Killer/Natural Killer (K/NK) cells are large granular lymphocytes (LGLs) which naturally protect one from cancers and infectious viruses by actively lysing diseased cells, thereby limiting their spread within an individual. The """"""""killer"""""""" function of K/NK cells is antibody-dependent cellular cytotoxicity (ADCC), whereas the """"""""natural killer"""""""" function is an antibody-independent cytolytic process. All K/NK cells bear Fc receptors (FcRs) on their surfaces which specifically bind to the Fc portions of antibodies on antibody-coated target cells. FcR/Fc interactions cause K/NK cells to form specific conjugates with their target cells which initiates the ensuing lysis of the targets. The FcRs interaction with antibody and the role of such interactions play in triggering the lytic processes are not presently understood. The primary objective of the experiments proposed here is to develop a simple and efficient method for preparatively purifying the FcRs of K/NK cells isolated from human peripheral blood leukocytes (PBL) using an anti-FcR monoclonal antibody and conventional biochemical and immunochemical strategies for membrane protein purification. The initial studies will focus on the structure of the FcR by determining the number of polypeptide subunits comprising the FcR and potential precursor/product relationships between different subunits. Purified FcR molecules will be fragmented by CNBr and the resulting peptide fragments will be characterized by gel filtration and by SDS polyacrylamide gel electrophoresis. The Phase II objective of this project would be to determine enough of the amino acid sequence of FcR molecules to make possible the development of a suitable oligonucleotide probe to be used for isolating the FcR gene(s) from the human genome. The isolated gene(s) would allow one to conduct molecular studies of FcR/Fc interactions and the lytic process. The sequence of the gene(s) could also be used to develop monoclonal antibody probes against selected portions of the FcR structure to clinically evaluate K/NK cell functions and modify K/NK functions response to disease.
