Abstract
Saccharomyces cerevisiae (S. cerevisiae) is a tractable yeast species for expression and coupling of heterologous G protein-coupled receptor (GPCRs) with the endogenous pheromone response pathway. Whilst 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 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 CCh 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. Importantly, these results were validated at the human M3K7.32E mAChR expressed in a mammalian (CHO) 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 (siRNA) 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
- Received February 17, 2010.
- Revision received May 11, 2010.
- Accepted May 12, 2010.
- The American Society for Pharmacology and Experimental Therapeutics