Tyr266 in the sixth transmembrane domain of the yeast alpha-factor receptor plays key roles in receptor activation and ligand specificity.

Date

2002-11-19

Author

Lee, BK
Lee, YH
Hauser, M
Son, Çağdaş Devrim
Khare, S
Naider, F
Becker, JM

Metadata

Show full item record

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Item Usage Stats

101
views

0
downloads

To identify interactions between Ste2p, a G protein-coupled receptor of the yeast Saccharomyces cerevisiae, and its tridecapepticle 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 cc-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 a-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.

Subject Keywords

Protein-coupled receptor, Cerevisiae tridecapeptide pheromone, Dominant-negative mutations, Saccharomyces-cerevisiae, Mating pheromone, Aromatic residues, Biological-activity, Peptide analogs, C-terminus, Ste2 gene

URI

https://hdl.handle.net/11511/42437

Journal

Biochemistry

DOI

https://doi.org/10.1021/bi026100u

Collections

Department of Biology, Article

Suggestions

OpenMETU
Core

Analysis of motifs in microRNA-transcription factor gene regulatory networks Sürün, Bilge; Acar, Aybar Can; Purutçuoğlu Gazi, Vilda; Department of Bioinformatics (2014) MicroRNAs are small non-coding RNA molecules which contain 21-25 nucleotides, and function in post transcriptional regulation by inhibiting the translation of mRNA targets. miRNAs typically affect gene regulation by forming composite feed forward circuits (cFFCs) which also comprise a transcription factor (TF) and a target gene. By analyzing these cFFCs, the contribution of miRNAs in altering TF networks can be revealed. These contributions could either be the de-escalation of the target gene repertoire or ...
Cloning and expression of periplasmic (clp p-like) and membrane-bound serine protease genes of thermoplasma volcanium in escherichia coli Demirok, Burçak; Kocabıyık, Semra; Department of Biology (2006) Serine proteases are a family of proteases that utilize an activated serine residue in the substrate-binding pocket to catalytically hydrolyze peptide bonds. Enzymes which belong to this family, with a diverse array of metabolic and regulatory functions, play critical roles in cell physiology and pathology. ءClp̕s are a class of ATP dependent serine proteases which are composed of a protease (ClpP) and an ATPase (ClpA or ClpX) component. Their involvements in degrading proteins are especially implicated und...
Transcription termination and RNA degradation contribute to silencing of RNA polymerase II transcription within heterochromatin. Vasiljeva, L; Kim, M; Terzi Çizmecioğlu, Nihal; Soares, LM; Buratowski, S (Elsevier BV, 2008-02-15) Within the heterochromatin of budding yeast, RNA polymerase II (RNAPII) transcription is repressed by the Sir2 deacetylase. Although heterochromatic silencing is generally thought to be due to limited accessibility of the underlying DNA, there are several reports of RNAPII and basal transcription factors within silenced regions. Analysis of the rDNA array revealed cryptic RNAPII transcription within the "non-transcribed" spacer region. These transcripts are terminated by the Nrd1 /Sen1 complex and degraded ...
Micelle Assisted Thin-Film Solid Phase Microextraction: A New Approach for Determination of Quaternary Ammonium Compounds in Environmental Samples Boyacı, Ezel (2014-09-16) Determination of quaternary ammonium compounds (QACs) often is considered to be a challenging undertaking owing to secondary interactions of the analytes permanently charged quaternary ammonium head or hydrophobic tail with the utilized labware. Here, for the first time, a micelle assisted thin-film solid phase microextraction (TF-SPME) using a zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) as a matrix modifier is introduced as a novel approach for in-laboratory sam...
Speciation of Selenium in Supplements by High Performance Liquid Chromatography-Inductively Coupled Plasma-Mass Spectrometry BAKIRDERE, Sezgin; Volkan, Mürvet; Ataman, Osman Yavuz (2015-06-13) Anion and cation exchange high-performance liquid chromatography (HPLC) combined with inductively coupled plasma-mass spectrometry (ICP-MS) were used for speciation of selenium in supplements. All the parameters in the extraction, separation, and determination procedures were optimized. Recovery studies for the selenium species from the anion and cation exchange columns were performed and there were no analyte losses. Limits of detection for selenium(IV), selenium(VI), Se in selenomethionine, and Se in sele...

Citation Formats

B. Lee et al., “Tyr266 in the sixth transmembrane domain of the yeast alpha-factor receptor plays key roles in receptor activation and ligand specificity.,” Biochemistry, pp. 13681–9, 2002, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/42437.