RT Journal Article
SR Electronic
T1 Prediction of Functionally Selective Allosteric Interactions at an M<sub>3</sub> Muscarinic Acetylcholine Receptor Mutant Using <span class="named-content genus-species">Saccharomyces cerevisiae</span>
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 205
OP 214
DO 10.1124/mol.110.064253
VO 78
IS 2
A1 Gregory D. Stewart
A1 Patrick M. Sexton
A1 Arthur Christopoulos
YR 2010
UL http://molpharm.aspetjournals.org/content/78/2/205.abstract
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 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.