The broad, long term objective of the proposed research is to understand the molecular basis of the control of neutrophil activation. This goal has particular health significance because it aids in understanding how these cells mount a microbicidal response and more significantly, how in a pathological state, they lose control of this response resulting in inappropriate inflammatory damage to healthy tissue. We propose to study control of neutrophil activation by focusing on N-formyl-chemotactic receptors which induce highly regulated responses that exhibit distinct activation and termination phases. The receptors clearly are under intricate cellular control since their abundance, distribution, and affinity on the cell surface changes during the course of cell activation. This control will be analysed by characterization of plasma membrane domains whose receptor content appears to be highly regulated. This characterization will entail purification of the domains by high resolution isopycnic ultracentrifugation and characterization of their protein and lipid compositions. It will involve identification of specific cytoskeletal and membrane proteins using immunological probes and will engage in extensive lipid analysis by conventional techniques. These characterizations will focus on components shown lo exhibit modulation in the domain during the activation and termination phases of the cellular response. The molecular interactions of the N-formyl peptide receptor that result in and stem from its differential residency in these domains will also be analysed. This analysis will be carried out by determining the relevant biochemical modifications to the receptor and by identifying the molecular species interacting with the receptor. Particular attention will be paid to modifications of cytoskeletal proteins shown to interact with the receptor. Significant effort will also be devoted to preparation of novel biochemical peptide and immunological probes of receptor structure and function. These probes will then be used to confirm the functional relevance of the molecular interactions observed.
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