HUMAN MYELOID CHEMOTACTIC RECEPTOR: STRUCTURE-FUNCTION

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The leukocyte form peptide receptor recognizes a product of bacterial metabolism and mediates leukocyte responses to microbial invasion. The long term objective is to define the biochemical pathway of this response. This proposal will focus on signal transduction via the formyl peptide receptor. Activation of phospholipase C appears to be the earliest measurable biochemical consequence of receptor occupancy. Diacylglycerol and inositol trisphosphate are generated; the former activates protein kinase C, the latter releases Ca2+ from intracellular stores. We have developed a novel assay to measure absolute levels of diacylglycerol in biological samples. This assay will be used to measure diacylglycerol levels in leukocytes after stimulation with formyl peptides and other agonists. The fatty acid composition, subcellular location and metabolism of the diacylglycerols will be determined. The role of diacylglycerol in receptor desensitization will be explored. An assay will be developed for measuring formyl peptide-stimulated diacylglycerol production in membranes and the role of a GTP binding protein in this process will be investigated. Synthetic dioctanoylglycerol causes both degranulation and oxidative burst, while didecanoylglycerol causes only degranulation. We will compare these compounds with regard to C kinase translocation and [32P] phosphoprotein changes induced in intact leukocytes. This may allow distinction between phosphoproteins involved in each response. The formyl peptide receptor will be purified by established techniques and the purified product compared with the receptor identified by affinity labeling. The proposal is health related because defects in leukocyte function result in recurrent infections. Many other cell surface receptors utilize diacylglycerol as a second messenger, so information obtained from these studies will have wide application to mechanisms of neoplastic transformation, wound healing and vascular disease.