MUTATIONS THAT ALTER THE 3RD CYTOPLASMIC LOOP OF THE A-FACTOR RECEPTOR LEAD TO A CONSTITUTIVE AND HYPERSENSITIVE PHENOTYPE

The STE3 gene of Saccharomyces cerevisiae encodes a G protein-coupled receptor that is specific for the mating pheromone a-factor. The ste3L 194Q mutation, which leads to the substitution of glutamine for leucin e-194 within the third cytoplasmic loop of the receptor, resulted in a 20-fold increase in pheromone sensitivity and also caused partial con stitutive activation of the response pathway. Moreover, other amino ac id substitutions at the 194 position and several deletion mutations th at collectively remove most of the third cytoplasmic loop resulted in hyperactive receptors. Therefore, we suggest that one role of the thir d cytoplasmic loop is to function as a negative regulatory domain invo lved in the maintenance of a nonsignaling state of the receptor. The c onstitutive activity and the pheromone hypersensitivity of ste3L194Q c ells were recessive, suggesting that the wild-type receptor can antago nize the signal associated with the activated receptor. The ste3DELTA3 06 mutation, which results in truncation of most of the C-terminal dom ain of the receptor, led to a 20-fold increase in pheromone sensitivit y, indicating that this domain also mediates negative regulation of th e receptor. The ste3L194Q and ste3DELTA306 mutations appear to affect receptor activity independently, because the double mutant was associa ted with a 400-fold increase in pheromone sensitivity.