title: Tyrosine sulfation of chemokine receptor CCR2 enhances interactions with both monomeric and dimeric forms of the chemokine monocyte chemoattractant protein-1 (MCP-1).
abstract: 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.
authors: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
authors: Joshua H Y
authors: JHY
authors: Tan
authors: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
authors: Justin P
authors: JP
authors: Ludeman
authors: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
authors: Jamie
authors: J
authors: Wedderburn
authors: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia.
authors: Meritxell
authors: M
authors: Canals
authors: Centre for Inflammatory Diseases, Monash University, Department of Medicine, Monash Medical Centre, Clayton, Victoria 3168, Australia.
authors: Pam
authors: P
authors: Hall
authors: School of Chemistry, The University of Sydney, Sydney, New South Wales 2006, Australia.
authors: Stephen J
authors: SJ
authors: Butler
authors: School of Chemistry, The University of Sydney, Sydney, New South Wales 2006, Australia.
authors: Deni
authors: D
authors: Taleski
authors: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia.
authors: Arthur
authors: A
authors: Christopoulos
authors: Centre for Inflammatory Diseases, Monash University, Department of Medicine, Monash Medical Centre, Clayton, Victoria 3168, Australia.
authors: Michael J
authors: MJ
authors: Hickey
authors: School of Chemistry, The University of Sydney, Sydney, New South Wales 2006, Australia.
authors: Richard J
authors: RJ
authors: Payne
authors: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia. Electronic address: martin.stone@monash.edu.
authors: Martin J
authors: MJ
authors: Stone
mesh_terms: D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D018932:Chemokine CCL2 / Q000378:metabolism*; D019281:Dimerization; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D054390:Receptors, CCR2 / Q000737:chemistry* / Q000502:physiology; D013455:Sulfur / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D018932:Chemokine CCL2; D010455:Peptides; D054390:Receptors, CCR2; D014443:Tyrosine; D013455:Sulfur; D048052:Mitogen-Activated Protein Kinase 3; D002118:Calcium