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 Gq/11, while even-numbered receptors prefer coupling to Gi/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 [3H]-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 M1 and M3 receptors also pre-couple with non-preferential Gi/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.
Highlights
G-protein coupled receptors (GPCR) represent the largest family of receptors, with more than 900 encoding genes [1]
Stable receptor-Gprotein complexes exist in the absence of agonist, agonist binds to this complex, induces change in the receptor conformation that leads to G-protein activation and dissociation of the complex [3]
Membranes from CHO cells containing from 1.4 to 2.5 fmol of M1 through M4 muscarinic receptors per mg of protein were exposed to carbachol in concentrations ranging from 0.1 mM to 1 mM and binding of [35S]GTPcS to G-protein classes was determined using a scintillation proximity assay (SPA) (Fig. 1)
Summary
G-protein coupled receptors (GPCR) represent the largest family of receptors, with more than 900 encoding genes [1] They process and transduce a multitude of signals elicited by hormones, neurotransmitter and odorants and are involved in a very wide array of physiological and pathological processes. Stable receptor-Gprotein complexes exist in the absence of agonist, agonist binds to this complex, induces change in the receptor conformation that leads to G-protein activation and dissociation of the complex [3] It should, be noted that the distinction between collision coupling and pre-coupling is rather a matter of kinetics of receptor-G-protein interaction, activation state and receptor to Gprotein stoichiometry [4]. Additional modes of interaction intermediate between pure collision coupling and pre-coupling, like transient receptor to G-protein complexing (‘‘dynamic scaffolding’’), have been observed [5]
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