Monocyte chemoattractant protein-1 (MCP-1), a member of the family of related proteins known as chemokines, is a mediator of leukocyte migration and activation that plays a number of significant roles in health and disease, including inflammation, atherosclerosis, cancer, wound healing and host defense. MCP-1 production and function constitute a target suitable for clinical intervention to treat a variety of such diseases. A detailed understanding of the structure and function of MCP-1 and its receptor is necessary to develop such therapeutic strategies. The MCP-1 structure can be modeled based on the structures of other chemokines and is likely to consist of a globular domain made up of an alpha helix and a three-strand beta sheet. The N-terminus of the protein extends as a rod from the globular domain. MCP-1 exerts its biological effects by binding to a number of receptors that are members of the seven transmembrane receptor family. Two of these receptors that have been characterized are the MCP-1 receptor B (MCP-1RB) (CC-CKR2B) and the product of the cytomegalovirus open reading frame US28 (CMV-US28). CC-CKR2B is specific for MCP-1 while CMV-US28 binds to MCP-1 plus a number of other chemokines. The long term goal of this research is to elucidate how MCP-1 binds to these receptors to transduce a signal into the target cell. A two-pronged approach, involving synergistic studies on both the receptors and MCP-1 will be used to build understanding of MCP-1/receptor interactions.
The specific aims to be accomplished are as follows: 1)Perform mutagenesis of MCP-1 to find the functionally important residues of the polypeptide. Determine the residues responsible for MCP-1/receptor interactions by making mutations in three regions: the N-terminal extended rod, the region at the base of the rod, and the globular domain, and determine the biological consequences by measuring binding, signaling and chemotaxis. Identify the residues in MCP-1 responsible for binding to glycosaminoglycans. 2) Make amino acid substitutions in evolutionarily conserved residues of MCP-I receptors CC-CKR2B to find which sites are involved in ligand binding and signal transduction. 3) Determine the sequence elements responsible for chemokine receptor specificity by creating two sets of chimeric receptors: measuring binding of MCP1 versus RANTES to CC-CKR2B/CMV-US28 chimeras and signaling triggered by the binding; and measuring binding and signal transduction parameters of MCP-1 versus IL8 to CMV-US28/IL8RB chimeras. 4) Find regions of the receptors that directly interact with specific residues of MCP-I by finding MCP-I mutations that differentially affect binding to different receptors or the consequent signal transduction, then characterizing the binding of this subset of MCP-1 mutants to chimeric receptors and the consequent signaling.
