title: Design and receptor interactions of obligate dimeric mutant of chemokine monocyte chemoattractant protein-1 (MCP-1).
abstract: Chemokine-receptor interactions regulate leukocyte trafficking during inflammation. CC chemokines exist in equilibrium between monomeric and dimeric forms. Although the monomers can activate chemokine receptors, dimerization is required for leukocyte recruitment in vivo, and it remains controversial whether dimeric CC chemokines can bind and activate their receptors. We have developed an obligate dimeric mutant of the chemokine monocyte chemoattractant protein-1 (MCP-1) by substituting Thr(10) at the dimer interface with Cys. Biophysical analysis showed that MCP-1(T10C) forms a covalent dimer with similar structure to the wild type MCP-1 dimer. Initial cell-based assays indicated that MCP-1(T10C) could activate chemokine receptor CCR2 with potency reduced 1 to 2 orders of magnitude relative to wild type MCP-1. However, analysis of size exclusion chromatography fractions demonstrated that the observed activity was due to a small proportion of MCP-1(T10C) being monomeric and highly potent, whereas the majority dimeric form could neither bind nor activate CCR2 at concentrations up to 1 μM. These observations help to reconcile previous conflicting results and indicate that dimeric CC chemokines do not bind to their receptors with affinities approaching those of the corresponding monomeric chemokines.
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
authors.forename: Joshua H Y
authors.initials: JH
authors.lastname: Tan
authors.forename: Meritxell
authors.initials: M
authors.lastname: Canals
authors.forename: Justin P
authors.initials: JP
authors.lastname: Ludeman
authors.forename: Jamie
authors.initials: J
authors.lastname: Wedderburn
authors.forename: Christopher
authors.initials: C
authors.lastname: Boston
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Butler
authors.forename: Ann Marie
authors.initials: AM
authors.lastname: Carrick
authors.forename: Todd R
authors.initials: TR
authors.lastname: Parody
authors.forename: Deni
authors.initials: D
authors.lastname: Taleski
authors.forename: Arthur
authors.initials: A
authors.lastname: Christopoulos
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Payne
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Stone
mesh_terms: D019943:Amino Acid Substitution; D002460:Cell Line; D018932:Chemokine CCL2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020125:Mutation, Missense; D055503:Protein Multimerization; D054390:Receptors, CCR2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: C111835:CCL2 protein, human; C515334:CCR2 protein, human; D018932:Chemokine CCL2; D054390:Receptors, CCR2