The role of ligand- and cell-specific parameters in influencing G protein coupled receptor states and cell responses.

Abstract

G protein coupled receptors (GPCRs) exist in multiple dynamic states (e.g. ligand-bound, active, internalized) that influence G-protein activation and ultimately response generation. This thesis integrates experimental measurements and mathematical models of GPCR signaling to examine ligand- and cell-specific parameters and temporal characteristics of signaling important in generating cellular responses. A particular GPCR system, the N-formyl peptide receptor on human neutrophils, and more general predictions for GPCR systems are examined. Binding of <super>[35</super>S<super>]</super>GTPgammaS to neutrophil membranes induced by six N-formyl peptide agonists was characterized and found to be strongly correlated with responses, suggesting that the character of N-formyl peptide-induced responses is determined early in the signal transduction cascade, at or near the receptor level. A well-characterized N-formyl peptide binding model, containing both a low-affinity and high-affinity bound receptor state, was found to predict antagonist-induced inhibition of the actin polymerization response elicited by agonist. Further, ligand-receptor binding rate constants for seven N-formyl peptide ligands were used to examine the number of receptor states required to describe the activation of neutrophils (actin polymerization and oxidant production). An additional receptor state, one not observed in kinetic binding assays, was required to account for these responses. This receptor state was interpreted as the number of low affinity bound receptors that are capable of activating G-proteins. Finally, parameter variation and sensitivity analysis were used to study a kinetic model of general GPCR signaling and determine the ligand- and cell-specific parameters important in determining response behavior. The character of response (i.e. positive/neutral/negative agonism) was significantly influenced both by a ligand's ability to bias the receptor into active conformations and by several cell-specific parameters, including the ratio of active to inactive receptor species, the rate constant for G protein activation, and expression levels of receptors and G proteins. This latter finding is significant in that receptor and G protein expression levels are routinely varied using molecular biology techniques and can be experimentally measured. Specifically, expressing either receptor or G protein in numbers several fold above or below endogenous levels may result in system behavior inconsistent with that measured in endogenous systems.