Ligand binding and ligand efficacy in the N-formyl peptide receptor system on the human neutrophil

Cellular communication is essential for organism survival and often is initiated by the binding of a ligand to a membrane-bound receptor. A variety of ligands may bind to the same receptor on the same cell type and thus activate the same signal transduction pathway. However, the rate and magnitude of the final response elicited may be different for each ligand. A question to be asked then is how does the cell distinguish between different ligands? Furthermore, what ligand-dependent parameters could account for differences in dynamic responses, which peak prior to receptor-ligand binding reaching equilibrium? Pharmacologists have defined a parameter, efficacy, to account for a bound ligand's ability to elicit a certain response. Although it is a useful parameter for comparing ligands, there is no molecular nor mechanistic basis for it.; In this thesis these questions are addressed by investigating the impact of the initial receptor-ligand binding and receptor processing kinetics on the final response. The model system utilized was the N-formyl peptide receptor system on the human neutrophil. This receptor is a member of the superclass of guanine nucleotide binding protein (G-protein) coupled receptor (GPCR) system and binds with multiple N-formyl peptide ligands, including fluorescently labeled peptides. This interaction between ligand and receptor results in the formation a low-affinity receptor-ligand complex that is believed to be a signaling complex, and in time converts to a high-affinity complex. Dynamic responses, such as actin polymerization and oxidant production, are elicited by ligand binding to the N-formyl peptide receptor with the time scale of maximum response occurring prior to receptor-ligand binding reaching equilibrium (about 10 seconds for actin polymerization and around 300 seconds for oxidant production).; Via quantitative measurements of ligand binding to the receptors by flow cytometry, the transient low-affinity complex was found to exist for six different N-formyl peptides. The methodology for monitoring ligand binding for unlabeled ligands by competitive binding protocols was validated and produced estimates for the initial rate constants describing the receptor-ligand interactions with no statistical difference from estimates evaluated by direct binding protocols. Based on the rate constant describing the conversion from low- to high-affinity complex, k_x, the ligands were classified into two groups. The ligand group with large values of k_x exhibited a clear dependence of the rate of formation of this low-affinity complex to the actin polymerization response. However, for the small k_x ligands the response was less dependent upon the presence of the low-affinity complex, as was the case for oxidant production. These results suggest that ligand-binding kinetics can only describe part of the response characteristics. In conclusion, the classification of ligands based on dynamic rate constants can be a useful tool in understanding cellular responses.