Monomeric monocyte chemoattractant protein-1 (MCP-1) binds and activates the MCP-1 receptor CCR2B

To address the role of dimerization in the function of the monocyte chemoat tractant protein-1, MCP-1, we mutated residues that comprise the core of th e dimerization interface and characterized the ability of these mutants to dimerize and to bind and activate the MCP-1 receptor, CCR2b, One mutant, P8 A*, does not dimerize. However, it has wild type binding affinity, stimulat es chemotaxis, inhibits adenylate cyclase, and stimulates calcium influx wi th wild type potency and efficacy. These data suggest that MCP-1 binds and activates its receptor as a monomer. In contrast, Y13A*, another monomeric mutant, has a 100-fold weaker binding affinity, is a much less potent inhib itor of adenylate cyclase and stimulator of calcium influx, and is unable t o stimulate chemotaxis. Thus Tyr(13) may make important contacts with the r eceptor that are required for high affinity binding and signal transduction , We also explored whether a mutant, [1+9-76]MCP-1 (MCP-1 lacking residues 2-8), antagonizes wild type MCP-1 by competitive inhibition, or by a domina nt negative mechanism wherein heterodimers of MCP-1 and [1+9-76]MCP-1 bind to the receptor but are signaling incompetent. Consistent with the finding that MCP-1 can bind and activate the receptor as a monomer, we demonstrate that binding of MCP-1 in the presence of [1+9-76]MCP-1 over a range of conc entrations of both ligands fits well to a simple model in which monomeric [ 1+9-76]MCP-I functions as a competitive inhibitor of monomeric MCP-1, These results are crucial for elucidating the molecular details of receptor bind ing and activation, for interpreting mutagenesis data, for understanding ho w antagonistic chemokine variants function, and for the design of receptor antagonists.