title: Exploring the mechanism of agonist efficacy: a relationship between efficacy and agonist dissociation rate at the muscarinic M3 receptor.
abstract: Although there are several empirical approaches that enable the comparison of relative agonist efficacy, the molecular basis that underlies differences in the ability of G protein-coupled receptor agonists to elicit a response is still largely unexplained. Several models have been described that incorporate the kinetics of receptor-mediated initiation of the G protein cycle, but these have not directly addressed the influence of agonist-binding kinetics. To test this, we investigated the relationship between the efficacy of seven M(3) muscarinic receptor agonists and their rate of dissociation (k(off)) from the M(3) receptor. The association and dissociation rate constants of the agonists were determined using a l-[N-methyl]-[(3)H]scopolamine methyl chloride competition binding assay in the presence of GTP. The agonists displayed a range of association and dissociation rates. Relative agonist efficacy was measured at two points after M(3) receptor activation: the stimulation of guanosine 5'-O-(3-[(35)S]thio)triphosphate binding to G alpha subunits, and the subsequent increase in intracellular calcium levels. These experiments revealed a range of intrinsic efficacy, from the low-efficacy pilocarpine and oxotremorine to high-efficacy acetylcholine. There was no relationship between agonist efficacy and the equilibrium binding affinity of each agonist (K(d)). When efficacy was compared with the dissociation rate constant, however, the two were highly correlated, suggesting a relationship between the duration of agonist binding at the receptor and the intrinsic efficacy. These data suggest that kinetic models incorporating the mean lifetime of specific complexes will be required to fully explain the nature of agonist efficacy.
authors.affiliation: Novartis Institutes for Biomedical Sciences, Wimblehurst Road, Horsham, West Sussex, UK. .
authors.forename: David A
authors.initials: DA
authors.lastname: Sykes
authors.forename: Mark R
authors.initials: MR
authors.lastname: Dowling
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Charlton
mesh_terms: D000109:Acetylcholine / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D018721:Muscarinic Agonists / Q000378:metabolism* / Q000494:pharmacology; D019832:N-Methylscopolamine / Q000378:metabolism / Q000494:pharmacology; D010095:Oxotremorine / Q000378:metabolism / Q000494:pharmacology; D043587:Receptor, Muscarinic M3 / Q000819:agonists* / Q000378:metabolism*
chemical_list: D018721:Muscarinic Agonists; D043587:Receptor, Muscarinic M3; D010095:Oxotremorine; D000109:Acetylcholine; D019832:N-Methylscopolamine