Classical and atypical agonists activate M1 muscarinic acetylcholine receptors through common mechanisms (original) (raw)
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Journal of Pharmacology and Experimental Therapeutics, 2006
A single asparagine-to-tyrosine point mutation in the human M 3 muscarinic acetylcholine (mACh) receptor at residue 514 (N514Y) resulted in a marked increase (ϳ300%) in agonistindependent [ 3 H]inositol phosphate ([ 3 H]IP x ) accumulation compared with the response observed for the wild-type (WT) receptor. All the antagonists tested were able to inhibit both the WT-M 3 and N514Y M 3 mACh receptor-mediated basal [ 3 H]IP x accumulation in a concentration-dependent manner. However, significant differences in both potency and binding affinity were only seen for those antagonists that possess greater receptor affinity. Despite being transfected with equivalent amounts of cDNA, cells expressed the N514Y M 3 mACh receptor at levels that were only 25 to 30% of those seen for the WT receptor. Differences in the ability of chronic antagonist exposure to up-regulate N514Y M 3 mACh receptor expression levels were also seen, with 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) producing only 50% of the receptor up-regulation produced by atropine or pirenzepine. Basal phosphorylation of the N514Y M 3 mACh receptor was approximately 100% greater than that seen for the WT-M 3 receptor. The ability of antagonists to decrease basal N514Y M 3 mACh receptor phosphorylation revealed differences in inverse-agonist efficacy. Atropine, 4-DAMP, and pirenzepine all reduced basal phosphorylation to similar levels, whereas methoctramine, a full inverse agonist with respect to reducing agonist-independent [ 3 H]IP x accumulation, produced no significant attenuation of basal receptor phosphorylation. This study shows that mACh receptor inverse agonists can exhibit differential signaling profiles, which are dependent on the specific pathway investigated, and therefore provides evidence that the molecular mechanism of inverse agonism is likely to be more complex than the stabilization of a single inactive receptor conformation.
IUPHAR/BPS Guide to Pharmacology CITE, 2019
Muscarinic acetylcholine receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Muscarinic Acetylcholine Receptors [45]) are GPCRs of the Class A, rhodopsin-like family where the endogenous agonist is acetylcholine. In addition to the agents listed in the table, AC-42, its structural analogues AC-260584 and 77-LH-28-1, N-desmethylclozapine, TBPB and LuAE51090 have been described as functionally selective agonists of the M1 receptor subtype via binding in a mode distinct from that utilized by non-selective agonists [243, 242, 253, 155, 154, 181, 137, 11, 230]. There are two pharmacologically characterised allosteric sites on muscarinic receptors, one defined by it binding gallamine, strychnine and brucine, and the other defined by the binding of KT 5720, WIN 62,577, WIN 51,708 and staurosporine [161, 162].
Pronounced pharmacologic deficits in M2 muscarinic acetylcholine receptor knockout mice
Proceedings of the National Academy of Sciences, 1999
Members of the muscarinic acetylcholine receptor family (M1-M5) are known to be involved in a great number of important central and peripheral physiological and pathophysiological processes. Because of the overlapping expression patterns of the M1-M5 muscarinic receptor subtypes and the lack of ligands endowed with sufficient subtype selectivity, the precise physiological functions of the individual receptor subtypes remain to be elucidated. To explore the physiological roles of the M2 muscarinic receptor, we have generated mice lacking functional M2 receptors by using targeted mutagenesis in mouse embryonic stem cells. The resulting mutant mice were analyzed in several behavioral and pharmacologic tests. These studies showed that the M2 muscarinic receptor subtype, besides its well documented involvement in the regulation of heart rate, plays a key role in mediating muscarinic receptor-dependent movement and temperature control as well as antinociceptive responses, three of the most prominent central muscarinic effects. These results offer a rational basis for the development of novel muscarinic drugs.
Functional and biochemical basis for multiple muscarinic acetylcholine receptors
Progress in Neuro-Psychopharmacology and Biological Psychiatry, 1985
The novel antimuscarinic compound pirenzepine (PZ) has generated considerable interest in the basis and the implications of muscarinic acetylcholine receptor (mAChR) heterogeneity. [3H]PZ has been used extensively to identify and characterize the putative M1 (high affinity for PZ) mAChR subtype, which predominates in central nervous system (CNS) and ganglia. The heterogeneity sensed by PZ is not identical to the heterogeneity sensed by agonists. Differences in effector coupling do not necessarily provide a simple explanation for the molecular basis of these putative M1 and M2 subtypes. Therapeutic and untoward effects of muscarinic drugs may be mediated by independent mAChR subpopulations which may be pharmacologically exploited to produce more highly selective as well as efficacious new drugs.
Journal of Biological Chemistry
Structure-function relationship studies of the m3 muscarinic acetylcholine receptor have recently identified a series of threonine and tyrosine residues (all located within the hydrophobic receptor core) that are critically involved in acetylcholine binding (Wess, J., Gdula, D., and Brann, M. R. (1991) EMBO J. 10,[3729][3730][3731][3732][3733][3734]. To gain further insight into the functional roles of these amino acids, the agonist binding properties of six rat m3 muscarinic receptor point mutants, in which the critical threonine and tyrosine residues had been individually replaced by alanine and phenylalanine, respectively, were studied in greater detail following their transient expression in COS-7 cells. The binding profiles of a series of acetylcholine derivatives suggest that the altered threonine and tyrosine residues are primarily involved in the interaction of the acetylcholine ester moiety with the receptor protein. The two m3 receptor point mutants, Thr234 + Ala and TyrSo6 4 Phe, which showed the most pronounced decreases in acetylcholine binding affinities (-40-60-fold as compared with the wild-type receptor), were stably expressed in CHO cells for further functional analysis. Both mutant receptors were found to be severely impaired in their ability to stimulate agonist-dependent phosphatidylinositol hydrolysis. Consistent with this observation, acetylcholine binding to the two mutant receptors was not significantly affected by addition of the GTP analog Gpp(NH)p (5'guanylyl imidodiphosphate). Our data suggest that Thr234 and TyrSo6 (located within transmembrane domains V and VI, respectively), which are conserved among all muscarinic receptors (ml-m5), may play an important role in agonist-induced muscarinic receptor activation.
European Journal of Pharmacology: Molecular Pharmacology, 1994
Activation of transfected muscarinic ml acetylcholine receptors (mlAChR) has been linked to several signal transduction pathways which include phosphoinositide hydrolysis, arachidonic acid release and cAMP accumulation. In Chinese hamster ovary cells stably transfected with the rat mlAChR gene, carbachol elicited all three responses with ECs0 values of 2.6, 3.8 and 76 p,M, respectively. However, pilocarpine and the selective muscarinic agonist AF102B activated phosphoinositide hydrolysis (by 94 and 27% vs. carbachol, respectively), while antagonizing carbachol-mediated cAMP accumulation. Carbachol also activated (by 4-fold) adenylyl cyclase in membranes prepared from these cells, indicating independence of this signal from intracellular mediators. Moreover, carbachol and AF102B similarly elevated cytosolic Ca 2+ in intact mlAChR-transfected cells. The ligand-selective cAMP accumulation, its independence from Ca 2+ and the carbachol-activated adenylyl cyclase in membranes suggest that it represents an independent mlAChR-mediated signal, unrelated to phosphoinositide hydrolysis. Selective muscarinic ligands such as AF102B may independently activate distinct signalling pathways, which may be important for designing cholinergic replacement therapy for treating Alzheimer's disease.
Different sensitivities to agonist of muscarinic acetylcholine receptor subtypes
FEBS Letters, 1988
Muscarinic acetylcholine receptor (mAChR) III expressed in Xenopus oocytes, like mAChR I, mediates activation of a Ca2 +-dependent Cl-current, whereas mAChR IV, like mAChR II, principally induces activation of Na+ and K + currents in a Ca2+-independent manner. mAChR III has a sensitivity to agonist of about one order of magnitude higher than that of mAChR I in mediating the Ca 2 +-dependent current response in Xenopus oocytes and in stimulating phosphoinositide hydrolysis in NGl08-15 neuroblastoma-glioma hybrid cells. The agonist-binding affinity of mAChR III is also about one order of magnitude higher than that of mAChR I.
Muscarinic acetylcholine receptors: novel opportunities for drug development
Nature Reviews Drug Discovery, 2014
The muscarinic acetylcholine receptors (mAChRs) comprise a family of five related G protein-coupled receptors (GPCRs) belonging to the α-branch of class A GPCRs 1 . The mAChR family consists of five distinct subtypes, denoted M 1 to M 5 (and encoded by the genes CHRM1 to CHRM5). Three of these receptor subtypes (M 1 , M 3 and M 5 ) have been shown to couple to G proteins of the G q/11 family, whereas the remaining two subtypes (M 2 and M 4 ) preferentially signal through the G i/o family of G proteins 2 . The mAChRs have a central role in human physiology, regulating heart rate, smooth muscle contraction, glandular secretion and many fundamental functions of the central nervous system (CNS) 3 .
Molecular Pharmacology, 2008
The M 4 muscarinic acetylcholine (ACh) receptor (mAChR) is a potential therapeutic target but characterized by a lack of subtype-selective ligands. We recently generated "designer receptors exclusively activated by a designer drug" (DREADDs), which contained mutations of two conserved orthosteric-site residues (Y 113 C/A 203 G in the M 4 mAChR) that caused a loss of ACh activity but a gain in responsiveness to clozapine-N-oxide (CNO). The current study characterized the interactions of the wild type and the M 4 DREADD with a range of agonists, antagonists, and the recently discovered M 4 mAChR allosteric potentiator, 3-amino-5-chloro-6-methoxy-4-methyl-thieno[2,3b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298). LY2033298 displayed positive binding cooperativity with ACh, neutral cooperativity with the antagonist, [ 3 H]quinuclidinyl benzilate, and agonism for activation of phosphorylated extracellular signal-regulated kinase (ERK) 1/2 at the wild-type M 4 mAChR. LY2033298's cooperativity with clozapine or CNO was weakly positive with respect to binding but profoundly negative with respect to LY2033298 signaling. Although the DREADD mutations increased the binding and function of clozapine-like compounds, all other agonists lost the ability to activate the mutant; for the orthosteric agonists ACh and pilocarpine, this was due partly to a reduced affinity, whereas the affinity of LY2033298 or the atypical agonist 4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride was unaltered. The interaction between LY2033298 and clozapine-like compounds reverted to neutral cooperativity on the DREADD, whereas LY2033298 caused a striking functional rescue of ACh potency and efficacy at the DREADD. These results provide conclusive evidence for the retention of a functional allosteric site on the M 4 DREADD and highlight a role for residues Tyr 113 and Ala 203 in the transmission of cooperativity.