Muscarinic M_2 Receptors Directly Activate G_(q/11) and G_s G-Proteins

摘要

Muscarinic M_2 receptors preferentially couple with the G_(i/o) class of G-proteins to inhibit cAMP synthesis. However, they can also stimulate net synthesis of cAMP and inositol phosphate (IP) accumulation. We investigated in intact Chinese hamster ovary (CHO) cells expressing human M_2 receptors (CHO-M_2 cells) whether direct interaction of M_2 receptors with G_s and G_(q/11) G-proteins is responsible for the latter effects. Suppression of the G_s alpha subunit using RNA interference abolished stimulation of cAMP synthesis induced by 1 mM carba-chol in both control and pertussis toxin-treated CHO-M_2 cells but had no effect on the inhibition of forskolin-stimulated cAMP synthesis. Carbachol stimulated accumulation of IP with an EC_(50) of 79 mu M Removal of the G_q, G_(11), or both alpha subunits reduced this response by 78, 54, and 92%, respectively, whereas suppression of the G_s alpha subunit had no effect. Similar results obtained in CHO cells expressing M_1 receptors that preferentially couple with G_s and G_(q/11) G-proteins confirmed the efficiency of siRNA treatments. Stimulation of M_2 receptors in control and pertussis toxin-treated cells by a series of full agonists with respect to inhibition of adenylyl cyclase displayed different efficacies in stimulating IP accumulation. Carbachol, acetylcholine, and oxotremorine-M [N,N,N-trimethyl-4-(2-oxo-1-pyrolidi-nyl)-2-butyn-1-ammonium] behaved as full agonists, furmethide (N,N,N-trimethyl-2-furanmethammonium) and methylfurmethide [(5-methyl-2-furyl)methyltrimethylammonium] were partial agonists, and oxotremorine (1 -[4-(1 -pyrrolidinyl)-2-butynyl]-2-pyrro-lidinone) had no effect. Our results provide direct evidence of M_2 receptor coupling with the alpha subunits of G_s and G_(q/11) G-proteins and demonstrate induction of multiple receptor conformational states dependent on both the concentration and the nature of the agonist used.