Identification of surface residues of the monocyte chemotactic protein 1 that affect signaling through the receptor CCR2

The CC chemokine, monocyte chemotactic protein, 1 (MCP-1) functions as a ma jor chemoattractant for T-cells and monocytes by interacting with the seven -transmembrane G protein-coupled receptor CCR2, To identify which residues of MCP-1 contribute to signaling though CCR2, we mutated all the surface-ex posed residues to alanine and other amino acids and made some selective lar ge changes at the amino terminus. We then characterized the impact of these mutations on three postreceptor pathways involving inhibition of cAMP synt hesis, stimulation of cytosolic calcium influx, and chemotaxis. The results highlight several important features of the signaling process and the con- elation between binding and signaling: The amino terminus of MCP-1 is essen tial as truncation of residues 2-8 ([1+9-76]hMCP-1) results in a protein th at cannot stimulate chemotaxis, However, the exact peptide sequence may be unimportant as individual alanine mutations or simultaneous replacement of residues 3-6 with alanine had little effect. Y13 is also important and must be a large nonpolar residue for chemotaxis to occur, Interestingly, both Y 13 and [1+9-76]hMCP-1 are high-affinity binders and thus affinity of these mutants is not correlated with ability to promote chemotaxis, For the other surface residues there is a strong correlation between binding affinity an d agonist potency in-all three signaling pathways. Perhaps the most interes ting observation is that although Y13A and [1+9-76]hMCP are antagonists of chemotaxis, they are agonists of pathways involving inhibition of cAMP synt hesis and, in the case of Y13A, calcium influx. These results demonstrate t hat these two well-known signaling events are not sufficient to drive chemo taxis, Furthermore, it suggests that specific molecular features of MCP-1 i nduce different conformations in CCR2 that are coupled to separate postrece ptor pathways. Therefore, by judicious design of antagonists, it should be possible to trap CCR2 in conformational states that are unable to stimulate all of the pathways required for chemotaxis.