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

Abstract: Analogs of the α-factor tridecapeptide mating pheromone (WHWLQLKPGQPMY) from Saccharomyces cerevisiae in which Tyr[sup 13] was replaced with Phe, p-F-Phe, m-F-Phe, p-NO[sub 2]-Phe, p-NH[sub 2]-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 α-factor to its receptor Ste2p. The results showed that the phenolic OH of Tyr[sup 13] is not required for either biological activity or receptor recognition. Analogs containing fluorine, amino, nitro or a hydrogen in place of OH had 80–120% of the biological activity of the parent pheromone in the gene induction assay and had receptor affinities from nearly equal to 6-fold lower than that of α-factor. In contrast, substitution of Ser or Ala at position 13 resulted in a > 100-fold decrease in receptor affinity suggesting that the aromatic ring is involved in binding to the receptor. The lack of a strict requirement for Tyr[sup 13] allowed the design of several multiple replacement analogs in which Phe or p-F-Phe were substituted at position 13 and Tyr was placed in other positions of the peptide. These analogs could then be iodinated and used in the development of a highly sensitive receptor-binding assay. One potential receptor ligand [Tyr([sup 125]I)[sup 1],Nle[sup 12], Phe[sup 13]]α-factor exhibited saturable binding with a K[sub D] of 81 n m and was competed by α-factor for binding in a whole-cell assay. Thus a new family of radioactive ligands for the α-factor receptor has been revealed. These ligands should be extremely useful in defining active site residues during mutagenesis and cross-linking studies. [ABSTRACT FROM AUTHOR]