Identification of a contact region between the tridecapeptide α-factor mating pheromone of Saccharomyces cerevisiae and its G protein-coupled receptor by photoaffinity labeling

Saccharomyces cerevisiae haploid cells communicate with their opposite mating type through peptide pheromones (alpha-factor and a-factor) that activate G protein-coupled receptors (GPCRs). S. cerevisiae was used as a model system for the study of peptide-responsive GPCRs. Here, we detail the synthesis and characterization of a number of a-factor (Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr) pheromone analogues containing the photo-cross-linkable group 4-benzoyl-L-phenylalanine (Bpa). Following characterization, one analogue, [Bpa(1), Tyr(3), Arg(7), Phe(13)]alpha-factor, was radioiodinated and used as a probe for Ste2p, the GPCR for alpha-factor. Binding of the di-iodinated probe was saturable (K-d = 200 nM) and competable by alpha-factor. Cross-linking into Ste2p was specific for this receptor and reversed by the wildtype pheromone. Chemical and enzymatic cleavage of the receptor/radioprobe complex indicated that cross-linking occurred on a portion of Ste2p spanning residues 251-294 which encompasses transmembrane domain 6, the extracellular loop between transmembrane domains 6 and 7, and transmembrane domain 7. This fragment was verified using T7-epitope-tagged Ste2p and a biotinylated, photoactivatable alpha-factor. After cross-linking with the biotinylated photoprobe and trypsin cleavage, the cross-linked receptor fragment was revealed by both an anti T7-epitope antibody and a biotin probe. This is the first determination of a specific contact region between a Class IV GPCR and its ligand. The results demonstrate that Bpa alpha-factor probes are useful in determining contacts between alpha-factor and Ste2p and initiate mapping of the ligand binding site of this GPCR.