Position 13 analogs of the tridecapeptide mating pheromone from Saccharomyces cerevisiae: design of an iodinatable ligand for receptor binding

Analogs of the alpha-factor tridecapeptide mating pheromone (WHWLQLKPGOPMY) from Saccharomyces cerevisiae in which Tyr(13) was replaced with Phe, p-F- Phe, m-F-Phe, p-NO2-Phe, p-NH2-Phe or Ser were synthesized and purified to >99% homogeneity. These analogs were bioassayed using a growth arrest assay and a gene induction assay and evaluated for their ability to compete with binding of tritiated alpha-factor to its receptor Ste2p. The results showe d that the phenolic OH of Tyr(13) is not required for either biological act ivity or receptor recognition. Analogs containing fluorine, amino, nitro or a hydrogen in place of OH had 80-120% of the biological activity of the pa rent pheromone in the gene induction assay and had receptor affinities from nearly equal to 6-fold lower than that of alpha-factor. In contrast, subst itution of Set or Ala at position 13 resulted in a >100-fold decrease in re ceptor affinity suggesting that the aromatic ring is involved in binding to the receptor. The lack of a strict requirement for Tyr(13) allowed the des ign of several multiple replacement analogs in which Phe or p-F-Phe were su bstituted at position 13 and Tyr was placed in other positions of the pepti de. These analogs could then be iodinated and used in the development of a highly sensitive receptor-binding assay. One potential receptor ligand [Tyr (I-125)(1), Nle(12), Phe(13)] alpha-factor exhibited saturable binding with a K-D of 81 nM and was competed by alpha-factor for binding in a whole-cel l assay. Thus a new family of radioactive ligands for the alpha-factor rece ptor has been revealed. These ligands should be extremely useful in definin g active site residues during mutagenesis and cross-linking studies.