RT Journal Article
SR Electronic
T1 Determination of Adenosine A<sub>1</sub> Receptor Agonist and Antagonist Pharmacology Using <em>Saccharomyces cerevisiae</em>: Implications for Ligand Screening and Functional Selectivity
JF Journal of Pharmacology and Experimental Therapeutics
JO J Pharmacol Exp Ther
FD American Society for Pharmacology and Experimental Therapeutics
SP 277
OP 286
DO 10.1124/jpet.109.158667
VO 331
IS 1
A1 Gregory D. Stewart
A1 Celine Valant
A1 Simon J. Dowell
A1 Dalibor Mijaljica
A1 Rodney J. Devenish
A1 Peter J. Scammells
A1 Patrick M. Sexton
A1 Arthur Christopoulos
YR 2009
UL http://jpet.aspetjournals.org/content/331/1/277.abstract
AB The budding yeast, Saccharomyces cerevisiae, is a convenient system for coupling heterologous G protein-coupled receptors (GPCRs) to the pheromone response pathway to facilitate empirical ligand screening and/or GPCR mutagenesis studies. However, few studies have applied this system to define GPCR-G protein-coupling preferences and furnish information on ligand affinities, efficacies, and functional selectivity. We thus used different S. cerevisiae strains, each expressing a specific human Gα/yeast Gpa1 protein chimera, and determined the pharmacology of various ligands of the coexpressed human adenosine A1 receptor. These assays, in conjunction with the application of quantitative models of agonism and antagonism, revealed that (−)-N6-(2-phenylisopropyl)adenosine was a high-efficacy agonist that selectively coupled to Gpa/1Gαo, Gpa1/Gαi1/2, and Gpa1/Gαi3, whereas the novel compound, 5′-deoxy-N6-(endo-norborn-2-yl)-5′-(2-fluorophenylthio)adenosine (VCP-189), was a lower-efficacy agonist that selectively coupled to Gpa1/Gαi proteins; the latter finding suggested that VCP-189 might be functionally selective. The affinity of the antagonist, 8-cyclopentyl-1,3-dipropylxanthine, was also determined at the various strains. Subsequent experiments performed in mammalian Chinese hamster ovary cells monitoring cAMP formation/inhibition, intracellular calcium mobilization, phosphorylation of extracellular signal-regulated kinase 1 and 2 or 35S-labeled guanosine 5′-(γ-thio)triphosphate binding, were in general agreement with the yeast data regarding agonist efficacy estimation and antagonist affinity estimation, but revealed that the apparent functional selectivity of VCP-189 could be explained by differences in stimulus-response coupling between yeast and mammalian cells. Our results suggest that this yeast system is a useful tool for quantifying ligand affinity and relative efficacy, but it may lack the sensitivity required to detect functional selectivity of low-efficacy agonists. © 2009 by The American Society for Pharmacology and Experimental Therapeutics