title: Receptor binding kinetics and cellular responses of six N-formyl peptide agonists in human neutrophils.
abstract: The goal of this study was to elucidate the relationships between early ligand binding/receptor processing events and cellular responses for the N-formyl peptide receptor system on human neutrophils as a model of a GPCR system in a physiologically relevant context. Binding kinetics of N-formyl-methionyl-leucyl-phenylalanyl-phenylalanyl-lysine-fluorescein and N-formyl-valyl-leucyl-phenylalanyl-lysine-fluorescein to the N-formyl peptide receptor on human neutrophils were characterized and combined with previously published binding data for four other ligands. Binding was best fit by an interconverting two-receptor state model that included a low affinity receptor state that converted to a high affinity state. Response behaviors elicited at 37 degrees C by the six different agonists for the N-formyl peptide receptor were measured. Dose response curves for oxidant production, actin polymerization, and G-protein activation were obtained for each ligand; whereas all ligands showed equal efficacy for all three responses, the ED(50) values varied as much as 7000-fold. The level of agonism and rank order of potencies of ligands for actin and oxidant responses were the same as for the G-protein activation assay, suggesting that the differences in abilities of ligands to mediate responses were determined upstream of G-protein activation at the level of ligand-receptor interactions. The rate constants governing ligand binding and receptor affinity conversion were ligand-dependent. Analysis of the forward and reverse rate constants governing binding to the proposed signaling receptor state showed that it was of a similar energy for all six ligands, suggesting the hypothesis that ligand efficacy is dictated by the energy state of this ligand-receptor complex. However, the interconverting two-receptor state model was not sufficient to predict response potency, suggesting the presence of receptor states not discriminated by the binding data.
authors.affiliation: Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan 48105, USA.
authors.forename: Anna
authors.initials: A
authors.lastname: Waller
authors.forename: Karyn L
authors.initials: KL
authors.lastname: Sutton
authors.forename: Tamara L
authors.initials: TL
authors.lastname: Kinzer-Ursem
authors.forename: Afaf
authors.initials: A
authors.lastname: Absood
authors.forename: John R
authors.initials: JR
authors.lastname: Traynor
authors.forename: Jennifer J
authors.initials: JJ
authors.lastname: Linderman
authors.forename: Geneva M
authors.initials: GM
authors.lastname: Omann
mesh_terms: D000199:Actins / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005452:Fluoresceins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D005453:Fluorescence; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D016877:Oxidants / Q000096:biosynthesis; D011869:Radioligand Assay; D044042:Receptors, Formyl Peptide / Q000819:agonists* / Q000378:metabolism*; D015398:Signal Transduction; D013462:Sulfur Radioisotopes
chemical_list: D000199:Actins; D005452:Fluoresceins; D008024:Ligands; D009842:Oligopeptides; D016877:Oxidants; D044042:Receptors, Formyl Peptide; D013462:Sulfur Radioisotopes; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins