HUMAN MYELOID CHEMOTACTIC RECEPTOR: STRUCTURE-FUNCTION

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This proposal explores several aspects of biochemical structure, metabolism and function of the formyl peptide chemotactic receptor present on human peripheral blood leukocytes. This is the beginning of a long-term study of human myeloid membrane development and function. This receptor, either native or covalently affinity-labeled, will be solubilized from human neutrophils or cultured myeloid cell lines (HL-60 and U937), purified by classical and affinity techniques and characterized with respect to protease domains and peptide maps, hydrophobicity, and molecular size. Anti-receptor antibodies will be produced and spontaneous anti-receptor antibodies will be sought in serum from patients with anti-granulocyte antibodies. The importance and mechanism of receptor-mediated ligand aggregation and endocytosis will be investigated using fluorescently-labeled agonists and antagoinists, multivalent antagonists and anti-receptor antibodies. The disposition of the receptor within the membrane and neighboring membrane proteins will be explored. The rate of receptor synthesis and turnover will be determined during myeloid maturation using heavy isotope amino acid labeling and CsCl2 gradient centrifugation. Chemotactic receptor structure and processing will be studied in patients with abnormal myeloid development (leukemias, myelodysplastic diseases, paroxysmal nocturnal hemoglobinuria, after bone marrow transplantation or cytotoxic chemotherapy), bacterial sepsis or recurrent bacterial infection and during anti-inflammatory drug use. Two scientific disciplines are involved. Hematology, because the proposal deals with blood leukocyte function, HL-60 differentiation is a model system for leukocyte development in the bone marrow and several hematologic diseases will be studied. And Immunology, because the receptor mediates immunologically important leukocyte responses.