« Previous Abstract"Projected changes in particulate matter concentrations in the South Coast Air Basin due to basin-wide reductions in nitrogen oxides, volatile organic compounds, and ammonia emissions"    Next AbstractA role for unsaturated fatty acids in mitochondrial movement and inheritance »

Mol Pharmacol

Title:		Prediction of functionally selective allosteric interactions at an M3 muscarinic acetylcholine receptor mutant using Saccharomyces cerevisiae
Author(s):		Stewart GD; Sexton PM; Christopoulos A;
Address:		"Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences & Department of Pharmacology, Monash University, Parkville, Victoria, Australia"
Journal Title:		Mol Pharmacol
Year:		2010
Volume:		20100513
Issue:		2
Page Number:		205 - 214
DOI:		10.1124/mol.110.064253
ISSN/ISBN:		1521-0111 (Electronic) 0026-895X (Linking)
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"
Keywords:		"Allosteric Regulation Animals Base Sequence CHO Cells Carbachol/metabolism/pharmacology Cricetinae Cricetulus DNA Primers Humans Radioligand Assay Rats Receptor, Muscarinic M3/genetics/*metabolism Saccharomyces cerevisiae/*genetics Strychnine/analogs & de;"
Notes:		"MedlineStewart, Gregory D Sexton, Patrick M Christopoulos, Arthur eng Research Support, Non-U.S. Gov't 2010/05/15 Mol Pharmacol. 2010 Aug; 78(2):205-14. doi: 10.1124/mol.110.064253. Epub 2010 May 13"