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Identification of orthosteric and allosteric site mutations in M2 muscarinic acetylcholine receptors that contribute to ligand-selective signaling bias.

ABSTRACT: Muscarinic acetylcholine receptors contain at least one allosteric site that is topographically distinct from the acetylcholine, orthosteric binding site. Although studies have investigated the basis of allosteric modulation at these receptors, less is known about putative allosteric ligands that activate the receptor in their own right. We generated M(2) muscarinic acetylcholine receptor mutations in either the orthosteric site in transmembrane helices 3 and 6 (TM3 and -6) or part of an allosteric site involving the top of TM2, the second extracellular (E2) loop, and the top of TM7 and investigated their effects on the binding and function of the novel selective (putative allosteric) agonists (AC-42 (4-n-butyl-1-(4-(2-methylphenyl)-4-oxo-1-butyl)piperidine HCl), 77-LH-28-1 (1-(3-(4-butyl-1-piperidinyl)propyl)-3,3-dihydro-2(1H)-quinolinone), and N-desmethylclozapine) as well as the bitopic orthosteric/allosteric ligand, McN-A-343 (4-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium). Four classes of agonists were identified, depending on their response to the mutations, suggesting multiple, distinct modes of agonist-receptor interaction. Interestingly, with the exception of 77-LH-28-1, allosteric site mutations had no effect on the affinity of any of the agonists tested, but some mutations in the E2 loop influenced the efficacy of both orthosteric and novel selective agonists, highlighting a role for this region of the receptor in modulating activation status. Two point mutations (Y104(3.33)A (Ballesteros and Weinstein numbers in superscript) in the orthosteric and Y177A in the allosteric site) unmasked ligand-selective and signaling pathway-selective effects, providing evidence for the existence of pathway-specific receptor conformations. Molecular modeling of 77-LH-28-1 and N-desmethylclozapine yielded novel binding poses consistent with the possibility that the functional selectivity of such agents may arise from a bitopic mechanism.

SUBMITTER: Gregory KJ

PROVIDER: S-EPMC2844194 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Identification of orthosteric and allosteric site mutations in M2 muscarinic acetylcholine receptors that contribute to ligand-selective signaling bias.

Gregory Karen J KJ Hall Nathan E NE Tobin Andrew B AB Sexton Patrick M PM Christopoulos Arthur A

The Journal of biological chemistry 20100105 10

Muscarinic acetylcholine receptors contain at least one allosteric site that is topographically distinct from the acetylcholine, orthosteric binding site. Although studies have investigated the basis of allosteric modulation at these receptors, less is known about putative allosteric ligands that activate the receptor in their own right. We generated M(2) muscarinic acetylcholine receptor mutations in either the orthosteric site in transmembrane helices 3 and 6 (TM3 and -6) or part of an alloste ...[more]

PMID: 20051519

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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.

Project description:Accumulating evidence suggests that selective M4 muscarinic acetylcholine receptor (mAChR) activators may offer a novel strategy for the treatment of psychosis. However, previous efforts to develop selective M4 activators were unsuccessful because of the lack of M4 mAChR subtype specificity and off-target muscarinic adverse effects. We recently developed VU0152100, a highly selective M4 positive allosteric modulator (PAM) that exerts central effects after systemic administration. We now report that VU0152100 dose-dependently reverses amphetamine-induced hyperlocomotion in rats and wild-type mice, but not in M4 KO mice. VU0152100 also blocks amphetamine-induced disruption of the acquisition of contextual fear conditioning and prepulse inhibition of the acoustic startle reflex. These effects were observed at doses that do not produce catalepsy or peripheral adverse effects associated with non-selective mAChR agonists. To further understand the effects of selective potentiation of M4 on region-specific brain activation, VU0152100 alone and in combination with amphetamine were evaluated using pharmacologic magnetic resonance imaging (phMRI). Key neural substrates of M4-mediated modulation of the amphetamine response included the nucleus accumbens (NAS), caudate-putamen (CP), hippocampus, and medial thalamus. Functional connectivity analysis of phMRI data, specifically assessing correlations in activation between regions, revealed several brain networks involved in the M4 modulation of amphetamine-induced brain activation, including the NAS and retrosplenial cortex with motor cortex, hippocampus, and medial thalamus. Using in vivo microdialysis, we found that VU0152100 reversed amphetamine-induced increases in extracellular dopamine levels in NAS and CP. The present data are consistent with an antipsychotic drug-like profile of activity for VU0152100. Taken together, these data support the development of selective M4 PAMs as a new approach to the treatment of psychosis and cognitive impairments associated with psychiatric disorders such as schizophrenia.

Dataset Information

Identification of orthosteric and allosteric site mutations in M2 muscarinic acetylcholine receptors that contribute to ligand-selective signaling bias.

Publications

Identification of orthosteric and allosteric site mutations in M2 muscarinic acetylcholine receptors that contribute to ligand-selective signaling bias.

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