When the mammalian organism is invaded by bacteria one critical aspect of the host response is the directional migration (chemotaxis) of leukocytes out of the bloodstream into sites of inflammation containing bacteria. Peptides such as CHO-Met-Leu-Phe-OH (FMLP) are present in culture filtrates from bacteria and can be shown to be chemoattractant for leukocytes. Chemotactic peptides, such as FMLP, can be shown to bind saturably and reversibly to a receptor on the surface of neutrophils and macrophages from a variety of species. The interaction of the peptide with the receptor is known to initiate a plethora of responses, including chemotaxis, secretion of cytolytic lysosomal granule enzymes, and the activation of metabolic pathways. Very little is known, particularly at the molecular level, about the receptor for chemotactic peptides. In the research work described in this proposal we intend to purify the rabbit neutrophil chemotaxis receptor to homegeneity. This will be accomplished by immunoaffinity procedures utilizing monoclonal antibodies reactive with the receptor. The molecular structure of the chemotaxis receptor will be studied to the extent of determination of partial amino acid sequence. These sequence studies will ultimately lead to the construction of a nucleotide probe that will be used to screen for the gene encoding the receptor. Radiochemical micromethods will be utilized to compare by peptide-mapping the structure of the murine chemotaxis receptor from different inbred strains of mice shown to possess chemotaxis receptors of differing affinities. We will evaluate the role of the intracellular domain of the receptor in signal transduction by perturbing this region of the molecule with a cytoplasmically """"""""injected"""""""" antibody specific for this region of the receptor. Finally, we will use DNA-mediated gene transfer experiments to evaluate whether or not it might ultimately be possible to put a modified gene for the FMLP-receptor back into a mammalian cell and then observe the biological effect of the new (altered) receptor. Our overall aims are to apply the molecular approaches used successfully to investigate the cell surface antigens and receptors of the cells of the immune system to cells of the inflammatory system. Detailed moleculr information about chemotaxis receptors is of direct health-relatedness. Thus, the information gained could aid in designing novel antiinflammatory reagents which might be applicable to the large numbers of patients with hypersensitivity and inflammatory diseases.
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