The binding of chemoattractants to receptors on phagocytes leads to biologically important responses such as directed motility, secretion of lysosomal enzymes, and production of toxic oxygen products. This research will characterize the biochemical pathways which regulate these processes during leukocyte activation. We previously identified the receptor for oligopeptide chemoattractants on human polymorphonuclear leukocytes (PMNs) and mononuclear phagocytes. We have also demonstrated GTP-dependent receptor-mediated degradation of phosphatidylinositol 4,5-bisphosphate in PMN plasma membranes. We proposed that chemoattractant receptors are coupled to a guanine nucleotide regulatory (N) protein which activates a phospholipase. This research will characterize the components of the transduction pathway of chemoattractant receptors and other leukocyte activators. It will characterize the mechanisms of phosphoinositide metabolism in phagocytic cells and identify how inflammatory mediators stimulate phosphoinositide degradation and Ca2+ mobilization. We have shown that activation of human PMNs is comprised of 3 processes: primining, activation, and desensitization. This research will characterize the molecular mechanisms of these responses. It will determine if priming and/or activation is due to alterations in the association of certain relevant enzymes with the plasma membranes of PMNs. It will determine if desensitization is located at the level of the receptor, the N protein, or phospholipase C. Moreover, it will determine whether the pathways responsible for chemotaxis vs. lysosomal enzyme secretion and superoxide anion production are distinct. An objective of these studies is to identify and reconstitute the initial steps of the oligopeptide chemo-attractant receptor transduction mechanism in a synthetic membrane. Purified receptor and N proteins will be reconstituted into phospholipid vesicles so that the specific N protein which interacts with the chemotactic factor receptor can be identified. The gene which codes for the oligopeptide chemoattractant receptor will be cloned allowing for the determination of its entire primary amino acid sequence. Finally, the nature of the defect responsible for dysfunctional chemotaxis associated with localized juvenile periodontitis will be determined. Information gained from these studies will be of importance, not only for understanding leukocyte activation, but also for understanding of the transduction mechanisms of the large group of receptors which mobilize Ca2+ through polyphosphoinositide hydrolysis.
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