The long term objectives are to define the role in immune and inflammatory reactions of receptors for the third component of complement, C3, that reside on lymphocytes, neutrophils, monocytes/macrophages, erythrocytes and other cell types. Two receptors are the focus of proposed studies. CR1, the C3b/C4b receptor that is relatively deficient on several cell types of patients with systemic lupus erythematosus (SLE), and CR2, the C3d receptor of B lymphocytes that is also the receptor for Epstein-Barr virus (EBV). These studies will elucidate further the structure of CR1 and CR2, the regulation of their biosynthesis, their roles in phagocytic reactions of neutrophils and proliferative reactions of lymphobytes, and the basis for their abnormalities in rheumatic disease. There are five specific aims by which these goals will be approached. First, for the molecular biology of CR1, full length cDNA will be obtained, the CR1 gene will be mapped with phage and cosmid clones, and the basis for variant forms of CR1 mRNA will be determined. Second, the relationship between phosphorylation of CR1 and its phagocytic function will be assessed by determining whether all agents that confer this capability on CR1 of neutrophils and monocytes also cause phosphorylation of CR1, by analyzing the phosphoamino acid(s) and phosphopeptide(s) of the receptor, and by constructing with molecular biologic techniques a hybrid molecule containing CR1 cytoplasmic and transmembrane regions and murine H-2 extracellular domains. The hybrid gene will be transfected into myelomonocytic cells and lines will be assayed for CR1-like functions. Third, the genetic basis for variable CR1 expression on cells of normals and SLE patients will be determined by analyzing cultured erythroid precursors for CR1 number, measuring CR1 mRNA in these cells and in B lymphocytes of selected deficient patients and relatives, and searching for restriction fragment length polymorphisms that correspond to structural and quantitative phenotypes of CR1. Fourth, cDNA clones specific for CR1 will be identified in a B cell library by hybridization with synthetic oligonucleotide probes based on sequence analysis of tryptic peptides generated from purified CR2, the clones will be used to identify cosmid clones containing the CR2 gene, and the gene will be modified and transfected into mouse or human cell lines to determine the C3d and EBV binding sites of the receptor. The fifth aim is to explore the unusual finding that certain monoclonal anti-CR2 antibodies induce T cell proliferation by defining the membrane protein on the T cell with which the monoclonal antibodies interact.
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