Functional coupling of mammalian receptors to the yeast mating pathway using novel yeast/mammalian G protein ?-subunit chimeras
Open Access
- 15 January 2000
Abstract
The expression of mammalian G protein coupled receptors (GPCRs) in S. cerevisiae provides a powerful assay system for functional analysis, ligand identification and pharmaceutical screening. However, relatively few receptors have been coupled to the pheromone response pathway via the yeast Gα, Gpa1p, or chimeric yeast/mammalian Gα subunits containing long C‐terminal regions of mammalian Gα proteins. We tested an extended range of seven such chimeras for Gα sub‐types of three major classes (Gαi/o, Gαs and Gαq), against eight human GPCRs (SST2, SST5, 5‐HT1A, 5‐HT1Dα, ML1B, P2Y1 and P2Y2). Although the Gαi/o chimeras increased the range of receptors that coupled efficiently, the Gαs and Gαq chimeras were inactive when expressed using the GPA1 promoter. We describe 10 novel Gpa1p chimeras, designated ‘transplants’, in which the C‐terminal five amino acids of Gpa1p were exchanged with mammalian residues. Coupling efficiency and ligand sensitivity improved significantly using the transplants. For the P2Y purinergic receptors, coupling could only be detected with the transplants; this is the first report of Gq specificity coupling in yeast. Thus, the transplants offer major advantages over previously described approaches, in terms of both the range of receptors coupled and the efficiency of coupling. Copyright © 2000 John Wiley & Sons, Ltd.Keywords
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