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Differential effects of allosteric M(1) muscarinic acetylcholine receptor agonists on receptor activation, arrestin 3 recruitment, and receptor downregulation.

ABSTRACT: Muscarinic acetylcholine receptors (mAChRs) are drug targets for multiple neurodegenerative and neuropsychiatric disorders, but the full therapeutic potential of mAChR-targeted drugs has not been realized, mainly because of a lack of subtype-selective agonists. Recent advances have allowed the development of highly selective agonists that bind to an allosteric site on the M(1) mAChR that is spatially distinct from the orthosteric acetylcholine binding site, but less is known about the profile of intracellular signals activated by orthosteric versus allosteric M(1) mAChR agonists. We investigated the activation and regulatory mechanisms of two structurally distinct allosteric M(1) mAChR agonists, AC260584 and TBPB. We show that allosteric agonists potently activate multiple signal transduction pathways linked to the M(1) mAChR receptor but, compared to orthosteric agonists, much less efficiently recruit arrestin 3, a protein involved in regulation of G-protein coupled receptor signaling. Consistent with decreased arrestin recruitment, both allosteric agonists showed blunted responses in measurements of receptor desensitization, internalization, and downregulation. These results advance the understanding of mAChR biology and may shed light on unanticipated differences in the pharmacology of orthosteric vs. allosteric agonists that might be capitalized upon for drug development for the treatment of CNS diseases.

SUBMITTER: Davis AA

PROVIDER: S-EPMC2936488 | biostudies-other | 2010 Aug

REPOSITORIES: biostudies-other

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Project description:Background and objectiveMuscarinic acetylcholine receptors (mAChRs) are 7-transmembrane, G protein-coupled receptors that regulate a variety of physiological processes and represent potentially important targets for therapeutic intervention. mAChRs can be stimulated by full and partial orthosteric and allosteric agonists, however the relative abilities of such ligands to induce conformational changes in the receptor remain unclear. To gain further insight into the actions of mAChR agonists, we have developed a fluorescently tagged M(1) mAChR that reports ligand-induced conformational changes in real-time by changes in Förster resonance energy transfer (FRET).MethodsVariants of CFP and YFP were inserted into the third intracellular loop and at the end of the C-terminus of the mouse M(1) mAChR, respectively. The optimized FRET receptor construct (M(1)-cam5) was expressed stably in HEK293 cells.ResultsThe variant CFP/YFP-receptor chimera expressed predominantly at the plasma membrane of HEK293 cells and displayed ligand-binding affinities comparable with those of the wild-type receptor. It also retained an ability to interact with G?(q/11) proteins and to stimulate phosphoinositide turnover, ERK1/2 phosphorylation and undergo agonist-dependent internalization. Addition of the full agonist methacholine caused a reversible decrease in M(1) FRET (F(EYFP)/F(ECFP)) that was prevented by atropine pre-addition and showed concentration-dependent amplitude and kinetics. Partial orthosteric agonists, arecoline and pilocarpine, as well as allosteric agonists, AC-42 and 77-LH-28-1, also caused atropine-sensitive decreases in the FRET signal, which were smaller in amplitude and significantly slower in onset compared to those evoked by methacholine.ConclusionThe M(1) FRET-based receptor chimera reports that allosteric and orthosteric agonists induce similar conformational changes in the third intracellular loop and/or C-terminus, and should prove to be a valuable molecular reagent for pharmacological and structural investigations of M(1) mAChR activation.

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Differential effects of allosteric M(1) muscarinic acetylcholine receptor agonists on receptor activation, arrestin 3 recruitment, and receptor downregulation.

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