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

Saccharomyces cerevisiae is a tractable yeast species for ex-pression and coupling of heterologous G protein-coupled re-ceptors 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 re-ceptor (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 G/yeast Gpa1 protein chi-mera, and probed for G protein-biased allosteric modulation. Subsequent assays performed in this system revealed that brucine was a partial allosteric agonist and positive modulato