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

Profiles of G protein activation have been assessed using a [35S]‐GTPγS binding/immunoprecipitation strategy in Chinese hamster ovary cells expressing either M1, M2, M3or M4muscarinic acetylcholine (mACh) receptor subtypes, where expression levels of M1and M3, or M2and M4receptors were approximately equal.Maximal [35S]‐GTPγS binding to Gq/11α stimulated by M1/M3receptors, or Gi1 – 3α stimulated by M2/M4receptors occurred within approximately 2 min of agonist addition. The increases in Gq/11α‐[35S]‐GTPγS binding after M1and M3receptor stimulation differed substantially, with M1receptors 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.Comparison of M2and M4receptor‐mediated Gi1 – 3α‐[35S]‐GTPγS binding also revealed differences, with M2receptors causing a greater increase in Gi1 – 3α activation and requiring 10 fold lower concentrations of methacholine to stimulate a half‐maximal response.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 M2and M4receptors. More marked agonist/partial agonist differences were observed with respect to M1/M3‐mediated stimulations of Gq/11α‐ and Gi1 – 3α‐[35S]‐GTPγS binding. Whereas coupling to these Gα subclasses decreased proportionately for M1receptor stimulation by these agonists, pilocarpine possesses a greater intrinsic activity at M3receptors for Giα versus Gq/11α activation.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.