Tyr266 in the sixth transmembrane domain of the yeast alpha-factor receptor plays key roles in receptor activation and ligand specificity.
To identify interactions between Ste2p, a G protein-coupled receptor of the yeast Saccharomyces cerevisiae, and its tridecapeptide ligand, alpha-factor (WHWLQLKPGQPMY), a variety of alpha-factor analogues were used in conjunction with site-directed mutagenesis of a targeted portion of Ste2p transmembrane domain six. Alanine substitution of residues in the 262-270 region of Ste2p did not affect pheromone binding or signal transduction, except for the Y266A mutant, which did not transduce signal yet exhibited only a small decrease in alpha-factor binding affinity. Substitutions with Ser, Leu, or Lys at Y266 also generated signaling-defective receptors. In contrast, Phe or Trp substitution at Y266 retained receptor function, suggesting that aromaticity at this position was critical. When coexpressed with WT receptor, the Y266A receptor exhibited a strong dominant-negative phenotype, indicating that this mutant bound G protein. A partial tryptic digest revealed that, in the presence of agonist, a different digestion profile for Y266A receptor was generated in comparison to that for WT receptor. The difference in trypsin-sensitive sites and their negative dominance indicated that the Y266A receptor was not able to switch into an "activated" conformation upon ligand binding. In comparison to WT Ste2p, the mutantY266A receptor showed increased binding affinity for N-terminal, alanine-substituted alpha-factor analogues (residues 1-4) and the antagonist [desW(1),desH(2)]alpha-factor. A substantial decrease in affinity was observed for alpha-factor analogues with Ala substitutions from residues 5-13. The results suggest that Y266 is part of the binding pocket that recognizes the N-terminal portion of alpha-factor and is involved in the transformation of Ste2p into an activated state upon agonist binding.
Lee
Byung-Kwon
BK
Department of Microbiology, University of Tennessee, Knoxville, Tennessee 37996, USA.
Lee
Yong-Hun
YH
Hauser
Melinda
M
Son
Cagdas D
CD
Khare
Sanjay
S
Naider
Fred
F
Becker
Jeffrey M
JM
D001665:Binding Sites; D001667:Binding, Competitive; D000072235:Mating Factor; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*
D010455:Peptides; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D014157:Transcription Factors; D014443:Tyrosine; D000072235:Mating Factor