The long term goal is the elucidation of the biochemical mechanisms which modify the function and expression of the receptor for leukotriene B4 (LTB4) on human polymorphonuclear leukocytes (PMNL)s and which consequently regulate the recruitment and activation of PMNLs at inflammatory sites. Initial studies provided evidence for two distinct components in the signal transduction pathway: a recognition unit which specifically binds LTB4 and a GTP-dependent regulatory (G) protein which transduces receptor occupancy into PMNL activation. Affinity labeling techniques have demonstrated that the receptor is a protein distinct from the G-protein, which interacts with the receptor and which mediates the effects of GTP on LTB4 binding to the receptor. The studies are directed towards characterizing the interaction of the LTB4 receptor with the associated G-protein, defining the functional integrity of these components under a variety of different conditions, and identifying biochemical modifications of the receptor which regulate the capacity of LTB4 to stimulate PMNL function. The extent of the LTB4 receptor interaction with G-protein will be analyzed by measuring the effects of GTP on LTB4 binding, by quantifying the exchange of guanine nucleotides induced by LTB4, and by demonstrating a LTB4-elicited increase in GTPase activity. Implantation of the solubilized LTB4 receptor and G-protein into deficient cells and membranes will permit an evaluation of the capacity of these components to participate in the signal transduction pathways which mediate increases in phosphatidylinositol metabolism and in cytosolic calcium, and thus elucidate some of the molecular mechanisms which regulate signal transduction events during desensitization of PMNL function. The LTB4 receptor will be isolated by chromatography on LTB4-affinity columns, and the binding properties of the isolated receptor will be defined. The G-protein will be isolated by chromatographic techniques and its physical properties compared to the other G-proteins which have already been purified and characterized. Antibodies to the LTB4 receptor, which will be used to assess changes in receptor expression on PMNLs, will be developed both by immunizing goats and by monoclonal techniques. Understanding how regulation of LTB4 receptor function controls the extent of PMNL activation will lead to a better understanding of PMNL dysfunction in pathogenic states.
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