A DOMINANT-NEGATIVE INHIBITOR INDICATES THAT MONOCYTE CHEMOATTRACTANTPROTEIN-1 FUNCTIONS AS A DIMER

Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokin e family of proinflammatory cytokines, all of which share a high degre e of amino acid sequence similarity, Aberrant expression of chemokines occurs in a variety of diseases that have an inflammatory component, such as atherosclerosis, Although structural analyses indicate that ch emokines form homodimers, there is controversy about whether dimerizat ion is necessary for activity. To address this question for MCP-1, we obtained evidence in four steps, First, coprecipitation experiments de monstrated that MCP-1 forms dimers at physiological concentrations, Se cond, chemically cross-linked MCP-1 dimers attract monocytes in vitro with a 50% effective concentration of 400 pM, identical to the activit y of non-cross-linked MCP-1. Third, an N-terminal deletion variant of MCP-1 (called 7ND) that inhibits MCP-1-mediated monocyte chemotaxis sp ecifically forms heterodimers with wild-type MCP-1, Finally, although 7ND inhibits wild-type MCP-1 activity, it has no effect on cross-linke d MCP-1, These results indicate that 7ND is a dominant negative inhibi tor, implying that MCP-1 activates its receptor as a dimer, In additio n, chemical cross-linking restores activity to an inactive N-terminal insertional variant of MCP-1, further supporting the need for dimeriza tion. Since the reported K-d for MCP-1 monomer dissociation is much hi gher than its 50% effective concentration or K-d for receptor binding, active dimer formation may require high local concentrations of MCP-1 . Our data further suggest that the dimer interface can be a target fo r MCP-1 inhibitory drugs.