title: Structural determinants of allosteric agonism and modulation at the M4 muscarinic acetylcholine receptor: identification of ligand-specific and global activation mechanisms.
abstract: The recently identified small molecule, 3-amino-5-chloro-6-methoxy-4-methylthieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298), is the first selective allosteric modulator of the muscarinic acetylcholine receptors (mAChRs) that mediates both receptor activation and positive modulation of the endogenous agonist, acetylcholine (ACh), via the same allosteric site on the M(4) mAChR. We thus utilized this novel chemical tool, as well as ACh, the bitopic (orthosteric/allosteric) agonist, McN-A-343, and the clinically efficacious M(1)/M(4) mAChR-preferring agonist, xanomeline, in conjunction with site-directed mutagenesis of four different regions of the M(4) mAChR (extracellular loops 1, 2, and 3, and transmembrane domain 7), to identify regions that govern ligand-specific modes of binding, signaling, and allosteric modulation. In the first extracellular loop (E1), we identified Ile(93) and Lys(95) as key residues that specifically govern the signaling efficacy of LY2033298 and its binding cooperativity with ACh, whereas Phe(186) in the E2 loop was identified as a key contributor to the binding affinity of the modulator for the allosteric site, and Asp(432) in the E3 loop appears to be involved in the functional (activation) cooperativity between the modulator and the endogenous agonist. In contrast, the highly conserved transmembrane domain 7 residues, Tyr(439) and Tyr(443), were identified as contributing to a key activation switch utilized by all classes of agonists. These results provide new insights into the existence of multiple activation switches in G protein-coupled receptors (GPCRs), some of which can be selectively exploited by allosteric agonists, whereas others represent global activation mechanisms for all classes of ligand.
authors.affiliation: Department of Pharmacology, Monash University, Monash Institute of Pharmaceutical Sciences, Parkville 3052, Victoria, Australia.
authors.forename: Vindhya
authors.initials: V
authors.lastname: Nawaratne
authors.forename: Katie
authors.initials: K
authors.lastname: Leach
authors.forename: Christian C
authors.initials: CC
authors.lastname: Felder
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Sexton
authors.forename: Arthur
authors.initials: A
authors.lastname: Christopoulos
mesh_terms: D008455:(4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride / Q000494:pharmacology; D000109:Acetylcholine / Q000737:chemistry*; D000495:Allosteric Site; D000818:Animals; D006224:Cricetinae; D006801:Humans; D008024:Ligands; D008954:Models, Biological; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011725:Pyridines / Q000737:chemistry / Q000494:pharmacology; D043588:Receptor, Muscarinic M4 / Q000819:agonists / Q000737:chemistry*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013830:Thiadiazoles / Q000737:chemistry / Q000494:pharmacology
chemical_list: D008024:Ligands; D011725:Pyridines; D043588:Receptor, Muscarinic M4; D043562:Receptors, G-Protein-Coupled; D013830:Thiadiazoles; D008455:(4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; C075257:xanomeline; D000109:Acetylcholine
references.citation: Pharmacol Rev. 2003 Dec;55(4):597-606
references.pmid: 14657418
references.citation: Curr Neuropharmacol. 2007 Sep;5(3):157-67
references.pmid: 19305798
references.citation: Proc Natl Acad Sci U S A. 2008 Aug 5;105(31):10978-83
references.pmid: 18678919
references.citation: Mol Pharmacol. 1999 Dec;56(6):1245-53
references.pmid: 10570052
references.citation: Trends Pharmacol Sci. 2006 Sep;27(9):475-81
references.pmid: 16889837
references.citation: Neuropsychopharmacology. 2010 Mar;35(4):855-69
references.pmid: 19940843
references.citation: Annu Rev Pharmacol Toxicol. 2006;46:481-519
references.pmid: 16402913
references.citation: Mol Pharmacol. 2005 Sep;68(3):769-78
references.pmid: 15937215
references.citation: Proc Natl Acad Sci U S A. 2009 Sep 15;106(37):15950-5
references.pmid: 19717450
references.citation: J Neurosci. 2008 Oct 8;28(41):10422-33
references.pmid: 18842902
references.citation: Proc Natl Acad Sci U S A. 2003 Nov 11;100(23):13674-9
references.pmid: 14595031
references.citation: J Biol Chem. 2010 Mar 5;285(10):7459-74
references.pmid: 20051519
references.citation: Proc R Soc Lond B Biol Sci. 1983 Dec 22;220(1219):141-62
references.pmid: 6141562
references.citation: Mol Pharmacol. 2001 Oct;60(4):761-7
references.pmid: 11562438
references.citation: Mol Pharmacol. 2007 Aug;72(2):463-76
references.pmid: 17525129
references.citation: J Biol Chem. 2001 Sep 7;276(36):34098-104
references.pmid: 11441014
references.citation: Am J Psychiatry. 2008 Aug;165(8):1033-9
references.pmid: 18593778
references.citation: J Biol Chem. 2008 Oct 24;283(43):29312-21
references.pmid: 18723515
references.citation: Mol Pharmacol. 1995 Aug;48(2):362-78
references.pmid: 7651370
references.citation: J Biol Chem. 2005 Apr 29;280(17):17135-41
references.pmid: 15722344
references.citation: Nucleic Acids Res. 2009 Jan;37(Database issue):D680-5
references.pmid: 18948278
references.citation: Mol Pharmacol. 2008 Oct;74(4):1119-31
references.pmid: 18628403
references.citation: Mol Pharmacol. 2002 Jun;61(6):1297-302
references.pmid: 12021390
references.citation: Biochem Soc Trans. 2003 Feb;31(Pt 1):29-34
references.pmid: 12546648
references.citation: Biochem Pharmacol. 1973 Dec 1;22(23):3099-108
references.pmid: 4202581
references.citation: Trends Pharmacol Sci. 2008 Dec;29(12):616-25
references.pmid: 18838178
references.citation: Trends Pharmacol Sci. 1998 Sep;19(9):351-7
references.pmid: 9786022
references.citation: EMBO J. 1991 Dec;10(12):3729-34
references.pmid: 1657592
references.citation: Nat Rev Drug Discov. 2002 Mar;1(3):198-210
references.pmid: 12120504