Mutations affecting ligand specificity of the G-protein-coupled receptor for the Saccharomyces cerevisiae tridecapeptide pheromone.
Random mutations were generated in the G-protein-coupled receptor (Ste2p) for the tridecapeptide pheromone (alpha-factor) of Saccharomyces cerevisiae. These mutants were screened for variants that responded to antagonists. Because multiple mutations were detected in each mutant receptor recovered from the screen, site-directed mutagenesis was used to create single-site mutant receptors. Three receptors containing mutations F55V, S219P, and S259P were analyzed for their biological responses to various alpha-factor analogs and for their ligand binding profiles. Cells expressing each of the mutant receptors responded to alpha-factor as well as or better than wild-type cells in a growth arrest assay. In contrast, the binding of alpha-factor to the F55V and S219P mutant receptors was at least 10-fold reduced in comparison to wild-type receptor indicating a complex non-linear correlation between binding affinity and biological activity. Cells expressing mutant receptors responded to some normally inactive analogs in biological assays, despite the fact that these analogs had a low affinity for Ste2p. The analysis of these mutant receptors confirms previous findings that the first and sixth transmembrane regions of Ste2p are important for ligand interaction, ligand specificity, and/or receptor activation to initiate the signal transduction pathway. Changes in binding affinity of pheromone analogs to wild-type and mutant receptors indicate that residue 55 of Ste2p is involved with both ligand binding and signal transduction.
Abel
M G
MG
Department of Microbiology, University of Tennessee, Knoxville, TN 37996, USA.
Lee
B K
BK
Naider
F
F
Becker
J M
JM
D001665:Binding Sites; D001667:Binding, Competitive; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000378:metabolism*; D000072235:Mating Factor; D016296:Mutagenesis; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D010675:Pheromones / Q000378:metabolism*; D011485:Protein Binding; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014157:Transcription Factors
D010455:Peptides; D010675:Pheromones; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D014157:Transcription Factors; D000072235:Mating Factor; D019204:GTP-Binding Proteins