title: Subtype differences in pre-coupling of muscarinic acetylcholine receptors.
abstract: Based on the kinetics of interaction between a receptor and G-protein, a myriad of possibilities may result. Two extreme cases are represented by: 1/Collision coupling, where an agonist binds to the free receptor and then the agonist-receptor complex "collides" with the free G-protein. 2/Pre-coupling, where stable receptor/G-protein complexes exist in the absence of agonist. Pre-coupling plays an important role in the kinetics of signal transduction. Odd-numbered muscarinic acetylcholine receptors preferentially couple to G(q/11), while even-numbered receptors prefer coupling to G(i/o). We analyzed the coupling status of the various subtypes of muscarinic receptors with preferential and non-preferential G-proteins. The magnitude of receptor-G-protein coupling was determined by the proportion of receptors existing in the agonist high-affinity binding conformation. Antibodies directed against the C-terminus of the α-subunits of the individual G-proteins were used to interfere with receptor-G-protein coupling. Effects of mutations and expression level on receptor-G-protein coupling were also investigated. Tested agonists displayed biphasic competition curves with the antagonist [(3)H]-N-methylscopolamine. Antibodies directed against the C-terminus of the α-subunits of the preferential G-protein decreased the proportion of high-affinity sites, and mutations at the receptor-G-protein interface abolished agonist high-affinity binding. In contrast, mutations that prevent receptor activation had no effect. Expression level of preferential G-proteins had no effect on pre-coupling to non-preferential G-proteins. Our data show that all subtypes of muscarinic receptors pre-couple with their preferential classes of G-proteins, but only M(1) and M(3) receptors also pre-couple with non-preferential G(i/o) G-proteins. Pre-coupling is not dependent on agonist efficacy nor on receptor activation. The ultimate mode of coupling is therefore dictated by a combination of the receptor subtype and the class of G-protein.
authors: Department of Neurochemistry, Institute of Physiology Academy of Sciences of the Czech Republic v.v.i., Prague, Czech Republic. jakubik@biomed.cas.cz
authors: Jan
authors: J
authors: Jakubík
authors: Helena
authors: H
authors: Janíčková
authors: Alena
authors: A
authors: Randáková
authors: Esam E
authors: EE
authors: El-Fakahany
authors: Vladimír
authors: V
authors: Doležal
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D002217:Carbachol / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009154:Mutation; D019832:N-Methylscopolamine / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000235:genetics / Q000378:metabolism*
chemical_list: D011976:Receptors, Muscarinic; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D002217:Carbachol; D019204:GTP-Binding Proteins; D019832:N-Methylscopolamine