Identification of a contact region between the tridecapeptide alpha-factor mating pheromone of Saccharomyces cerevisiae and its G protein-coupled receptor by photoaffinity labeling.

Date

2002-05-14

Author

Henry, Lk
Khare, S
Son, Çağdaş Devrim
Babu, Vv
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

179
views

0
downloads

Saccharomyces cerevisiae haploid cells communicate with their opposite mating type through peptide pheromones (alpha-factor and a-factor) that activate G protein-coupled receptors (GPCRs). S. cerevisiae was used as a model system for the study of peptide-responsive GPCRs. Here, we detail the synthesis and characterization of a number of a-factor (Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr) pheromone analogues containing the photo-cross-linkable group 4-benzoyl-L-phenylalanine (Bpa). Following characterization, one analogue, [Bpa(1), Tyr(3), Arg(7), Phe(13)]alpha-factor, was radioiodinated and used as a probe for Ste2p, the GPCR for alpha-factor. Binding of the di-iodinated probe was saturable (K-d = 200 nM) and competable by alpha-factor. Cross-linking into Ste2p was specific for this receptor and reversed by the wildtype pheromone. Chemical and enzymatic cleavage of the receptor/radioprobe complex indicated that cross-linking occurred on a portion of Ste2p spanning residues 251-294 which encompasses transmembrane domain 6, the extracellular loop between transmembrane domains 6 and 7, and transmembrane domain 7. This fragment was verified using T7-epitope-tagged Ste2p and a biotinylated, photoactivatable alpha-factor. After cross-linking with the biotinylated photoprobe and trypsin cleavage, the cross-linked receptor fragment was revealed by both an anti T7-epitope antibody and a biotin probe. This is the first determination of a specific contact region between a Class IV GPCR and its ligand. The results demonstrate that Bpa alpha-factor probes are useful in determining contacts between alpha-factor and Ste2p and initiate mapping of the ligand binding site of this GPCR.

Subject Keywords

Analogs, Binding, Ligand, Expression, Sites

URI

https://hdl.handle.net/11511/34531

Journal

Biochemistry

DOI

https://doi.org/10.1021/bi015863z

Collections

Department of Biology, Article

Suggestions

OpenMETU
Core

Detecting g-protein coupled receptor interactions using enhanced green fluorescent protein reassembly Kumaş, Gözde; Son, Çağdaş Devrim; Yanık, Tülin; Department of Biotechnology (2012) The largest class of cell surface receptors in mammalian genomes is the superfamily of G protein-coupled receptors (GPCRs) which are activated by a wide range of extracellular responses such as hormones, pheromones, odorants, and neurotransmitters. Drugs which have therapeutic effects on a wide range of diseases are act on GPCRs. In contrast to traditional idea, it is recently getting accepted that G-protein coupled receptors can form homo- and hetero-dimers and this interaction could have important role on...
Characterization and analysis of the antioxidant capacity of functional phenolics oxidized by Scytalidium thermophilum catalase phenol oxidase (CATPO) Söyler, Ulviye Betül; Ögel, Zümrüt Begüm; Şensoy, İlkay; Department of Food Engineering (2012) Scytalidium thermophilum is a termophilic fungus that effectively produces the extracellular enzyme catalase phenol oxidase (CATPO). The enzyme is distinct among catalases with its bifunctionality of oxidising phenolic compounds in the absence of H2O2. CATPO is capable of oxidizing catechol, chlorogenic acid, caffeic acid and catechin which are ortho –diphenolic compounds. Diphenolic compounds are known as strong antioxidants. Catalase is one of the important antioxidant enzymes. Therefore, in this thesis t...
Identification of interaction sites of G protein-coupled receptors using machine learning techniques Şahin, Mehmet Emre; Can, Tolga; Department of Computer Engineering (2014) G protein-coupled receptors (GPCRs), which play a crucial role in a host of pathophysiological pathways, form the largest and most divergent receptor family. Typically, they transmit outer signals to the inner cell by interacting with G-proteins. The emerging concept of GPCR dimerization has unsettled the classical idea that GPCRs function as monomeric units. Prediction of the interface residues of GPCR dimers is a challenging topic. The method proposed in this thesis trains itself with known interfaces fro...
Detection of physically interacting proteins with the CC and NB-ARC domains of a putative yellow rust resistance protein, Yr10, in wheat Yildirim-Ersoy, Figen; Ridout, Christopher J.; Akkaya, Mahinur (Springer Science and Business Media LLC, 2011-08-01) Gene-for-gene (GFG) resistance is a potent defense mechanism in plants, that is mediated by resistance (R) proteins. In GFG resistance, pathogen effector or avirulence (Avr) proteins are recognised by R-proteins which initiate a series of signal transduction events that lead to hypersensitive cell death. In cereals, many R-proteins are comprised of an N-terminal coiled-coiled (CC) domain, a Nucleotide Binding (NB) domain and a Leucine Rich Repeats (LRR) region associated with effector recognition. NB-LRR im...
Characterization of extracellular beta-lactamases from penicillin G-resistant cells of Streptococcus thermophilus Chirica, LC; Güray, Nülüfer Tülün; Gültekin, Güzin Candan; Bozoglu, F (International Association for Food Protection, 1998-07-01) In this study, biochemical properties of two extracellular beta-lactamases produced by penicillin-resistant Streptococcus thermophilus cells were investigated. Both beta-lactamases showed specificity for penicillins but not for cephaloridins. The p-lactamases exhibited different affinities for penicillin G. The one with the higher molecular weight (F1) had a K(m) value of 3.44 mu M and a V(max), value of 8.33, mu mol/min/mg of protein, whereas the beta-lactamase with the lower molecular weight (FII) had a K...

Citation Formats

L. Henry, S. Khare, Ç. D. Son, V. Babu, F. Naider, and J. Becker, “Identification of a contact region between the tridecapeptide alpha-factor mating pheromone of Saccharomyces cerevisiae and its G protein-coupled receptor by photoaffinity labeling.,” Biochemistry, pp. 6128–39, 2002, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/34531.