Dominant negative mutations in the alpha-factor receptor, a G protein-coupled receptor encoded by the STE2 gene of the yeast Saccharomyces cerevisiae

The alpha-mating pheromone receptor encoded by the STE2 gene of the yeast S accharomyces cevevisiae is a G protein-coupled receptor (GPCR) that is homo logous to the large family of GPCRs that mediate multiple types of signal t ransduction in mammals. We have screened libraries of mutant receptors to i dentify dominant negative alleles that are capable of interfering with the function of a co-expressed normal receptor. Two dominant negative alleles h ave been recovered in this manner. In addition, we find that previously iso lated loss-of-function mutations in the alpha-factor receptor exhibit domin ant negative effects. Detection of the dominant effects requires high-level expression of the mutant receptors but does not require a high ratio of mu tant to normal receptors. Cellular levels of the normal receptors are not a ffected by co-expression of the dominant negative alleles. Expression of th e mutant receptors does not interfere with constitutive signaling in a stra in that lacks the G protein alpha subunit encoded by GPA1, indicating that interference with signaling occurs at the level of the receptor or the inte racting G protein. Expression of increased levels of G protein subunits par tially reverses the dominant negative effects. The dominant negative behavi or of the mutant receptors is diminished by deletion of the SST2 gene, whic h encodes an RGS (Regulator of G protein Signaling) protein involved in des ensitization of pheromone signaling. The most likely explanation for the do minant negative effects of the mutations appears to be the existence of an interaction between unactivated receptors and the trimeric G protein that t itrates the G protein away from the normal receptors or renders the G prote in insensitive to receptor activation. This interaction appears to be media ted by the SST2 gene product.