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Molecular Pharmacology

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Research ArticleArticle

Prediction of Functionally Selective Allosteric Interactions at an M3 Muscarinic Acetylcholine Receptor Mutant Using Saccharomyces cerevisiae

Gregory D. Stewart, Patrick M. Sexton and Arthur Christopoulos
Molecular Pharmacology August 2010, 78 (2) 205-214; DOI: https://doi.org/10.1124/mol.110.064253
Gregory D. Stewart
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Patrick M. Sexton
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Arthur Christopoulos
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Abstract

Saccharomyces cerevisiae is a tractable yeast species for expression and coupling of heterologous G protein-coupled receptors with the endogenous pheromone response pathway. Although this platform has been used for ligand screening, no studies have probed its ability to predict novel pharmacology and functional selectivity of allosteric ligands. As a proof of concept, we expressed a rat M3 muscarinic acetylcholine receptor (mAChR) bearing a mutation (K7.32E) recently identified to confer positive cooperativity between acetylcholine and the allosteric modulator brucine in various strains of S. cerevisiae, each expressing a different human Gα/yeast Gpa1 protein chimera, and probed for G protein-biased allosteric modulation. Subsequent assays performed in this system revealed that brucine was a partial allosteric agonist and positive modulator of carbachol when coupled to Gpa1/Gq proteins, a positive modulator (no agonism) when coupled to Gpa1/G12 proteins, and a neutral modulator when coupled to Gpa1/Gi proteins. It is noteworthy that these results were validated at the human M3K7.32E mAChR expressed in a mammalian (Chinese hamster ovary) cell background by determination of calcium mobilization and membrane ruffling as surrogate measures of Gq and G12 protein activation, respectively. Furthermore, the combination of this functionally selective allosteric modulator with G protein-biased yeast screens allowed us to ascribe a potential G protein candidate (G12) as a key mediator for allosteric modulation of M3K7.32E mAChR-mediated ERK1/2 phosphorylation, which was confirmed by small interfering RNA knockdown experiments. These results highlight how the yeast platform can be used to identify functional selectivity of allosteric ligands and to facilitate dissection of convergent signaling pathways.

Footnotes

  • This work was funded by the National Health and Medical Research Council (NHMRC) of Australia [Program Grant 519461]. Arthur Christopoulos is a Senior, and Patrick Sexton a Principal, Research Fellow of the NHMRC. Gregory Stewart is the recipient of an Australian Postgraduate Award (Industry) from the Australian Research Council.

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

    doi:10.1124/mol.110.064253.

  • ABBREVIATIONS:

    mAChR
    muscarinic acetylcholine receptor
    GPCR
    G protein-coupled receptor
    TM
    transmembrane
    CCh
    carbachol
    NMS
    N-methyl scopolamine
    CHO
    Chinese hamster ovary
    AM
    acetoxymethyl
    DMEM
    Dulbecco's modified Eagle's medium
    FBS
    fetal bovine serum
    siRNA
    small interfering RNA
    ERK
    extracellular signal-regulated kinase
    PTX
    pertussis toxin
    r
    rat
    ANOVA
    analysis of variance.

  • Received February 17, 2010.
  • Accepted May 12, 2010.
  • Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 78 (2)
Molecular Pharmacology
Vol. 78, Issue 2
1 Aug 2010
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Research ArticleArticle

Prediction of Functionally Selective Allosteric Interactions at an M3 Muscarinic Acetylcholine Receptor Mutant Using Saccharomyces cerevisiae

Gregory D. Stewart, Patrick M. Sexton and Arthur Christopoulos
Molecular Pharmacology August 1, 2010, 78 (2) 205-214; DOI: https://doi.org/10.1124/mol.110.064253

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Research ArticleArticle

Prediction of Functionally Selective Allosteric Interactions at an M3 Muscarinic Acetylcholine Receptor Mutant Using Saccharomyces cerevisiae

Gregory D. Stewart, Patrick M. Sexton and Arthur Christopoulos
Molecular Pharmacology August 1, 2010, 78 (2) 205-214; DOI: https://doi.org/10.1124/mol.110.064253
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