Abstract
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.
- GPCR, G protein-coupled receptor
- ADA, adenosine deaminase
- BSA, bovine serum albumin
- CHO, Chinese hamster ovary
- DMEM, Dulbecco's modified Eagle's medium
- DPCPX, 8-cyclopentyl-1,3-dipropylxanthine
- ERK1/2, extracellular signal-regulated kinase 1 and 2
- FBS, fetal bovine serum
- FSCPX, 8-cyclopentyl-3-(3-((4-(fluorosulfonylbenzoyl)oxy)propyl)-1-propylxanthine
- GTPγS, guanosine 5′(γ-thio)triphosphate
- PBS, phosphate-buffered saline
- PTX, pertussis toxin
- R-PIA, (−)-N6-(2-phenylisopropyl)adenosine
- VCP-189, 5′-deoxy-N6-(endo-norborn-2-yl)-5′-(2-fluorophenylthio)adenosine.
Footnotes
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This work was supported by the National Health and Medical Research Council (NHMRC) [Program Grant 519461]. A.C. is an NHMRC Senior Research Fellow, and P.S. is an NHMRC Principal Research Fellow. G.S. received an Australian Postgraduate Award (Industry) from the Australian Research Council.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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ABBREVIATIONS:
- Received July 7, 2009.
- Accepted July 27, 2009.
- © 2009 by The American Society for Pharmacology and Experimental Therapeutics
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