A dominant negative inhibitor indicates that monocyte chemoattractant protein 1 functions as a dimer.
Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine family of proinflammatory cytokines, all of which share a high degree 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 chemokines form homodimers, there is controversy about whether dimerization is necessary for activity. To address this question for MCP-1, we obtained evidence in four steps. First, coprecipitation experiments demonstrated that MCP-1 forms dimers at physiological concentrations. Second, chemically cross-linked MCP-1 dimers attract monocytes in vitro with a 50% effective concentration of 400 pM, identical to the activity of non-cross-linked MCP-1. Third, an N-terminal deletion variant of MCP-1 (called 7ND) that inhibits MCP-1-mediated monocyte chemotaxis specifically forms heterodimers with wild-type MCP-1. Finally, although 7ND inhibits wild-type MCP-1 activity, it has no effect on cross-linked MCP-1. These results indicate that 7ND is a dominant negative inhibitor, implying that MCP-1 activates its receptor as a dimer. In addition, chemical cross-linking restores activity to an inactive N-terminal insertional variant of MCP-1, further supporting the need for dimerization. Since the reported Kd for MCP-1 monomer dissociation is much higher than its 50% effective concentration or Kd 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 for MCP-1 inhibitory drugs.
Zhang
Y
Y
Department of Medicine, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
Rollins
B J
BJ
D001483:Base Sequence; D001667:Binding, Competitive; D018932:Chemokine CCL2; D002630:Chemotactic Factors / Q000235:genetics / Q000378:metabolism*; D002634:Chemotaxis, Leukocyte / Q000502:physiology*; D003432:Cross-Linking Reagents; D016207:Cytokines / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017384:Sequence Deletion; D012996:Solutions
D018932:Chemokine CCL2; D002630:Chemotactic Factors; D003432:Cross-Linking Reagents; D016207:Cytokines; D012996:Solutions