Functional coupling of mammalian receptors to the yeast mating pathway using novel yeast/mammalian G protein...

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 G(alpha), Gpa1p, or chimeric yeast/mammalian G(alpha) subunits containing long C-terminal regions of mammalian G(alpha) proteins. We tested an extended range of seven such chimeras for G(alpha) sub-types of three major classes (G(alphai/o), G(alphas) and G(alphaq)), against eight human GPCRs (SST(2), SST(5), 5-HT(1A), 5-HT(1Dalpha), ML(1B), P2Y(1) and P2Y(2)). Although the G(alphai/o) chimeras increased the range of receptors that coupled efficiently, the G(alphas) and G(alphaq) 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 G(q) 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.