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Research ArticleArticle

The Role of Transmembrane Domain 3 in the Actions of Orthosteric, Allosteric, and Atypical Agonists of the M4 Muscarinic Acetylcholine Receptor

Katie Leach, Anna E. Davey, Christian C. Felder, Patrick M. Sexton and Arthur Christopoulos
Molecular Pharmacology May 2011, 79 (5) 855-865; DOI: https://doi.org/10.1124/mol.111.070938
Katie Leach
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Anna E. Davey
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Christian C. Felder
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Patrick M. Sexton
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Arthur Christopoulos
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Abstract

Despite the discovery of a diverse range of novel agonists and allosteric modulators of the M4 muscarinic acetylcholine (ACh) receptor (mAChR), little is known about how such ligands activate the receptor. We used site-directed mutagenesis of conserved residues in transmembrane 3 (TMIII), a key region involved in G protein-coupled receptor activation, to probe the binding and function of prototypical orthosteric mAChR agonists, allosteric modulators, and “atypical” agonists. We found that most mutations did not affect the binding of the allosteric modulators, with the exception of W1083.28A and L1093.29A (which may contribute directly to the interface between allosteric and orthosteric sites) and mutation D1123.32N (which may cause a global disruption of a hydrogen bond network). Although numerous mutations affected signaling, we did not identify amino acids that were important for the functional activity of any one class of agonist (orthosteric, allosteric, or atypical) to the exclusion of any others, suggesting that TMIII is key for the transmission of stimulus irrespective of the agonist. We also identified two key residues, Trp1083.28 and Asp1123.32, that are essential for the transmission of binding cooperativity between 3-amino-5-chloro-6-methoxy-4-methyl-thieno[2,3-b]pyridine- 2-carboxylic acid cyclopropylamide (LY2033298) and ACh. Finally, we found that LY2033298 was able to rescue functionally impaired signaling of ACh at the majority of mutants tested in a manner that was inversely correlated with the ACh signaling efficacy, indicating that a key part of the mechanism of the positive cooperativity mediated by LY2033298 on the endogenous agonist involves a global drive of the receptor toward an active conformation.

Footnotes

  • ↵Embedded Image The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

  • This work was supported by the National Health and Medical Research Council (NHMRC) of Australia [Grant 519461]. A.C. is a Senior and P.S. a Principal Research Fellow of the NHMRC.

  • A.C. is a consultant for Johnson and Johnson and Alchemia. C.C.F. is an employee of Eli Lilly and Co.

  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

    doi:10.1124/mol.111.070938.

  • ABBREVIATIONS:

    GPCR
    G protein-coupled receptor
    ACh
    acetylcholine
    C7/3-phth
    heptane-1,7-bis-(dimethyl-3′-phthalimidopropyl) ammonium bromide
    CHO
    Chinese hamster ovary
    FBS
    fetal bovine serum
    LY2033298
    3-amino-5-chloro-6-methoxy-4-methyl-thieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide
    mAChR
    muscarinic acetylcholine receptor
    McN-A-343
    4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride
    NDMC
    N-desmethylclozapine
    [3H]NMS
    [3H]N-methylscopolamine
    [3H]QNB
    [3H]quinuclidinyl benzilate
    TM
    transmembrane domain
    ERK1/2
    extracellular signal-regulated kinase 1/2
    PBS
    phosphate-buffered saline
    WT
    wild type
    HA
    hemagglutinin.

  • Received January 2, 2011.
  • Accepted February 7, 2011.
  • Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 79 (5)
Molecular Pharmacology
Vol. 79, Issue 5
1 May 2011
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Research ArticleArticle

The Role of Transmembrane Domain 3 in the Actions of Orthosteric, Allosteric, and Atypical Agonists of the M4 Muscarinic Acetylcholine Receptor

Katie Leach, Anna E. Davey, Christian C. Felder, Patrick M. Sexton and Arthur Christopoulos
Molecular Pharmacology May 1, 2011, 79 (5) 855-865; DOI: https://doi.org/10.1124/mol.111.070938

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Research ArticleArticle

The Role of Transmembrane Domain 3 in the Actions of Orthosteric, Allosteric, and Atypical Agonists of the M4 Muscarinic Acetylcholine Receptor

Katie Leach, Anna E. Davey, Christian C. Felder, Patrick M. Sexton and Arthur Christopoulos
Molecular Pharmacology May 1, 2011, 79 (5) 855-865; DOI: https://doi.org/10.1124/mol.111.070938
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