Functional coupling of mammalian receptors to the yeast mating pathway using novel yeast/mammalian G protein alpha-subunit chimeras

The expression of mammalian G protein coupled receptors (GPCRs) in S. cerev isiae provides a powerful assay system for functional analysis, ligand iden tification and pharmaceutical screening. However, relatively few receptors have been coupled to the pheromone response pathway via the yeast G(proport ional to), Gpa1p, or chimeric yeast/mammalian G(proportional to) subunits c ontaining long C-terminal regions of mammalian G(proportional to) proteins. We tested an extended range of seven such chimeras for G(proportional to) sub-types of three major classes (G(proportional to i/o), G(proportional to s) and G(proportional to q)), against eight human GPCRs (SST2, SST5, 5-HT1 A, 5-HT1D proportional to, ML1B, P2Y(1) and P2Y(2)). Although the G(proport ional to i/o) chimeras increased the range of receptors that coupled effici ently, the G(proportional to s) and G(proportional to q) chimeras were inac tive when expressed using the GPA1 promoter. We describe 10 novel Gpa1p chi meras, designated 'transplants', in which the C-terminal five amino acids o f Gpa1p were exchanged with mammalian residues. Coupling efficiency and lig and sensitivity improved significantly using the transplants. For the P2Y p urinergic receptors, coupling could only be detected with the transplants; this is the first report of G(q) specificity coupling in yeast. Thus, the t ransplants offer major advantages over previously described approaches, in terms of both the range of receptors coupled and the efficiency of coupling . Copyright (C) 2000 John Wiley & Sons, Ltd.