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• W2037331574 abstract "Fractalkine/CX3CL1 is a membrane-tethered chemokine that functions as a chemoattractant and adhesion protein by interacting with the receptor CX3CR1. To understand the molecular basis for the interaction, an extensive mutagenesis study of fractalkine's chemokine domain was undertaken. The results reveal a cluster of basic residues (Lys-8, Lys-15, Lys-37, Arg-45, and Arg-48) and one aromatic (Phe-50) that are critical for binding and/or signaling. The mutant R48A could bind but not induce chemotaxis, demonstrating that Arg-48 is a signaling trigger. This result also shows that signaling residues are not confined to chemokine N termini, as generally thought. F50A showed no detectable binding, underscoring its importance to the stability of the complex. K15A displayed unique signaling characteristics, eliciting a wild-type calcium flux but minimal chemotaxis, suggesting that this mutant can activate some, but not all, pathways required for migration. Fractalkine also binds the human cytomegalovirus receptor US28, and analysis of the mutants indicates that US28 recognizes many of the same epitopes of fractalkine as CX3CR1. Comparison of the binding surfaces of fractalkine and the CC chemokine MCP-1 reveals structural details that may account for their dual recognition by US28 and their selective recognition by host receptors. Fractalkine/CX3CL1 is a membrane-tethered chemokine that functions as a chemoattractant and adhesion protein by interacting with the receptor CX3CR1. To understand the molecular basis for the interaction, an extensive mutagenesis study of fractalkine's chemokine domain was undertaken. The results reveal a cluster of basic residues (Lys-8, Lys-15, Lys-37, Arg-45, and Arg-48) and one aromatic (Phe-50) that are critical for binding and/or signaling. The mutant R48A could bind but not induce chemotaxis, demonstrating that Arg-48 is a signaling trigger. This result also shows that signaling residues are not confined to chemokine N termini, as generally thought. F50A showed no detectable binding, underscoring its importance to the stability of the complex. K15A displayed unique signaling characteristics, eliciting a wild-type calcium flux but minimal chemotaxis, suggesting that this mutant can activate some, but not all, pathways required for migration. Fractalkine also binds the human cytomegalovirus receptor US28, and analysis of the mutants indicates that US28 recognizes many of the same epitopes of fractalkine as CX3CR1. Comparison of the binding surfaces of fractalkine and the CC chemokine MCP-1 reveals structural details that may account for their dual recognition by US28 and their selective recognition by host receptors. seven-transmembrane G-protein-coupled receptor wild type high performance liquid chromatography bovine serum albumin Dulbecco's modified Eagle's medium 4-morpholinepropanesulfonic acid heteronuclear single quantum coherence regulated on activation normal T cell expressed and secreted Chemokines are proinflammatory proteins that coordinate the immune response by directing the migration of leukocytes (1Mackay C.R. Nat. Immunol. 2001; 2: 95-101Crossref PubMed Scopus (707) Google Scholar, 2Murdoch C. Finn A. Blood. 2000; 95: 3032-3043Crossref PubMed Google Scholar, 3Moser B. Loetscher P. Nat. Immunol. 2001; 2: 123-128Crossref PubMed Scopus (1027) Google Scholar). For humans alone, the chemokine superfamily comprises over 40 members that cluster into four families (CC, CXC, C, and CX3C) according to the number and spacing of conserved cysteines (4Zlotnik A. Yoshie O. Immunity. 2000; 12: 121-127Abstract Full Text Full Text PDF PubMed Scopus (3245) Google Scholar). Chemokines mediate their effects by binding and signaling through seven-transmembrane G-protein-coupled receptors (7TMRs),1 of which 18 human receptors have been identified thus far (2Murdoch C. Finn A. Blood. 2000; 95: 3032-3043Crossref PubMed Google Scholar, 5Thelen M. Nat. Immunol. 2001; 2: 129-134Crossref PubMed Scopus (513) Google Scholar). The patterns of ligand-receptor recognition are complex since chemokines and chemokine receptors typically interact with multiple partners. Furthermore, a given chemokine can act as an agonist, antagonist, or inverse agonist in the context of different receptors (6Weng Y. Siciliano S.J. Waldburger K.E. Sirotina-Meisher A. Staruch M.J. Daugherty B.L. Gould S.L. Springer M.S. DeMartino J.A. J. Biol. Chem. 1998; 273: 18288-18291Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 7Blanpain C. Migeotte I. Lee B. Vakili J. Doranz B.J. Govaerts C. Vassart G. Doms R.W. Parmentier M. Blood. 1999; 94: 1899-1905Crossref PubMed Google Scholar, 8Rosenkilde M.M. Schwartz T.W. Mol. Pharmacol. 2000; 57: 602-609Crossref PubMed Scopus (57) Google Scholar, 9Rosenkilde M.M. Kledal T.N. Brauner-Osborne H. Schwartz T.W. J. Biol. Chem. 1999; 274: 956-961Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 10Loetscher P. Pellegrino A. Gong J.-H. Mattioli I. Loetscher M. Bardi G. Baggiolini M. Clark-Lewis I. J. Biol. Chem. 2001; 276: 2986-2991Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Despite the high level of promiscuity, interactions generally occur between receptors and ligands within a particular subfamily, an important exception being viral proteins that typically show broad spectrum recognition profiles (11Damon I. Murphy P.M. Moss B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6403-6407Crossref PubMed Scopus (105) Google Scholar, 12Lusso P. Virology. 2000; 273: 228-240Crossref PubMed Scopus (59) Google Scholar, 13Kledal T.N. Rosenkilde M.M. Coulin F. Simmons G. Johnsen A.H. Alouani S. Power C.A. Luttichau H.R. Gerstoft J. Clapham P.R. Clark-Lewis I. Wells T.N.C. Schwartz T.W. Science. 1997; 277: 1656-1659Crossref PubMed Scopus (424) Google Scholar, 14Kledal T.N. Rosenkilde M.M. Schwartz T.W. FEBS Lett. 1998; 441: 209-214Crossref PubMed Scopus (169) Google Scholar). A large number of studies have aimed to dissect the molecular determinants of chemokine receptor binding, signaling, and specificity (15Crump M.P. Gong J.H. Loetscher P. Rajarathnam K. Amara A. Arenzana-Seisdedos F. Virelizier J.L. Baggiolini M. Sykes B.D. Clark-Lewis I. EMBO J. 1997; 16: 6996-7007Crossref PubMed Scopus (629) Google Scholar, 16Jones S.A. Dewald B. Clark-Lewis I. Baggiolini M. J. Biol. Chem. 1997; 272: 16166-16169Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 17Clark-Lewis I. Kim K.S. Rajarathnam K. Gong J.H. Dewald B. Moser B. Baggiolini M. Sykes B.D. J. Leukocyte Biol. 1995; 57: 703-711Crossref PubMed Scopus (332) Google Scholar, 18Gong J.H. Clark-Lewis I. J. Exp. Med. 1995; 181: 631-640Crossref PubMed Scopus (266) Google Scholar, 19Pakianathan D.R. Kuta E.G. Artis D.R. Skelton N.J. Hebert C.A. Biochemistry. 1997; 36: 9642-9648Crossref PubMed Scopus (148) Google Scholar, 20Hebert C.A. Vitangcol R.V. Baker J.B. J. Biol. Chem. 1991; 266: 18989-18994Abstract Full Text PDF PubMed Google Scholar). However, these details remain poorly understood because structural information on the receptors is difficult to obtain, and the important features of the ligands are encrypted in remarkably conserved tertiary structures.Fractalkine/CX3CL1 and its receptor CX3CR1 are currently the only known members of the CX3C family (21Combadiere C. Salzwedel K. Smith E.D. Tiffany H.L. Berger E.A. Murphy P.M. J. Biol. Chem. 1998; 273: 23799-23804Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 22Imai T. Hieshima K. Haskell C. Baba M. Nagira M. Nishimura M. Kakizaki M. Takagi S. Nomiyama H. Schall T.J. Yoshie O. Cell. 1997; 91: 521-530Abstract Full Text Full Text PDF PubMed Scopus (1150) Google Scholar). Unlike other chemokines except CXCL16 (23Wilbanks A. Zondlo S.C. Murphy K. Mak S. Soler D. Langdon P. Andrew D.P. Wu L. Briskin M. J. Immunol. 2001; 166: 5145-5154Crossref PubMed Scopus (259) Google Scholar), fractalkine is a membrane-anchored protein consisting of a chemokine module attached to the cell membrane via a mucin-like stalk, which serves as a presentation vehicle (24Fong A.M. Erickson H.P. Zachariah J.P. Poon S. Schamberg N.J. Imai T. Patel D.D. J. Biol. Chem. 2000; 275: 3781-3786Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). A soluble form, generated by protease cleavage near the membrane, has been observed in the supernatants of fractalkine cell transfectants (25Bazan J.F. Bacon K.B. Hardiman G. Wang W. Soo K. Rossi D. Greaves D.R. Zlotnik A. Schall T.J. Nature. 1997; 385: 640-644Crossref PubMed Scopus (1686) Google Scholar). This form induces chemotaxis of monocytes, T cells, and natural killer cells. No difference in binding or signaling is observed between fractalkine variants with different lengths of mucin stalk (26Harrison J.K. Fong A.M. Swain P.A. Chen S., Yu, Y.R. Salafranca M.N. Greenleaf W.B. Imai T. Patel D.D. J. Biol. Chem. 2001; 276: 21632-21641Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), and functional responses can be stimulated by the chemokine module alone, which indicates that it is the effector domain (22Imai T. Hieshima K. Haskell C. Baba M. Nagira M. Nishimura M. Kakizaki M. Takagi S. Nomiyama H. Schall T.J. Yoshie O. Cell. 1997; 91: 521-530Abstract Full Text Full Text PDF PubMed Scopus (1150) Google Scholar, 25Bazan J.F. Bacon K.B. Hardiman G. Wang W. Soo K. Rossi D. Greaves D.R. Zlotnik A. Schall T.J. Nature. 1997; 385: 640-644Crossref PubMed Scopus (1686) Google Scholar). Fractalkine is up-regulated on the surface of activated endothelial cells and can induce firm adhesion of CX3CR1-expressing cells, independent of G-protein activation (27Haskell C.A. Cleary M.D. Charo I.F. J. Biol. Chem. 1999; 274: 10053-10058Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). These observations suggest that fractalkine and CX3CR1 fulfill special roles in leukocyte trafficking at the endothelium. Fractalkine and CX3CR1 probably also have important homeostatic and developmental roles, given their high constitutive expression in a variety of non-hematopoietic tissues, including the brain (25Bazan J.F. Bacon K.B. Hardiman G. Wang W. Soo K. Rossi D. Greaves D.R. Zlotnik A. Schall T.J. Nature. 1997; 385: 640-644Crossref PubMed Scopus (1686) Google Scholar, 28Pan Y. Lloyd C. Zhou H. Dolich S. Deeds J. Gonzalo J.A. Vath J. Gosselin M. Ma J. Dussault B. Woolf E. Alperin G. Culpepper J. Gutierrez-Ramos J.C. Gearing D. Nature. 1997; 387: 611-617Crossref PubMed Scopus (569) Google Scholar). Their functions in the central nervous system are not fully defined, but there is recent evidence that fractalkine serves as an anti-apoptotic factor promoting microglial and hippocampal neuron survival (29Boehme S.A. Lio F.M. Maciejewski-Lenoir D. Bacon K.B. Conlon P.J. J. Immunol. 2000; 165: 397-403Crossref PubMed Scopus (166) Google Scholar, 30Meucci O. Fatatis A. Simen A.A. Miller R.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8075-8080Crossref PubMed Scopus (319) Google Scholar). Like many other chemokine receptors, CX3CR1 can function as an human immunodeficiency virus cofactor in vitro, and fractalkine can inhibit viral entry into cells, but it is unclear whether these activities are relevant in vivo. Although it has been reported that a variant haplotype of CX3CR1 (CX3CR1-I249 M280) is associated with accelerated progression to AIDS (31Faure S. Meyer L. Costagliola D. Vaneensberghe C. Genin E. Autran B. Delfraissy J.F. McDermott D.H. Murphy P.M. Debre P. Theodorou I. Combadiere C. Science. 2000; 287: 2274-2277Crossref PubMed Scopus (286) Google Scholar), these data were recently called into question (32McDermott D.H. Colla J.S. Kleeberger C.A. Plankey M. Rosenberg P.S. Smith E.D. Zimmerman P.A. Combadiere C. Leitman S.F. Kaslow R.A. Goedert J.J. Berger E.A. O'Brien T.R. Murphy P.M. Science. 2000; 290: 2031Crossref PubMed Scopus (79) Google Scholar).We previously solved the NMR structure of fractalkine's chemokine domain and described its interaction with an N-terminal fragment of CX3CR1 (33Mizoue L.S. Bazan J.F. Johnson E.C. Handel T.M. Biochemistry. 1999; 38: 1402-1414Crossref PubMed Scopus (124) Google Scholar). In the present study, we use mutagenesis to identify residues that are important for receptor binding and signaling. Although CX3CR1 is the endogenous receptor, fractalkine also binds with subnanomolar affinity to US28, a 7TMR encoded by human cytomegalovirus (14Kledal T.N. Rosenkilde M.M. Schwartz T.W. FEBS Lett. 1998; 441: 209-214Crossref PubMed Scopus (169) Google Scholar). Accordingly, we examined binding of the mutants to US28 to determine whether the viral receptor recognizes the same “hotspots” on fractalkine as CX3CR1. These studies further define the structural and chemical elements necessary for fractalkine:CX3CR1 interactions and provide insight into the molecular basis of chemokine:receptor specificity. Since chemokines and their receptors have been implicated in a number of diseases (34Segerer S. Nelson P.J. Schlondorff D. J. Am. Soc. Nephrol. 2000; 11: 152-176PubMed Google Scholar, 35Homey B. Zlotnik A. Curr. Opin. Immunol. 1999; 11: 626-634Crossref PubMed Scopus (102) Google Scholar, 36McDermott D.H. Murphy P.M. Springer Semin. Immunopathol. 2000; 22: 393-415Crossref PubMed Scopus (17) Google Scholar, 37Gerard C. Rollins B.J. Nat. Immunol. 2001; 2: 108-115Crossref PubMed Scopus (1199) Google Scholar), the results may also aid in the development of agents that block their interaction (38Proudfoot A.E. Power C.A. Wells T.N. Immunol. Rev. 2000; 177: 246-256Crossref PubMed Scopus (79) Google Scholar).DISCUSSIONOur goal is to understand the molecular details of how chemokines bind and activate their receptors. Given that chemokines have remarkably similar folds, biochemical studies are necessary to determine what features control the specific recognition of some receptors over others. Understanding how viral receptors like US28 achieve high affinity interactions with a broad spectrum of chemokines is also an important endeavor. To this end, we identified residues of fractalkine that contribute to the interaction with the host receptor CX3CR1, and with the cytomegalovirus receptor US28. Fig.8 (left) shows a GRASP model of fractalkine highlighting residues that, when mutated, had a large (>10-fold) impact on CX3CR1 binding and/or signaling (Lys-8, Lys-15, Lys-37, Arg-45, Arg-48, and Phe-50). The identification of Lys-8 and Arg-48 as important for the interaction with CX3CR1 is also consistent with a recent mutagenesis analysis of basic residues in fractalkine (26Harrison J.K. Fong A.M. Swain P.A. Chen S., Yu, Y.R. Salafranca M.N. Greenleaf W.B. Imai T. Patel D.D. J. Biol. Chem. 2001; 276: 21632-21641Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Below, we discuss the results in more detail and compare them to what is known about other chemokine receptor systems, particularly MCP-1:CCR2.The N Terminus Contributes to Binding Affinity and Signaling SpecificityLike other chemokines, the N terminus of fractalkine is disordered in the solution structure (33Mizoue L.S. Bazan J.F. Johnson E.C. Handel T.M. Biochemistry. 1999; 38: 1402-1414Crossref PubMed Scopus (124) Google Scholar) and probably becomes ordered only upon receptor engagement. The importance of chemokine N termini in receptor activation is well established (15Crump M.P. Gong J.H. Loetscher P. Rajarathnam K. Amara A. Arenzana-Seisdedos F. Virelizier J.L. Baggiolini M. Sykes B.D. Clark-Lewis I. EMBO J. 1997; 16: 6996-7007Crossref PubMed Scopus (629) Google Scholar, 18Gong J.H. Clark-Lewis I. J. Exp. Med. 1995; 181: 631-640Crossref PubMed Scopus (266) Google Scholar, 51Gong J.H. Uguccioni M. Dewald B. Baggiolini M. Clark-Lewis I. J. Biol. Chem. 1996; 271: 10521-10527Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar), and fractalkine is no exception, as removal of the first 8 residues abolishes function. Although the contribution of chemokine N termini to binding varies considerably, deletion of fractalkine's N terminus caused a loss in affinity of more than 2 orders of magnitude. By comparison, N-terminal deletion of MCP-1 causes only a 7-fold reduction in affinity for CCR2 (45Hemmerich S. Paavola C. Bloom A. Bhakta S. Freedman R. Grunberger D. Krstenansky J. Lee S. McCarley D. Mulkins M. Wong B. Pease J. Mizoue L. Mirzadegan T. Polsky I. Thompson K. Handel T.M. Jarnagin K. Biochemistry. 1999; 38: 13013-13025Crossref PubMed Scopus (141) Google Scholar, 52Paavola C.D. Hemmerich S. Grunberger D. Polsky I. Bloom A. Freedman R. Mulkins M. Bhakta S. McCarley D. Wiesent L. Wong B. Jarnagin K. Handel T.M. J. Biol. Chem. 1998; 273: 33157-33165Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar), whereas short peptides derived from the N terminus of SDF-1 have sufficient affinity to bind CXCR4 and stimulate chemotaxis in the absence of the rest of the chemokine (53Elisseeva E.L. Slupsky C.M. Crump M.P. Clark-Lewis I. Sykes B.D. J. Biol. Chem. 2000; 275: 26799-26805Abstract Full Text Full Text PDF PubMed Google Scholar).The importance of specific residues within the N termini also varies significantly among chemokines, thereby encoding functional specificity. For fractalkine, extension of the N terminus with a Met had no effect on binding or function whereas similar extensions of MCP-1 (18Gong J.H. Clark-Lewis I. J. Exp. Med. 1995; 181: 631-640Crossref PubMed Scopus (266) Google Scholar) and RANTES (47Proudfoot A.E. Power C.A. Hoogewerf A.J. Montjovent M.O. Borlat F. Offord R.E. Wells T.N. J. Biol. Chem. 1996; 271: 2599-2603Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar) produce receptor antagonists. Among the N-terminal residues, Lys-8 is particularly crucial, somewhat akin to the ELR motif of interleukin-8 (20Hebert C.A. Vitangcol R.V. Baker J.B. J. Biol. Chem. 1991; 266: 18989-18994Abstract Full Text PDF PubMed Google Scholar, 54Clark-Lewis I. Dewald B. Loetscher M. Moser B. Baggiolini M. J. Biol. Chem. 1994; 269: 16075-16081Abstract Full Text PDF PubMed Google Scholar), and specific residues identified as critical to receptor activation in RANTES (19Pakianathan D.R. Kuta E.G. Artis D.R. Skelton N.J. Hebert C.A. Biochemistry. 1997; 36: 9642-9648Crossref PubMed Scopus (148) Google Scholar), SDF-1(15), and eotaxin (55Mayer M.R. Stone M.J. J. Biol. Chem. 2001; 276: 13911-13916Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). In contrast, the length rather than the amino acid composition of the N terminus is important for MCP-1 (45Hemmerich S. Paavola C. Bloom A. Bhakta S. Freedman R. Grunberger D. Krstenansky J. Lee S. McCarley D. Mulkins M. Wong B. Pease J. Mizoue L. Mirzadegan T. Polsky I. Thompson K. Handel T.M. Jarnagin K. Biochemistry. 1999; 38: 13013-13025Crossref PubMed Scopus (141) Google Scholar).K15A Displays Unique Signaling CharacteristicsThe functional responses of CX3CR1 to the fractalkine variant K15A are particularly instructive from the standpoint of general issues of 7TMR signaling. In contrast to mutants that showed marked effects on both calcium and chemotaxis (ΔNT, F50A, and R48A), K15A produced a normal calcium flux and could desensitize to further stimulation, but had a greatly diminished chemotaxis response. For certain chemokines including MCP-1 (56Yen H. Zhang Y. Penfold S. Rollins B.J. J. Leukocyte Biol. 1997; 61: 529-532Crossref PubMed Scopus (89) Google Scholar) and fractalkine (43Maciejewski-Lenoir D. Chen S. Feng L. Maki R. Bacon K.B. J. Immunol. 1999; 163: 1628-1635PubMed Google Scholar), receptor binding has been shown to lead to mitogen-activated protein kinase activation, a critical event controlling actin polymerization and cytoskeletal reorganization, which in turn are necessary for migration. In preliminary analysis of mitogen-activated protein kinase activation stimulation, we noted no appreciable activity induced by K15A compared with WT fractalkine (data not shown), suggesting a possible cause for the impaired chemotaxis. Differential activation of pathways was observed previously with MCP-1, where the Y13A mutant was able to inhibit adenylate cyclase and stimulate calcium flux, but not chemotaxis (46Jarnagin K. Grunberger D. Mulkins M. Wong B. Hemmerich S. Paavola C. Bloom A. Bhakta S. Diehl F. Freedman R. McCarley D. Polsky I. Ping-Tsou A. Kosaka A. Handel T.M. Biochemistry. 1999; 38: 16167-16177Crossref PubMed Scopus (93) Google Scholar). Based on these findings, we suggested that certain molecular features of chemokines are required for activation of some pathways but not others, and fractalkine's K15A provides additional support for this hypothesis. Interestingly, the location of Lys-15 on fractalkine's surface is similar to that of Tyr-13 on MCP-1 (Fig. 8).Phe-50 and Arg-48 Are Essential Residues That Are Spatially Removed from the N TerminusMany mutagenesis studies of chemokines have targeted the N terminus and the extended loop (the “N-loop”) following the second cysteine because of their importance for binding and particularly signaling (15Crump M.P. Gong J.H. Loetscher P. Rajarathnam K. Amara A. Arenzana-Seisdedos F. Virelizier J.L. Baggiolini M. Sykes B.D. Clark-Lewis I. EMBO J. 1997; 16: 6996-7007Crossref PubMed Scopus (629) Google Scholar, 19Pakianathan D.R. Kuta E.G. Artis D.R. Skelton N.J. 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Consequently, it is interesting that the two most critical residues of fractalkine, Arg-48 and Phe-50, lie outside of these regions. Like the N terminus and Lys-8, Arg-48 is essential for signaling because mutation to Ala completely eliminated calcium flux and chemotaxis, but not binding. Mutation of Arg-48 to Glu eliminated binding whereas mutation to Gln restored much of the affinity, presumably due to the size and hydrogen bonding capacity of Gln compared with Ala. Since calcium and chemotaxis are still markedly affected by R48Q, it appears that a basic charge is required for receptor activation. The significant difference between R48Q and R48E also suggest that Arg-48 interacts with an acidic region of the receptor (see below). For the mutant F50A, a 489-fold reduction in affinity was observed on transfectants and no binding could be detected on microglia. The binding and signaling of F50L was much improved over the alanine mutant; however, it was still significantly less active than the WT protein, suggesting that an aromatic side chain at this position is important. These data provide further evidence that residues critical for signaling are not confined to the N termini of chemokines but can be dispersed over the receptor-binding surface (46Jarnagin K. Grunberger D. Mulkins M. Wong B. Hemmerich S. Paavola C. Bloom A. Bhakta S. Diehl F. Freedman R. McCarley D. Polsky I. Ping-Tsou A. Kosaka A. Handel T.M. Biochemistry. 1999; 38: 16167-16177Crossref PubMed Scopus (93) Google Scholar,55Mayer M.R. Stone M.J. J. Biol. Chem. 2001; 276: 13911-13916Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 59Laurence J.S. Blanpain C. Burgner J.W. Parmentier M. LiWang P.J. Biochemistry. 2000; 39: 3401-3409Crossref PubMed Scopus (102) Google Scholar). Thus, there may be significant variability in the mechanisms involved in activation of different chemokine receptor systems.Similarities in the Recognition Surfaces That May Account for Broad Spectrum Recognition by US28US28 not only interacts with fractalkine, but also binds CC chemokines such as MCP-1 with subnanomolar affinity (14Kledal T.N. Rosenkilde M.M. Schwartz T.W. FEBS Lett. 1998; 441: 209-214Crossref PubMed Scopus (169) Google Scholar). Our previous mutagenesis studies identified residues important for binding of MCP-1 to CCR2, its cognate receptor (45Hemmerich S. Paavola C. Bloom A. Bhakta S. Freedman R. Grunberger D. Krstenansky J. Lee S. McCarley D. Mulkins M. Wong B. Pease J. Mizoue L. Mirzadegan T. Polsky I. Thompson K. Handel T.M. Jarnagin K. Biochemistry. 1999; 38: 13013-13025Crossref PubMed Scopus (141) Google Scholar, 46Jarnagin K. Grunberger D. Mulkins M. Wong B. Hemmerich S. Paavola C. Bloom A. Bhakta S. Diehl F. Freedman R. McCarley D. Polsky I. Ping-Tsou A. Kosaka A. Handel T.M. Biochemistry. 1999; 38: 16167-16177Crossref PubMed Scopus (93) Google Scholar). Preliminary data on a subset of those mutants suggest that, as with fractalkine, US28 uses the same binding epitopes on MCP-1 as the host receptor (data not shown). Therefore, a comparison of the binding surfaces of fractalkine and MCP-1 may shed light on what enables the viral receptor to engage both chemokines.At low resolution, the receptor binding surfaces of fractalkine and MCP-1 have a lot in common, as they consist of a largely basic surface with one aromatic residue (Fig. 8). In contrast, the CXC chemokine interleukin-8, which does not bind US28, has a much more hydrophobic surface (45Hemmerich S. Paavola C. Bloom A. Bhakta S. Freedman R. Grunberger D. Krstenansky J. Lee S. McCarley D. Mulkins M. Wong B. Pease J. Mizoue L. Mirzadegan T. Polsky I. Thompson K. Handel T.M. Jarnagin K. Biochemistry. 1999; 38: 13013-13025Crossref PubMed Scopus (141) Google Scholar, 60Hammond M.E. Shyamala V. Siani M.A. Gallegos C.A. Feucht P.H. Abbott J. Lapointe G.R. Moghadam M. Khoja H. Zakel J. Tekamp-Olson P. J. Biol. Chem. 1996; 271: 8228-8235Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The distribution of basic residues on the tertiary structures of fractalkine and MCP-1 are also similar (Fig. 8). Furthermore, both chemokines have residues that are highly sensitive to charge swap mutations (Arg-48 in fractalkine, Arg-24 in MCP-1), suggesting that they may interact with acidic regions of their receptors. Many studies have demonstrated that the receptor N termini are important for binding chemokines (61LaRosa G.J. Thomas K.M. Kaufmann M.E. Mark R. White M. Taylor L. Gray G. Witt D. Navarro J. J. Biol. Chem. 1992; 267: 25402-25406Abstract Full Text PDF PubMed Google Scholar, 62Monteclaro F.S. Charo I.F. J. Biol. Chem. 1996; 271: 19084-19092Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 63Rosenkilde M.M. Kledal T.N. Holst P.J. Schwartz T.W. J. Biol. Chem. 2000; 275: 26309-26315Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 64Lu Z.H. Wang Z.X. Horuk R. Hesselgesser J. Lou Y.C. Hadley T.J. Peiper S.C. J. Biol. Chem. 1995; 270: 26239-26245Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 65Ho H.H. Du D. Gershengorn M.C. J. Biol. Chem. 1999; 274: 31327-31332Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). We showed previously that a DYDY sequence in the N terminus of CCR2 was critical for binding MCP-1 (45Hemmerich S. Paavola C. Bloom A. Bhakta S. Freedman R. Grunberger D. Krstenansky J. Lee S. McCarley D. Mulkins M. Wong B. Pease J. Mizoue L. Mirzadegan T. Polsky I. Thompson K. Handel T.M. Jarnagin K. Biochemistry. 1999; 38: 13013-13025Crossref PubMed Scopus (141) Google Scholar), and thus may be involved in contacting MCP-1's R24. Others have shown that this motif is tyrosine-sulfated in vivo, and the post-translational modification is important for ligand binding (66Preobrazhensky A.A. Dragan S. Kawano T. Gavrilin M.A. Gulina I.V. Chakravarty L. Kolattukudy P.E. J. Immunol. 2000; 165: 5295-5303Crossref PubMed Scopus (95) Google Scholar). Interestingly, CX3CR1 and US28 also contain potential tyrosine sulfation sites (EYDD and DYDD, respectively) in their N termini (67Farzan M. Mirzabekov T. Kolchinsky P. Wyatt R. Cayabyab M. Gerard N.P. Gerard C. Sodroski J. Choe H. Cell. 1999; 96: 667-676Abstract Full Text Full Text PDF PubMed Scopus (601) Google Scholar) that could be involved in interactions with basic epitopes on the chemokines. We plan to pursue these questions in future studies.Differences in the Recognition Surfaces That Contribute to SpecificityDespite the similarities in the binding epitopes, closer inspection of the structures reveals sufficient differences to account for the class-specific recognition of fractalkine by CX3CR1 and MCP-1 by CCR2. Previously, we noted that subtle structural and chemical features create different topological landscapes on the two chemokines (33Mizoue L.S. Bazan J.F. Johnson E.C. Handel T.M. Biochemistry. 1999; 38: 1402-1414Crossref PubMed Scopus (124) Google Scholar). Other salient features emerge from the mutagenesis data. Lys-8 and another critical residue, Lys-15, flank fractalkine's characteristic CX3C motif (residues 9–13). In contrast, the corresponding region of MCP-1 contains an aromatic residue, Tyr-13. Tyr-13 was the single most important residue for binding and activation of CCR2, as mutation to Ala completely eliminated chemotaxis. It is also one of the most important features distinguishing MCP-1 and fractalkine, as it produces a large hydrophobic bump on MCP-1's surface that is not present in fractalkine. Like MCP-1, fractalkine's single most important residue is aromatic, Phe-50. Since it is located in a different region of fractalkine's surface relative to Tyr-13 in MCP-1, it is probably also a critical specificity determinant.In summary, we have id" @default.
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• W2037331574 title "Molecular Determinants of Receptor Binding and Signaling by the CX3C Chemokine Fractalkine" @default.
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