MCP--MV/C3B/C4B BINDING SITES AND ISOFORM VARIATION

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DESCRIPTION (Adapted from the applicant's abstract): For more than twenty years, the applicant has examined the complement system in an effort to understand its workings in health and disease. Earlier studies focused on the biology of guinea pig, mouse and human C4. Since the 1980's, attention has centered on complement receptors and regulatory proteins. One such regulator, Membrane Cofactor Protein (MCP; CD46), protects host tissue by acting as a cofactor, along with factor I, to inactivate C3b and C4b that deposit on autologous cells. Since the applicant identified MCP as a C3-binding protein in 1984, its role has progressively expanded beyond that of only a complement inhibitor. MCP was recently identified as the measles virus (MV) receptor and is being used as a probe for related virus receptors (rinderpest and canine distemper). Additionally, MCP is of interest in reproductive biology due to its expression on sperm and at the maternal-fetal interface. As an inhibitor of complement activation, MCP is being engineered into xenografts and produced as a soluble therapeutic agent. Because of these and other remarkable interactions of MCP, three specific aims are proposed. First, to identify the binding sites on MCP of MV, C3b and C4b, deletion/substitution constructs will be expressed and evaluated for ligand interaction. Second, the cytoprotective properties of MCP isoforms will be compared by using stably transfected clones bearing equivalent copy numbers of isoforms in functional analyses of ligand binding, cofactor activity and cytoprotection. Third, the functional significance of the two cytoplasmic tails of MCP will be analyzed as they relate to processing of high mannose precursors (pro-MCP) and to phosphorylation. The mechanism governing the tail-mediated fourfold difference in processing of pro-MCP will be determined by using MCP transfectants expressing each tail, tail chimeras and tail mutants to determine if an endoplasmic reticulum (ER) transport accelerating factor, oligomerization in the ER or another process accounts for this difference. The cytoplasmic tails of MCP possess several putative phosphorylation sites. To determine if MCP is phosphorylated on the tail, MCP transfectants, human cells and cell lines will be evaluated following challenge by a complement activating (or MCP upregulating) strategy. Completion of the specific goals of this proposal will provide relevant information not only for the complement system, but also for other areas such as reproductive immunology, xenotransplantation and infectious diseases.