Crossref PubMed Scopus (160) Google Scholar, 15.Signoret N. Christophe T. Oppermann M. Marsh M. Traffic. 2004; 5: 529-543Crossref PubMed Scopus (21) Google Scholar), but little is currently known about how the receptor resensitization process is triggered. Finally, although some progress has been made (10.Mack 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 (375) Google Scholar, 13.Signoret N. Pelchen-Matthews A. Mack M. Proudfoot A.E. Marsh M. J. Cell Biol. 2000; 151: 1281-1294Crossref PubMed Scopus (160) Google Scholar, 18.Pastore C. Picchio G.R. Galimi F. Fish R. Hartley O. Offord R.E. Mosier D.E. Antimicrob. Agents Chemother. 2003; 47: 509-517Crossref PubMed Scopus (73) Google Scholar), the mechanism by which anti-HIV chemokine analogs interfere with the desensitization process to achieve prolonged receptor down-modulation has yet to be elucidated in detail.In this study we used both native chemokine ligands and anti-HIV chemokine analogs to study CCR5 trafficking in response to ligands. Through a series of experiments using Chinese hamster ovary (CHO)-CCR5 cell lines and primary T lymphocytes, we present data that help explain the differences seen in previous work on the location of sequestered CCR5 during desensitization, as well as refining the existing mechanistic model to explain physiological CCR5 trafficking during desensitization and how it is disrupted by anti-HIV chemokine
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Potent Inhibition of HIV-1 Infectivity in Macrophages and Lymphocytes by a Novel CCR5 Antagonist
Graham Simmons, Paul R. Clapham, Laurent Picard et al. · Science · 1997 · 614 citations
Medicine, Immunology, Pharmacology +17
Ali Amara, Sylvie Le Gall, Olivier Schwartz et al. · The Journal of Experimental Medicine · 1997 · 552 citations · Full text