Gq/11 and Gi/o activation profiles in CHO cells expressing human muscarinic acetylcholine receptors:: dependence on agonist as well as receptor-subtype

1 Profiles of G protein activation have been assessed using a [S-35]-GTP gammaS binding/immunoprecipitation strategy in Chinese hamster ovary cells expressing either M-1, M-2, M-3 or M-4 muscarinic acetylcholine (mACh) receptor subtypes, where expression levels of M-1 and M-3, or M-2 and M-4 receptors were approximately equal. 2 Maximal [S-35]-GTP gammaS binding to G(q 11)alpha stimulated by M-1/M-3 receptors, or G(11-3)alpha stimulated by M-2/M-4 receptors occurred within approximately 2 min of agonist addition. The increases in G(q 11)alpha-[S-35]-GTP gammaS binding after M-1 and M-3 receptor stimulation differed substantially, with M1 receptors causing a 2-3 fold greater increase in [S-35]-GTP gammaS binding and requiring 5 fold lower concentrations of methacholine to stimulate a half-maximal response. 3 Comparison of M-2 and M-4 receptor-mediated G(il) (3)alpha[S-35]-GTP gammaS binding also revealed differences, with M-2 receptors causing a greater increase in G(il-3)alpha activation and requiring 10 fold lower concentrations of methacholine to stimulate a half-maximal response. 4 Comparison of methacholine- and pilocarpine-mediated effects revealed that the latter partial agonist is more effective in activating G(i3)alpha compared to G(il) (2)alpha for both M-2 and M-4 receptors. More marked agonist/partial agonist differences were observed with respect to M-1/M-3-mediated stimulations of G(q) (11)alpha- and G(il-3)alpha-[S-35]-GTP gammaS binding. Whereas coupling to these G alpha subclasses decreased proportionately for M-1 receptor stimulation by these agonists, pilocarpine possesses a greater intrinsic activity at M-3 receptors for G(i)alpha versus G(q 11)alpha activation. 5 These data demonstrate that mACh receptor subtype and the nature of the agonist used govern the repertoire of G proteins activated. They also provide insights into how the diversity of coupling can be pharmacologically exploited, and provide a basis for a better understanding of how multiple receptor subtypes can be differentially regulated.