The central focus of this proposal is the elucidation of the biochemistry and the role in cellular activation of human Fe gamma receptors (FcR) expressed on monocyt-es, neutroohi lvmphocvtes and human leukemic cells as defined by a monoclonal antibodv KuFc-9. In addit two new clones (02 and 19 have been demonstrated to have similar cellular dist-ibution patt as KuFc79 and will be analyzed coneurrently as potential anti-FcR antibodies. The questio structural heterogeneitv of FcR defined by KuFc79 on var!olis cell types will be addresse extensive bio- and physiochemical analysis including SDS-gel electrophoresis, isoelee focusing, tryptic peptide mar)ping, pulse-chase experiments, Western blotting, analvsis of s residues and amino acid sequencing. Initial steps will be taken to isolate the specific o, coding for FcR using a gtll expression vector EDNA librarv of the FcR-positive U93'4' cell I T,he number of FcR, their binding affinities and specificiiies will be calcultated by Scate analvsis usincr direct binding of the 1251-labeled Fab fragments of KuFc79 to purified populat of cells isolated from blood and etablished cell lines in cultures. The ability of KuFc79 to inh the binding of radiolabeled human IgG subclasses will be determined for each cell type in o to define the isotype specificitv of FcR binding. Particular attention will be made to pl various cell lineages unde prolfferative and differentiative signals as well as human leuke cells to determine the number and the type of FcR species expressed in conjunction with o immunologically relevant receptors such as complement receptors, transferrin receptors HLA-DR antigens. Furtherrrore, it will be determined whether FcR interaction is transduced hydrolysis of inositol phosdholipids. Functional consequences of FcR activation by receptor and (KuFc79) interaction will be assessed in immune systems by lymphokine-mononkine production and in non-immune systems by monitoring releaseof lysosomal enzymes, elaboration oxvgen metabolites and prostaglandin production. The long-term objective of this proposal is to use KuFc79 to isolate and biochemically characterize the FcR species expressed on different normal an neoplastic cell types and to determine the role of these receptors in the activation and differentiation of the cells which bear them.
|Alevy, Y G; Tucker, J; Naziruddin, B et al. (1993) CD32C (Fc gamma RIIC) mRNA expression and regulation. Mol Immunol 30:775-82|
|Alevy, Y G; Tucker, J; Mohanakumar, T (1992) CD32A (Fc gamma RIIa) mRNA expression and regulation in blood monocytes and cell lines. Mol Immunol 29:1289-97|
|Naziruddin, B; Duffy, B F; Tucker, J et al. (1992) Evidence for cross-regulation of Fc gamma RIIIB (CD16) receptor-mediated signaling by Fc gamma RII (CD32) expressed on polymorphonuclear neutrophils. J Immunol 149:3702-9|
|Willis, H E; Browder, B; Feister, A J et al. (1988) Monoclonal antibody to human IgG Fc receptors. Cross-linking of receptors induces lysosomal enzyme release and superoxide generation by neutrophils. J Immunol 140:234-9|