The third cytoplasmic loop of a yeast G-protein-coupled receptor controls pathway activation, ligand discrimination, and receptor internalization

To identify functional domains of G-protein-coupled receptors that control pathway activation, ligand discrimination, and receptor regulation, we have used as a model the α-factor receptor (STE2 gene product) of the yeast Saccharomyces cerevisiae. From a collection of random mutations introduced in the region coding for the third cytoplasmic loop of Ste2p, six ste2(sst) alleles were identified by genetic screening methods that increased α- factor sensitivity 2.5- to 15-fold. The phenotypic effects of ste2(sst) and sst2 mutations were not additive, consistent with models in which the third cytoplasmic loop of the α-factor receptor and the regulatory protein Sst2p control related aspects of pheromone response and/or desensitization. Four ste2(sst) mutations did not dramatically alter cell surface expression or agonist binding affinity of the receptor; however, they did permit detectable responses to an α-factor antagonist. One ste2(sst) allele increased receptor binding affinity for α-factor and elicited stronger responses to antagonist. Results of competition binding experiments indicated that wild-type and representative mutant receptors bound antagonist with similar affinities. The antagonist-responsive phenotypes caused by ste2(sst) alleles were therefore due to defects in the ability of receptors to discriminate between agonist and antagonist peptides. One ste2(sst) mutation caused rapid, ligand- independent internalization of the receptor. These results demonstrate that the third cytoplasmic loop of the α-factor receptor is a multifunctional regulatory domain that controls pathway activation and/or desensitization and influences the processes of receptor activation, ligand discrimination, and internalization.