Prediction of functionally selective allosteric interactions at an M 3 muscarinic acetylcholine receptor mutant using Saccharomyces cerevisiae

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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 M(3) muscarinic acetylcholine receptor (mAChR) bearing a mutation (K(7.32)E) recently identified to confer positive cooperativity between acetylcholine and the allosteric modulator brucine in various strains of S. cerevisiae, each expressing a different human Galpha/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/G(q) proteins, a positive modulator (no agonism) when coupled to Gpa1/G(12) proteins, and a neutral modulator when coupled to Gpa1/G(i) proteins. It is noteworthy that these results were validated at the human M(3)K(7.32)E mAChR expressed in a mammalian (Chinese hamster ovary) cell background by determination of calcium mobilization and membrane ruffling as surrogate measures of G(q) and G(12) 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 (G(12)) as a key mediator for allosteric modulation of M(3)K(7.32)E 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.
Original languageEnglish
Pages (from-to)205 - 214
Number of pages10
JournalMolecular Pharmacology
Volume78
Issue number2
Publication statusPublished - 2010

Cite this

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title = "Prediction of functionally selective allosteric interactions at an M 3 muscarinic acetylcholine receptor mutant using Saccharomyces cerevisiae",
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 M(3) muscarinic acetylcholine receptor (mAChR) bearing a mutation (K(7.32)E) recently identified to confer positive cooperativity between acetylcholine and the allosteric modulator brucine in various strains of S. cerevisiae, each expressing a different human Galpha/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/G(q) proteins, a positive modulator (no agonism) when coupled to Gpa1/G(12) proteins, and a neutral modulator when coupled to Gpa1/G(i) proteins. It is noteworthy that these results were validated at the human M(3)K(7.32)E mAChR expressed in a mammalian (Chinese hamster ovary) cell background by determination of calcium mobilization and membrane ruffling as surrogate measures of G(q) and G(12) 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 (G(12)) as a key mediator for allosteric modulation of M(3)K(7.32)E 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.",
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AU - Stewart, Gregory D

AU - Sexton, Patrick

AU - Christopoulos, Arthur

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N2 - 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 M(3) muscarinic acetylcholine receptor (mAChR) bearing a mutation (K(7.32)E) recently identified to confer positive cooperativity between acetylcholine and the allosteric modulator brucine in various strains of S. cerevisiae, each expressing a different human Galpha/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/G(q) proteins, a positive modulator (no agonism) when coupled to Gpa1/G(12) proteins, and a neutral modulator when coupled to Gpa1/G(i) proteins. It is noteworthy that these results were validated at the human M(3)K(7.32)E mAChR expressed in a mammalian (Chinese hamster ovary) cell background by determination of calcium mobilization and membrane ruffling as surrogate measures of G(q) and G(12) 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 (G(12)) as a key mediator for allosteric modulation of M(3)K(7.32)E 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.

AB - 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 M(3) muscarinic acetylcholine receptor (mAChR) bearing a mutation (K(7.32)E) recently identified to confer positive cooperativity between acetylcholine and the allosteric modulator brucine in various strains of S. cerevisiae, each expressing a different human Galpha/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/G(q) proteins, a positive modulator (no agonism) when coupled to Gpa1/G(12) proteins, and a neutral modulator when coupled to Gpa1/G(i) proteins. It is noteworthy that these results were validated at the human M(3)K(7.32)E mAChR expressed in a mammalian (Chinese hamster ovary) cell background by determination of calcium mobilization and membrane ruffling as surrogate measures of G(q) and G(12) 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 (G(12)) as a key mediator for allosteric modulation of M(3)K(7.32)E 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.

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