Numerous hormones and neurotransmitters act by initiating the turnover of polyphosphoinositides and subsequent release of intracellular calcium, yet very little is known about how the receptors for these agents couple to the phospholipase C effector system. The neutrophil is a cell which participates in many aspects of the inflammatory response, both in a defensive capacity and also as a cause of many of the pathological responses to inflammation. Neutrophil activation by several chemotactic agents occurs through receptor-mediated phospholipase C activation, leading to release of intracellular calcium and the activation of protein kinase C. Recent evidence implicates the GTP-binding regulatory proteins (G proteins) in this signal transduction process. We plan to identify and characterize neutrophil G proteins using both immunological and biochemical techniques. We will assess the interaction of the N-formyl peptide chemotactic factor receptor with G proteins in both permeabilized neutrophil preparations and in membranes using fluorescent analysis of ligand binding in real time. We will prepare an in vitro reconstitution system in which we can directly examine the interaction of the NFP receptor and G protein(s). Our ability to prepare purified G protein subunits, in combination with reconstitution methodology, will allow us to develop a system in which the R-G interaction can be evaluated both qualitatively and quantitatively for the first time. We will then examine mechanisms of G activation by R, subunit requirements, kinetics of interaction, specificity and efficacy of various G proteins for the interactions, results of covalent modificaiton upon R-G dynamics, etc. Such knowledge of signal transduction in the neutrophil should have profound implications in the regulation of inflammatory processes involved in such disease states as rheumatoid arthritis, atherosclerosis, myocardial infarct damage, autoimmune disorders, and acute inflammation.
Showing the most recent 10 out of 68 publications
