Neutrophils are stimulated by specific ligands that interact with distinct classes of membrane receptors. In response, these cells migrate, phagocytize particles, generate injurious oxidants and discharge granule enzymes. These processes constitute fundamental facets of inflammation and contribute to a variety of human diseases. The goal of this proposed research is to describe quantitatively how the interactions of a model ligand, a fluorescent N-formyl peptide, with is receptor leads to the observed cellular responses, particularly the secretion of proteases and the production of free radicals of oxygen. Stimulatory ligands bind to the receptor with a half time (less than 1 minute) which is considerably longer than the period required to initiate the cellular responses (less than 5 seconds). The binding of the ligands appears to involve a change in receptor affinity which occurs prior to or during the course of the cellular response. The observed cellular responses depend both upon the rate and number of receptors occupied and may depend in part upon the proportion of receptors in the available affinity states. Internalization of the ligand-receptor complex is slower than the cellular responses noted above and appears to be separable, in time, from the binding events and the change in receptor affinity. We propose to describe in detail the quantitative relationships among receptor occupancy, cellular responses, and those biochemical events which initiate the cellular responses. In particular, we will: 1) define the interactions of stimulatory and non-stimulatory ligands with the receptor; 2) analyze the residence characteristics of these ligands and relate these characteristics to the cellular responses; 3) examine the dependence of the cellular responses on the rate and extent of receptor occupancy. To accomplish these goals we have available: 1) a series of novel real-time spectroscopic procedures with which to analyze ligand binding, ligand dissociation, ligand internalization, and cellular responses, all with a time resolution of 1 to 5 seconds; 2) methods to control independently the rate and the extent of receptor occupancy; 3) computerized analyses of the kinetics of ligand binding and dissociation and competitive binding interactions. Our work will not only yield unique information concerning the stimulation of neutrophils but should have applicability to many related areas of receptor-mediated cell stimulation.
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