Identification of residues of the Saccharomyces cerevisiae G protein-coupled receptor contributing to alpha-factor pheromone binding

The Saccharomyces cerevisiae pheromone, a-factor (WHWLQLKPGQPMY), and Ste2p , its G protein-coupled receptor, were studied as a model for peptide ligan d-receptor interaction. The affinities and activities of various synthetic position-10 alpha -factor analogs with Ste2p expressing mutations at residu es Ser(47) and Thr(48) were investigated. All mutant receptors were express ed at a similar level in the cytoplasmic membrane, and their efficacies of signal transduction were similar to that of the wild-type receptor. Mutant receptors differed in binding affinity (K-d) and potency (EC50) for gene in duction by alpha -factor. One mutant receptor (S47K,T48K) had dramatically reduced affinity and activity for [Lys(10)]- and [Orn(10)] alpha -factor, w hereas the affinity for Saccharomyces kluyveri alpha -factor (WHWLSFSKGEPMY ) was increased over 20-fold compared with that of wild-type receptor. In c ontrast, the affinity of [Lys(10)]- and [Orn(10)] alpha -factor was increas ed greatly in a S47E,T48E mutant receptor, whereas the binding of the S. kl uyveri alpha -factor was abolished. The affinity of [Lys(10)]- and [Orn(10) ] alpha -factor for the S47E,T48E receptor dropped 4-6fold in the presence of 1 M NaCl, whereas the affinity of alpha -factor was not affected by this treatment. These results demonstrate that when bound to its receptor the 1 0th residue (Gln) of the S. cerevisiae alpha -factor is adjacent to Ser(47) and Thr(48) residues in the receptor and that the 10th residue of alpha -f actors from two Saccharomyces species is responsible for the ligand selecti vity to their cognate receptors. Based on these data, we have developed a t wo-dimensional model of alpha -factor binding to its receptor.