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 (WHWLQLKPGQPMY) from Saccharomyces cerevisiae in which Tyr13 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 showed that the phenolic OH of Tyr13 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 alpha-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 Tyr13 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(125I)1,Nle12, Phe13] alpha-factor exhibited saturable binding with a KD of 81 nM and was competed by alpha-factor for binding in a whole-cell assay. Thus a new family of radioactive ligands for the alpha-factor receptor has been revealed. These ligands should be extremely useful in defining active site residues during mutagenesis and cross-linking studies.
Department of Chemistry, The College of Staten Island and The Graduate School of The City University of New York, 10314, USA.
S
S
Liu
L K
LK
Henry
B K
BK
Lee
S H
SH
Wang
B
B
Arshava
J M
JM
Becker
F
F
Naider
D001667:Binding, Competitive; D002455:Cell Division / Q000187:drug effects; D002462:Cell Membrane / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D007457:Iodine Radioisotopes / Q000737:chemistry; D008024:Ligands; D000072235:Mating Factor; D008956:Models, Chemical; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D018000:Receptors, Peptide / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry; D001616:beta-Galactosidase / Q000378:metabolism
D005656:Fungal Proteins; D007457:Iodine Radioisotopes; D008024:Ligands; D010455:Peptides; D018000:Receptors, Peptide; D014443:Tyrosine; D000072235:Mating Factor; D001616:beta-Galactosidase