FRET-Based Detection of M1 Muscarinic Acetylcholine Receptor Activation by Orthosteric and Allosteric Agonists

Danijela Markovic,Cornelius Krasel,Suleiman Al-Sabah,R. A. John Challiss,Jonathan Holdich,Rajendra Mistry,Martyn P. Mahaut-Smith,David Holowka

doi:10.1371/journal.pone.0029946

Danijela Markovic, Cornelius Krasel + Show 6 more

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https://doi.org/10.1371/journal.pone.0029946

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Abstract

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 M1 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 M1 mAChR, respectively. The optimized FRET receptor construct (M1-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 Gαq/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 M1 FRET (FEYFP/FECFP) 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 M1 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 M1 mAChR activation.

Highlights

Muscarinic acetylcholine receptors are 7-transmembrane domain proteins that belong to the rhodopsin family of G protein-coupled receptors (GPCRs). mAChRs are widely distributed and are responsible for the metabotropic effects of acetylcholine. mAChR subtypes are encoded by 5 distinct genes in mammals, referred to as M1–M5
Design of M1-cam mAChR Forster resonance energy transfer (FRET) conformational sensors An overview of the M1-cameleon receptor constructs is given in Cellular localization of the constructs In order to assess the cellular localization of the cameleon receptors, Human embryonic kidney 293 (HEK293) cells were transiently transfected with the appropriate cDNA
Confocal microscopy revealed that addition of the EYFPF46L to the C-terminus of full length M1 mAChR did not affect plasma membrane receptor expression

Summary

Introduction

Muscarinic acetylcholine receptors (mAChRs) are 7-transmembrane domain proteins that belong to the rhodopsin family of G protein-coupled receptors (GPCRs). mAChRs are widely distributed and are responsible for the metabotropic effects of acetylcholine. mAChR subtypes are encoded by 5 distinct genes in mammals, referred to as M1–M5. The majority of the newly reported compounds appear to interact with the receptor at sites distinct from the orthosteric binding pocket, which has been shown to be highly conserved across the M1–M5 mAChRs [7,8]. Binding and activation of M1–M5 mAChRs by orthosteric ligands is mediated largely though interactions with a small number of key residues (e.g. Y381 and Q382 in TM6 (numbering refers to M1 receptor) [9]), leading to a relative change in the orientation of TM3 and TM6 of the receptor and a consequent change in the distance between the C-terminus and third intracellular (i3) loop [10]. Muscarinic 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. To gain further insight into the actions of mAChR agonists, we have developed a fluorescently tagged M1 mAChR that reports ligand-induced conformational changes in real-time by changes in Forster resonance energy transfer (FRET)

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