Exploring agonist dependency of receptor-G protein-grouping and constitutive activity at muscarinic acetylcholine receptors
2014-12-15T10:34:47Z (GMT) by
The effects of structurally diverse agonists were assessed on receptor-mediated activation of G-proteins using in Chinese hamster ovary cells stably expressing similar densities of M1 and M3 mACh receptors. Using a total [35S]-GTPgammaS binding protocol both receptor subtypes were shown to couple to both pertussis toxin-sensitive and -insensitive G-proteins. M1 mACh receptors coupled with greater potency and intrinsic efficacy than M3 receptors, indicating that in this cell-line that M1 mACh receptor may be more efficiently coupled to its complement of G-proteins. Total [36S]-GTPgammaS binding concentration-response curves for both receptor subtypes were shallow and, in the case of the M1 receptor, could be readily resolved into high and low affinity components. The heterogeneity of Galpha subunits activated was further investigated using an immunoprecipitation technique. This strategy revealed that agonist binding to M1 receptor caused the activation of a heterogeneous population of G-proteins, which was directly related to agonist efficacy. Full agonists were able to activate both Galphaq/11 and Galphail-3 subtypes, whereas partial agonists activated only the most efficiently coupled G-protein Galpha q/11. In contrast, the complement of G-proteins activated after M3 receptor stimulation was not related to the efficacy of the agonist and suggested that agonists may form active conformations of the M 3 receptor that possess different G-protein coupling profiles.;To investigate the G-protein coupling of M1 and M3 receptors further, homologous point mutations were introduced in both subtypes, which conferred agonist-independent constitutive activity. All of the antagonists tested were able to concentration-dependently inhibit basal [3H]-inositol phosphate accumulation and were therefore classified as 'inverse agonists'. However, subtle differences in the results obtained for different inverse agonists via different functional and binding readouts suggested the existence of more than one conformation of inactive receptor.