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

Abstract

1. Profiles of G protein activation have been assessed using a [35S]-GTPγS binding/immunoprecipitation strategy in Chinese hamster ovary cells expressing either M1, M2, M3 or M4 muscarinic acetylcholine (mACh) receptor subtypes, where expression levels of M1 and M3, or M2 and M4 receptors were approximately equal. 2. Maximal [35S]-GTPγS binding to Gq/11α stimulated by M1/M3 receptors, or Gi1-3α stimulated by M2/M4 receptors occurred within approximately 2 min of agonist addition. The increases in Gq/11α-[35S]-GTPγS binding after M1 and M3 receptor stimulation differed substantially, with M1 receptors causing a 2-3 fold greater increase in [35S]-GTPγS binding and requiring 5 fold lower concentrations of methacholine to stimulate a half-maximal response. 3. Comparison of M2 and M4 receptor-mediated Gi1-3α-[35S]-GTPγS binding also revealed differences, with M2 receptors causing a greater increase in Gi1-3α 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 Gi3α compared to Gi1/2α for both M2 and M4 receptors. More marked agonist/partial agonist differences were observed with respect to M1M3-mediated stimulations of Gq/11α- and Gi1-3α-[35S]-GTPγS binding. Whereas coupling to these Gα subclasses decreased proportionately for M1 receptor stimulation by these agonists, pilocarpine possesses a greater intrinsic activity at M3 receptors for Giα versus Gq/11α 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.