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Tyrosine Sulfation of Chemokine Receptor CCR2 Enhances Interactions with Both Monomeric and Dimeric Forms of the Chemokine Monocyte Chemoattractant Protein-1 (MCP-1)*

Open AccessPublished:April 05, 2013DOI:https://doi.org/10.1074/jbc.M112.447359
      Chemokine receptors are commonly post-translationally sulfated on tyrosine residues in their N-terminal regions, the initial site of binding to chemokine ligands. We have investigated the effect of tyrosine sulfation of the chemokine receptor CCR2 on its interactions with the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). Inhibition of CCR2 sulfation, by growth of expressing cells in the presence of sodium chlorate, significantly reduced the potency for MCP-1 activation of CCR2. MCP-1 exists in equilibrium between monomeric and dimeric forms. The obligate monomeric mutant MCP-1(P8A) was similar to wild type MCP-1 in its ability to induce leukocyte recruitment in vivo, whereas the obligate dimeric mutant MCP-1(T10C) was less effective at inducing leukocyte recruitment in vivo. In two-dimensional NMR experiments, sulfated peptides derived from the N-terminal region of CCR2 bound to both the monomeric and dimeric forms of wild type MCP-1 and shifted the equilibrium to favor the monomeric form. Similarly, MCP-1(P8A) bound more tightly than MCP-1(T10C) to the CCR2-derived sulfopeptides. NMR chemical shift mapping using the MCP-1 mutants showed that the sulfated N-terminal region of CCR2 binds to the same region (N-loop and β3-strand) of both monomeric and dimeric MCP-1 but that binding to the dimeric form also influences the environment of chemokine N-terminal residues, which are involved in dimer formation. We conclude that interaction with the sulfated N terminus of CCR2 destabilizes the dimerization interface of inactive dimeric MCP-1, thus inducing dissociation to the active monomeric state.

      Introduction

      Chemokines, or chemoattractant cytokines, are a family of small (8–10 kDa) globular proteins that function to direct leukocyte migration (
      • Thelen M.
      • Stein J.V.
      How chemokines invite leukocytes to dance.
      ). The ability of chemokines to recruit leukocytes is mediated by high affinity interactions with G protein-coupled receptors expressed in leukocyte membranes (
      • Thelen M.
      • Stein J.V.
      How chemokines invite leukocytes to dance.
      ). According to the prevailing two-site model of these interactions (
      • Crump 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.
      Solution structure and basis for functional activity of stromal cell-derived factor-1. Dissociation of CXCR4 activation from binding and inhibition of HIV-1.
      ), chemokines first use residues in the N-loop region (following the CC or CXC motif) and the second and third β-strands to bind to the receptor N terminus. Subsequently, the chemokine N terminus activates the receptor by binding to its extracellular loops and/or transmembrane helices.
      A key factor that regulates chemokine-receptor interactions is the sulfation of tyrosine residues on the receptor N terminus. Tyrosine sulfation is a post-translational modification prevalent among membrane-bound proteins such as chemokine receptors, as well as among secreted proteins such as peptide hormones, enzymes, blood coagulants, and complement proteins (
      • Stone M.J.
      • Chuang S.
      • Hou X.
      • Shoham M.
      • Zhu J.Z.
      Tyrosine sulfation. An increasingly recognised post-translational modification of secreted proteins.
      ). This modification is catalyzed in the Golgi apparatus by the enzymes tyrosylprotein sulfotransferases 1 and 2, which preferentially sulfate tyrosine residues located near acidic residues (
      • Liu J.
      • Louie S.
      • Hsu W.
      • Yu K.M.
      • Nicholas Jr., H.B.
      • Rosenquist G.L.
      Tyrosine sulfation is prevalent in human chemokine receptors important in lung disease.
      ), a motif found in the N-terminal regions of most chemokine receptors (
      • Stone M.J.
      • Chuang S.
      • Hou X.
      • Shoham M.
      • Zhu J.Z.
      Tyrosine sulfation. An increasingly recognised post-translational modification of secreted proteins.
      ). The chemokine receptors CCR2, CCR5, CCR8, CXCR3, CXCR4, CX3CR1, and Duffy antigen and receptor for chemokines (DARC) have been demonstrated to contain sulfated tyrosine residues that modulate chemokine binding (
      • Preobrazhensky A.A.
      • Dragan S.
      • Kawano T.
      • Gavrilin M.A.
      • Gulina I.V.
      • Chakravarty L.
      • Kolattukudy P.E.
      Monocyte chemotactic protein-1 receptor CCR2B is a glycoprotein that has tyrosine sulfation in a conserved extracellular N-terminal region.
      ,
      • Fong A.M.
      • Alam S.M.
      • Imai T.
      • Haribabu B.
      • Patel D.D.
      CX3CR1 tyrosine sulfation enhances fractalkine-induced cell adhesion.
      ,
      • Farzan M.
      • Mirzabekov T.
      • Kolchinsky P.
      • Wyatt R.
      • Cayabyab M.
      • Gerard N.P.
      • Gerard C.
      • Sodroski J.
      • Choe H.
      Tyrosine sulfation of the amino terminus of CCR5 facilitates HIV-1 entry.
      ,
      • Gutiérrez J.
      • Kremer L.
      • Zaballos A.
      • Goya I.
      • Martínez-A. C.
      • Márquez G.
      Analysis of post-translational CCR8 modifications and their influence on receptor activity.
      ,
      • Colvin R.A.
      • Campanella G.S.
      • Manice L.A.
      • Luster A.D.
      CXCR3 requires tyrosine sulfation for ligand binding and a second extracellular loop arginine residue for ligand-induced chemotaxis.
      ,
      • Farzan M.
      • Babcock G.J.
      • Vasilieva N.
      • Wright P.L.
      • Kiprilov E.
      • Mirzabekov T.
      • Choe H.
      The role of post-translational modifications of the CXCR4 amino terminus in stromal-derived factor 1α association and HIV-1 entry.
      ,
      • Choe H.
      • Moore M.J.
      • Owens C.M.
      • Wright P.L.
      • Vasilieva N.
      • Li W.
      • Singh A.P.
      • Shakri R.
      • Chitnis C.E.
      • Farzan M.
      Sulphated tyrosines mediate association of chemokines and Plasmodium vivax Duffy binding protein with the Duffy antigen/receptor for chemokines (DARC).
      ). Moreover, studies using differentially sulfated N-terminal peptides from various chemokine receptors have shown that tyrosine sulfation increases the binding affinity of the receptor peptides to their cognate chemokines (
      • Veldkamp C.T.
      • Seibert C.
      • Peterson F.C.
      • Sakmar T.P.
      • Volkman B.F.
      Recognition of a CXCR4 sulfotyrosine by the chemokine stromal cell-derived factor-1α (SDF-1α/CXCL12).
      ,
      • Zhu J.Z.
      • Millard C.J.
      • Ludeman J.P.
      • Simpson L.S.
      • Clayton D.J.
      • Payne R.J.
      • Widlanski T.S.
      • Stone M.J.
      Tyrosine sulfation influences the chemokine binding selectivity of peptides derived from chemokine receptor CCR3.
      ,
      • Duma L.
      • Häussinger D.
      • Rogowski M.
      • Lusso P.
      • Grzesiek S.
      Recognition of RANTES by extracellular parts of the CCR5 receptor.
      ,
      • Simpson L.S.
      • Zhu J.Z.
      • Widlanski T.S.
      • Stone M.J.
      Regulation of chemokine recognition by site-specific tyrosine sulfation of receptor peptides.
      ,
      • Seibert C.
      • Veldkamp C.T.
      • Peterson F.C.
      • Chait B.T.
      • Volkman B.F.
      • Sakmar T.P.
      Sequential tyrosine sulfation of CXCR4 by tyrosylprotein sulfotransferases.
      ).
      Among the chemokine receptors demonstrated to possess a tyrosine sulfation motif, CCR2 has received considerable attention because of its role in inflammatory diseases such as atherosclerosis and multiple sclerosis (
      • Charo I.F.
      • Peters W.
      Chemokine receptor 2 (CCR2) in atherosclerosis, infectious diseases, and regulation of T-cell polarization.
      ,
      • Izikson L.
      • Klein R.S.
      • Charo I.F.
      • Weiner H.L.
      • Luster A.D.
      Resistance to experimental autoimmune encephalomyelitis in mice lacking the CC chemokine receptor (CCR)2.
      ). CCR2 is a major chemokine receptor expressed on monocytes, and activation by its chemokine ligands monocyte chemoattractant proteins 1 to 4 (MCP-1 to -4; systematic names CCL2, CCL8, CCL7, and CCL13, respectively)
      The abbreviations used are: MCP
      monocyte chemoattractant protein
      GAG
      glycosaminoglycan
      HSQC
      heteronuclear single quantum coherence spectrum
      SDF-1
      stromal cell-derived factor-1.
      stimulates the migration of monocytes across the vascular wall into tissues where they mediate chronic inflammation (
      • Mestas J.
      • Ley K.
      Monocyte-endothelial cell interactions in the development of atherosclerosis.
      ). The tyrosine sulfation motif of CCR2 has the sequence 25DYDY28. In a HEK293 cell line transfected to express CCR2, mutation of Tyr26 resulted in a dramatic decrease in sulfation, a 10-fold decrease in binding affinity for MCP-1 and almost complete loss of activation in response to MCP-1 (
      • Preobrazhensky A.A.
      • Dragan S.
      • Kawano T.
      • Gavrilin M.A.
      • Gulina I.V.
      • Chakravarty L.
      • Kolattukudy P.E.
      Monocyte chemotactic protein-1 receptor CCR2B is a glycoprotein that has tyrosine sulfation in a conserved extracellular N-terminal region.
      ). Because the Tyr26 mutant had no detectable sulfation, the authors concluded that Tyr28 on CCR2 is not sulfated. However, it is possible that Tyr28 is at least partially sulfated in the wild type receptor. An independent study reported that a double Asp → Ala mutation in the DYDY motif, which is likely to decrease the extent of tyrosine sulfation, resulted in a large (>50-fold) decrease in MCP-1 binding affinity (
      • Hemmerich 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.
      Identification of residues in the monocyte chemotactic protein-1 that contact the MCP-1 receptor, CCR2.
      ).
      Although the above mutational experiments strongly suggest that sulfation of Tyr26 in CCR2 can modulate the strength of the interaction with MCP-1, they do not directly compare sulfated with nonsulfated forms of the wild type receptor. Thus, it is instructive to complement the mutational approach with experiments that examine the influence of post-translational sulfation without introducing receptor mutations. In the current study, we have used two independent approaches to achieve this. First, we measured CCR2 activation by MCP-1 using cells in which tyrosine sulfation was inhibited by sodium chlorate. Second, we used NMR spectroscopy to monitor the binding of MCP-1 to CCR2 N-terminal peptides possessing defined patterns of sulfation on Tyr26 and/or Tyr28. Because MCP-1 exists in a monomer-dimer equilibrium in solution, we distinguished the binding abilities of the monomer and dimer by using a previously characterized obligate monomer, MCP-1(P8A) (
      • Paavola 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.
      Monomeric monocyte chemoattractant protein-1 (MCP-1) binds and activates the MCP-1 receptor CCR2B.
      ), and obligate dimer, MCP-1(T10C) (
      • Tan J.H.
      • Canals M.
      • Ludeman J.P.
      • Wedderburn J.
      • Boston C.
      • Butler S.J.
      • Carrick A.M.
      • Parody T.R.
      • Taleski D.
      • Christopoulos A.
      • Payne R.J.
      • Stone M.J.
      Design and receptor interactions of obligate dimeric mutant of chemokine monocyte chemoattractant protein-1 (MCP-1).
      ). Our results indicate that tyrosine sulfation of the CCR2 N terminus increases the binding affinity for both the MCP-1 monomer and dimer and, moreover, induces dissociation of the dimer into its functional monomeric units. These findings suggest that the sulfation state of CCR2 influences its ability to be activated by MCP-1 and is therefore likely to play a role in the physiological regulation of MCP-1/CCR2 interactions.

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