A quantitative characterization of ligand, receptor, and G-protein in signal transduction in the human neutrophil.

Abstract

Competent function of living cells requires cellular communication and the transmission of chemical signals across cell membranes. One of the most prevalent eukaryotic signal transduction mechanisms involves the interaction of a ligand, a receptor, and a guanine nucleotide binding protein (G-protein). In this thesis the quantitative relationship among ligand, receptor and G-protein interactions that leading to cellular responses in the human neutrophil was explored. Existing mathematical models were found to be inadequate for describing high resolution flow cytometric N-formyl peptide binding data at 37$\sp\circ$C, and a binding model including the events of receptor upregulation, internalization, affinity conversion of the receptor, and fluorescence quenching of the tracer ligand was developed. A two-site model was also developed to describe ligand binding at 4$\sp\circ$C, and was used to determine binding rate constants for five N-formyl peptide ligands. The rate constant for the conversion of receptor from low to high affinity form was found to be ligand-dependent, suggesting that differences in the conversion rate constants can, in part, explain observed differences in agonist potency. A graded reduction in receptor number, affected by Cyclosporin H, an N-formyl peptide antagonist, produced a graded actin polymerization response. In contrast, a graded reduction in G-protein number, affected by pertussis toxin treatment, produced not a graded actin polymerization response in all cells, but an all-or-none response characterized by the conversion of cells from a responding to a non-responding population. The all-or-none response was observed for maximal and submaximal stimulus concentrations of two stimuli (N-formyl peptides and leukotriene B$\sb4$) which utilize the same G$\rm\sb{i2}$-protein to transduce the extracellular signal. An assay for quantifying the number of active plasma membrane-associated G$\rm\sb{i2}$-proteins was developed. This assay utilizes avidin-biotin technology and a magnetic solid support to quickly and efficiently isolate plasma membrane fraction from 10$\sp5$ cells. Identification and quantification of the G-protein is accomplished with a combination of autoradiography and immunoblotting. Protocols for the quantitative determination of active G-protein in responding and non-responding cells are described.