Abstract
Muscarinic M2 receptors preferentially couple with the Gi/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 M2 receptors (CHO-M2 cells) whether direct interaction of M2 receptors with Gs and Gq/11 G-proteins is responsible for the latter effects. Suppression of the Gsα subunit using RNA interference abolished stimulation of cAMP synthesis induced by 1 mM carbachol in both control and pertussis toxin-treated CHO-M2 cells but had no effect on the inhibition of forskolin-stimulated cAMP synthesis. Carbachol stimulated accumulation of IP with an EC50 of 79 μM. Removal of the Gq,G11, or both α subunits reduced this response by 78, 54, and 92%, respectively, whereas suppression of the Gsα subunit had no effect. Similar results obtained in CHO cells expressing M1 receptors that preferentially couple with Gs and Gq/11 G-proteins confirmed the efficiency of siRNA treatments. Stimulation of M2 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-pyrolidinyl)-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-pyrrolidinone) had no effect. Our results provide direct evidence of M2 receptor coupling with the α subunits of Gs and Gq/11 G-proteins and demonstrate induction of multiple receptor conformational states dependent on both the concentration and the nature of the agonist used.
Footnotes
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This work was supported by Project AV0Z50110509 and by Grants GACR305/05/P209, GACR305/05/0452, National Institutes of Health Grant NS25743, and MSMT CR LC554.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.106.114314.
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ABBREVIATIONS: CHO, Chinese hamster ovary cells; IP, inositol phosphate(s); CHO-M1, CHO-M2, Chinese hamster ovary cells expressing the M1 or M2 subtype of muscarinic receptors; GTPγS, guanosine 5′-O-(3-thio)triphosphate; ANOVA, analysis of variance.
- Received September 19, 2006.
- Accepted October 24, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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