Structure-function studies of allosteric agonism at M2 muscarinic acetylcholine receptors

Lauren Therese May, Vimesh Ashvinkumar Avlani, Christopher J Langmead, Hugh J Herdon, Martyn D Wood, Patrick Sexton, Arthur Christopoulos

Research output: Contribution to journalArticleResearchpeer-review

Abstract

The M2 muscarinic acetylcholine receptor (mAChR) possesses at least one binding site for allosteric modulators that is dependent on the residues (172)EDGE(175), Y(177) and T(423). However, the contribution of these residues to actions of allosteric agonists, as opposed to modulators, is unknown. We created mutant M2 mAChRs where the charge of the (172)EDGE(175) sequence had been neutralized and each of Y(177) and T(423) were substituted with alanine. Radioligand binding experiments revealed that these mutations had a profound inhibitory effect on the prototypical modulators gallamine, alcuronium and C7/3-phth, but minimal effects on the orthosteric antagonist, [(3)H]NMS. In contrast, the allosteric agonists, McN-A-343, AC-42 and a novel AC-42 derivative, 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 ERK1/2 phosphorylation and [(35)S]GTPgammaS binding revealed minimal effects of the mutations on the orthosteric agonists, 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 Y(177) and T(423), rather than the (172)EDGE(175) 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.
Original languageEnglish
Pages (from-to)463 - 476
Number of pages14
JournalMolecular Pharmacology
Volume72
Issue number2
Publication statusPublished - 2007

Cite this

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title = "Structure-function studies of allosteric agonism at M2 muscarinic acetylcholine receptors",
abstract = "The M2 muscarinic acetylcholine receptor (mAChR) possesses at least one binding site for allosteric modulators that is dependent on the residues (172)EDGE(175), Y(177) and T(423). However, the contribution of these residues to actions of allosteric agonists, as opposed to modulators, is unknown. We created mutant M2 mAChRs where the charge of the (172)EDGE(175) sequence had been neutralized and each of Y(177) and T(423) were substituted with alanine. Radioligand binding experiments revealed that these mutations had a profound inhibitory effect on the prototypical modulators gallamine, alcuronium and C7/3-phth, but minimal effects on the orthosteric antagonist, [(3)H]NMS. In contrast, the allosteric agonists, McN-A-343, AC-42 and a novel AC-42 derivative, 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 ERK1/2 phosphorylation and [(35)S]GTPgammaS binding revealed minimal effects of the mutations on the orthosteric agonists, 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 Y(177) and T(423), rather than the (172)EDGE(175) 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.",
author = "May, {Lauren Therese} and Avlani, {Vimesh Ashvinkumar} and Langmead, {Christopher J} and Herdon, {Hugh J} and Wood, {Martyn D} and Patrick Sexton and Arthur Christopoulos",
year = "2007",
language = "English",
volume = "72",
pages = "463 -- 476",
journal = "Molecular Pharmacology",
issn = "1521-0111",
publisher = "ACS Books",
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Structure-function studies of allosteric agonism at M2 muscarinic acetylcholine receptors. / May, Lauren Therese; Avlani, Vimesh Ashvinkumar; Langmead, Christopher J; Herdon, Hugh J; Wood, Martyn D; Sexton, Patrick; Christopoulos, Arthur.

In: Molecular Pharmacology, Vol. 72, No. 2, 2007, p. 463 - 476.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Structure-function studies of allosteric agonism at M2 muscarinic acetylcholine receptors

AU - May, Lauren Therese

AU - Avlani, Vimesh Ashvinkumar

AU - Langmead, Christopher J

AU - Herdon, Hugh J

AU - Wood, Martyn D

AU - Sexton, Patrick

AU - Christopoulos, Arthur

PY - 2007

Y1 - 2007

N2 - The M2 muscarinic acetylcholine receptor (mAChR) possesses at least one binding site for allosteric modulators that is dependent on the residues (172)EDGE(175), Y(177) and T(423). However, the contribution of these residues to actions of allosteric agonists, as opposed to modulators, is unknown. We created mutant M2 mAChRs where the charge of the (172)EDGE(175) sequence had been neutralized and each of Y(177) and T(423) were substituted with alanine. Radioligand binding experiments revealed that these mutations had a profound inhibitory effect on the prototypical modulators gallamine, alcuronium and C7/3-phth, but minimal effects on the orthosteric antagonist, [(3)H]NMS. In contrast, the allosteric agonists, McN-A-343, AC-42 and a novel AC-42 derivative, 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 ERK1/2 phosphorylation and [(35)S]GTPgammaS binding revealed minimal effects of the mutations on the orthosteric agonists, 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 Y(177) and T(423), rather than the (172)EDGE(175) 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.

AB - The M2 muscarinic acetylcholine receptor (mAChR) possesses at least one binding site for allosteric modulators that is dependent on the residues (172)EDGE(175), Y(177) and T(423). However, the contribution of these residues to actions of allosteric agonists, as opposed to modulators, is unknown. We created mutant M2 mAChRs where the charge of the (172)EDGE(175) sequence had been neutralized and each of Y(177) and T(423) were substituted with alanine. Radioligand binding experiments revealed that these mutations had a profound inhibitory effect on the prototypical modulators gallamine, alcuronium and C7/3-phth, but minimal effects on the orthosteric antagonist, [(3)H]NMS. In contrast, the allosteric agonists, McN-A-343, AC-42 and a novel AC-42 derivative, 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 ERK1/2 phosphorylation and [(35)S]GTPgammaS binding revealed minimal effects of the mutations on the orthosteric agonists, 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 Y(177) and T(423), rather than the (172)EDGE(175) 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.

UR - http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17525129

M3 - Article

VL - 72

SP - 463

EP - 476

JO - Molecular Pharmacology

JF - Molecular Pharmacology

SN - 1521-0111

IS - 2

ER -