QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: mol.107.037630v1 72/2/463    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by May, L. T. Articles by Christopoulos, A. Search for Related Content PubMed PubMed Citation Articles by May, L. T. Articles by Christopoulos, A.

Structure-Function Studies of Allosteric Agonism at M₂ Muscarinic Acetylcholine Receptors

Drug Discovery Biology Laboratory, Department of Pharmacology, Monash University, Clayton, Victoria, Australia (L.T.M., V.A.A., P.M.S., A.C.) and Psychiatry Centre of Excellence for Drug Discovery, GlaxoSmithKline, Harlow, Essex, United Kingdom (C.J.L., H.J.H., M.D.W.)

The M₂ muscarinic acetylcholine receptor (mAChR) possesses at least one binding site for allosteric modulators that is dependent on the residues ¹⁷²EDGE¹⁷⁵, Tyr¹⁷⁷, and Thr⁴²³. However, the contribution of these residues to actions of allosteric agonists, as opposed to modulators, is unknown. We created mutant M₂ mAChRs in which the charge of the ¹⁷²EDGE¹⁷⁵ sequence had been neutralized and each Tyr¹⁷⁷ and Thr⁴²³ was substituted with alanine. Radioligand binding experiments revealed that these mutations had a profound inhibitory effect on the prototypical modulators gallamine, alcuronium, and heptane-1,7-bis-[dimethyl-3'-phthalimidopropyl]-ammonium bromide (C₇/3-phth) but minimal effects on the orthosteric antagonist [³H]N-methyl scopolamine. In contrast, the allosteric agonists 4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride (McN-A-343), 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine hydrogen chloride (AC-42), and the novel AC-42 derivative 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1) demonstrated an increased affinity or proportion of high-affinity sites at the combined EDGE-YT mutation, indicating a different mode of binding to the prototypical modulators. Subsequent functional assays of extracellular signal-regulated kinase (ERK)1/2 phosphorylation and guanosine 5'-(-[³⁵S]thio)triphosphate ([³⁵S]GTPS) binding revealed minimal effects of the mutations on the orthosteric agonists acetylcholine (ACh) and pilocarpine but a significant increase in the efficacy of McN-A-343 and potency of 77-LH-28-1. Additional mutagenesis experiments found that these effects were predominantly mediated by Tyr¹⁷⁷ and Thr⁴²³, rather than the ¹⁷²EDGE¹⁷⁵ sequence. The functional interaction between each of the allosteric agonists and ACh was characterized by high negative cooperativity but was consistent with an increased allosteric agonist affinity at the combined EDGE-YT mutant M₂ mAChR. This study has thus revealed a differential role of critical allosteric site residues on the binding and function of allosteric agonists versus allosteric modulators of M₂ mAChRs.

Received for publication April 30, 2007.

Accepted for publication May 24, 2007.

Address correspondence to: Prof Arthur Christopoulos, Drug Discovery Biology Laboratory, Department of Pharmacology, Monash University, Clayton, Victoria 3800, Australia. E-mail: arthur.christopoulos{at}med.monash.edu.au

This article has been cited by other articles:

				L. Ma, M. A. Seager, M. Wittmann, M. Jacobson, D. Bickel, M. Burno, K. Jones, V. K. Graufelds, G. Xu, M. Pearson, et al. Selective activation of the M1 muscarinic acetylcholine receptor achieved by allosteric potentiation PNAS, September 15, 2009; 106(37): 15950 - 15955. [Abstract] [Full Text] [PDF]

				C. Valant, P. M. Sexton, and A. Christopoulos Orthosteric/Allosteric Bitopic Ligands: Going Hybrid at GPCRs Mol. Interv., June 1, 2009; 9(3): 125 - 135. [Abstract] [Full Text] [PDF]

				K. J. Mansfield, J. J. Chandran, K. J. Vaux, R. J. Millard, A. Christopoulos, F. J. Mitchelson, and E. Burcher Comparison of Receptor Binding Characteristics of Commonly Used Muscarinic Antagonists in Human Bladder Detrusor and Mucosa J. Pharmacol. Exp. Ther., March 1, 2009; 328(3): 893 - 899. [Abstract] [Full Text] [PDF]

				G. Lebon, C. J. Langmead, B. G. Tehan, and E. C. Hulme Mutagenic Mapping Suggests a Novel Binding Mode for Selective Agonists of M1 Muscarinic Acetylcholine Receptors Mol. Pharmacol., February 1, 2009; 75(2): 331 - 341. [Abstract] [Full Text] [PDF]

				B. Holst, T. M. Frimurer, J. Mokrosinski, T. Halkjaer, K. B. Cullberg, C. R. Underwood, and T. W. Schwartz Overlapping Binding Site for the Endogenous Agonist, Small-Molecule Agonists, and Ago-allosteric Modulators on the Ghrelin Receptor Mol. Pharmacol., January 1, 2009; 75(1): 44 - 59. [Abstract] [Full Text] [PDF]

				R. L. Thomas, R. Mistry, C. J. Langmead, M. D. Wood, and R. A. J. Challiss G Protein Coupling and Signaling Pathway Activation by M1 Muscarinic Acetylcholine Receptor Orthosteric and Allosteric Agonists J. Pharmacol. Exp. Ther., November 1, 2008; 327(2): 365 - 374. [Abstract] [Full Text] [PDF]

				H. Suga, K. W. Figueroa, and F. J. Ehlert Use of Acetylcholine Mustard to Study Allosteric Interactions at the M2 Muscarinic Receptor J. Pharmacol. Exp. Ther., November 1, 2008; 327(2): 518 - 528. [Abstract] [Full Text] [PDF]

				C. Valant, K. J. Gregory, N. E. Hall, P. J. Scammells, M. J. Lew, P. M. Sexton, and A. Christopoulos A Novel Mechanism of G Protein-coupled Receptor Functional Selectivity: MUSCARINIC PARTIAL AGONIST McN-A-343 AS A BITOPIC ORTHOSTERIC/ALLOSTERIC LIGAND J. Biol. Chem., October 24, 2008; 283(43): 29312 - 29321. [Abstract] [Full Text] [PDF]

				V. Nawaratne, K. Leach, N. Suratman, R. E. Loiacono, C. C. Felder, B. N. Armbruster, B. L. Roth, P. M. Sexton, and A. Christopoulos New Insights into the Function of M4 Muscarinic Acetylcholine Receptors Gained Using a Novel Allosteric Modulator and a DREADD (Designer Receptor Exclusively Activated by a Designer Drug) Mol. Pharmacol., October 1, 2008; 74(4): 1119 - 1131. [Abstract] [Full Text] [PDF]

				L. Zhang, A. Hu, H. Yuan, L. Cui, G. Miao, X. Yang, L. Wang, J. Liu, X. Liu, S. Wang, et al. A Missense Mutation in the CHRM2 Gene Is Associated With Familial Dilated Cardiomyopathy Circ. Res., June 6, 2008; 102(11): 1426 - 1432. [Abstract] [Full Text] [PDF]

				F. J. Ehlert and M. T. Griffin Two-State Models and the Analysis of the Allosteric Effect of Gallamine at the M2 Muscarinic Receptor J. Pharmacol. Exp. Ther., June 1, 2008; 325(3): 1039 - 1060. [Abstract] [Full Text] [PDF]