Identification of residues of the Saccharomyces cerevisiae G protein-coupled receptor contributing to alpha-factor pheromone binding.
The Saccharomyces cerevisiae pheromone, alpha-factor (WHWLQLKPGQPMY), and Ste2p, its G protein-coupled receptor, were studied as a model for peptide ligand-receptor interaction. The affinities and activities of various synthetic position-10 alpha-factor analogs with Ste2p expressing mutations at residues Ser47 and Thr48 were investigated. All mutant receptors were expressed 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 (Kd) and potency (EC50) for gene induction by alpha-factor. One mutant receptor (S47K,T48K) had dramatically reduced affinity and activity for [Lys10]- and [Orn10]alpha-factor, whereas the affinity for Saccharomyces kluyveri alpha-factor (WHWLSFSKGEPMY) was increased over 20-fold compared with that of wild-type receptor. In contrast, the affinity of [Lys10]- and [Orn10]alpha-factor was increased greatly in a S47E,T48E mutant receptor, whereas the binding of the S. kluyveri alpha-factor was abolished. The affinity of [Lys10]- and [Orn10]alpha-factor for the S47E,T48E receptor dropped 4-6-fold 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 10th residue (Gln) of the S. cerevisiae alpha-factor is adjacent to Ser47 and Thr48 residues in the receptor and that the 10th residue of alpha-factors from two Saccharomyces species is responsible for the ligand selectivity to their cognate receptors. Based on these data, we have developed a two-dimensional model of alpha-factor binding to its receptor.
Lee
B K
BK
Department of Microbiology, University of Tennessee, Knoxville, 37996, USA.
Khare
S
S
Naider
F
F
Becker
J M
JM
D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D019204:GTP-Binding Proteins / Q000378:metabolism*; D000072235:Mating Factor; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D015398:Signal Transduction
D017931:DNA Primers; D010455:Peptides; D011956:Receptors, Cell Surface; D000072235:Mating Factor; D019204:GTP-Binding Proteins