Signaling through the N-formyl peptide receptor on human neutrophils: G-protein quantification and thresholds.

摘要

Proper functioning of neutrophils requires the regulation of oxidant production in response to bacterial infection. This regulation involves N-formyl peptide receptors which are G-protein coupled receptors. One powerful heuristic to understand this regulation is combining quantitative data with mathematical models to predict changes in the signaling pathways responsible for oxidant production. In this thesis, this heuristic was used to elucidate the role G-proteins play in generating a threshold type response in the oxidant production in human neutrophils.; A G-protein quantification assay was developed to measure G-proteins from isolated plasma membrane fragments. This method consists of using magnetic beads conjugated with antibody for CD15, a cell surface protein, as an anchor. Plasma membrane fragments were free of contaminating secretory vesicles, primary and secondary granules. Unlike conventional methods for isolating plasma membrane fragments, this assay could be used to quantify G-protein in small numbers of cells (106 cells).; Human neutrophil responses to N-formyl peptides, including oxidant production and release, exhibit threshold behavior with respect to the number of G-proteins available for signaling: progressive treatment of neutrophils with pertussis toxin causes the conversion of responding cells to non-responding cells. To quantify the threshold level of G-proteins required for signaling of N-formyl peptide stimulated oxidant production in a neutrophil population, the developed G-protein quantification assay was used in conjunction with a sorting flow cytometer to measure differences in the average number of G-proteins available for signaling per cell (GAS) in responding and non-responding subpopulations after pertussis toxin treatment. Although there appeared to be a threshold separating responding and non-responding cells for a given sample, no discrete threshold was measured across multiple treatment conditions. A mathematical model of the early steps in signaling suggests that cell-to-cell variability in signal parameters such as numbers of signal components and values of kinetic rate constants obscures the measurement of a discrete threshold and leads to an apparent decrease in the GAS threshold as the total G-protein is decreased. However, further modeling and experimentation will be required to develop a mechanistic relationship between receptor and G-protein level events and oxidant production.