Cross-Linking of a DOPA-Containing Peptide Ligand into its G Protein-Coupled Receptor

Download

index.pdf

Date

2009-04-01

Author

Umanah, George E.
Son, Çağdaş Devrim
Ding, FaXiang
Naider, Fred
Becker, Jeffrey M.

Metadata

Show full item record

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

Item Usage Stats

199
views

96
downloads

The interaction between a 3,4-dihydroxylphenylalanine (DOPA) labeled analog of the tridecapeptide α-factor (W-H-W-L-Q-L-K-P-G-Q-P-M-Y) and Ste2p, a Saccharomyces cerevisiae model G protein-coupled receptor (GPCR), has been analyzed by periodate-mediated cross-linking. Chemically synthesized α-factor with DOPA substituting for tyrosine at position 13 and biotin tagged onto lysine7 ([Lys7 (BioACA),-Nle12,DOPA13]α-factor; Bio-DOPA-α-factor) was used for crosslinking into Ste2p. The biological activity of Bio-DOPA-α-factor was about one-third that of native α-factor as determined by growth arrest assay and exhibited about a ten-fold lower binding affinity to Ste2p. Bio-DOPA-α-factor cross-linked into Ste2p as demonstrated by western blot analysis using a Neutravidin-HRP conjugate to detect Bio-DOPA-α-factor. Cross-linking was inhibited by excess native α-factor and an α-factor antagonist. The Ste2p-ligand complex was purified using a metal ion affinity column, and after cyanogen bromide treatment, avidin affinity purification was used to capture Bio-DOPA-α-factor-Ste2p cross-linked peptides. MALDI-TOF spectrometric analyses of the cross-linked fragments showed that Bio-DOPA-α-factor reacted with the Phe55-Met69 region of Ste2p. Cross-linking of Bio-DOPA-α-factor was reduced by 80% using a cysteine-less Ste2p (Cys59Ser). These results suggest an interaction between position thirteen of α-factor and residue Cys59 of Ste2p. This study is the first to report DOPA cross-linking of a peptide hormone to a GPCR and the first to identify a residue-to-residue cross-link between Ste2p and α-factor thereby defining a specific contact point between the bound ligand and its receptor.

Subject Keywords

Biochemistry

URI

https://hdl.handle.net/11511/34294

Journal

FASEB JOURNAL

DOI

https://doi.org/10.1021/bi802061z

Collections

Department of Biology, Article

Suggestions

OpenMETU
Core

Sequences in the intracellular loops of the yeast pheromone receptor Ste2p required for G protein activation. Celić, A; Martin, NP; Son, Çağdaş Devrim; Becker, JM; Naider, F; Dumont, ME (American Chemical Society (ACS), 2003-03-18) The α-factor receptor of the yeast Saccharomyces cerevisiae encoded by the STE2 gene is a member of the large family of G protein-coupled receptors (GPCRs) that mediate multiple signal transduction pathways. The third intracellular loop of GPCRs has been identified as a likely site of interaction with G proteins. To determine the extent of allowed substitutions within this loop, we subjected a stretch of 21 amino acids (Leu228−Leu248) to intensive random mutagenesis and screened multiply substituted alleles...
Unnatural amino acid replacement in a yeast G protein-coupled receptor in its native environment. Huang, LY; Umanah, G; Hauser, M; Son, Çağdaş Devrim; Arshava, B; Naider, F; Becker, JM (American Chemical Society (ACS), 2008-05-20) Ste2p is the G protein-coupled receptor (GPCR) for the tridecapeptide pheromone cc factor of Saccharomyces cerevisiae. This receptor- pheromone pair has been used extensively as a paradigm for investigating GPCR structure and function. Expression in yeast harboring a cognate tRNA/aminoacyl-tRNA synthetase pair specifically evolved to incorporate p-benzoyl-L-phenylalanine (Bpa) in response to the amber codon allowed the biosynthesis of Bpa-substituted Ste2p in its native cell. We replaced natural amino acid ...
Isolation and immunological characterization of theta class glutathione-s-transferase gstt2-2 from bovine liver İşgör, Sultan Belgin; Çoruh, Nursen; Department of Biochemistry (2004) The glutathione-S-transferases (GSTs) (EC.2.5.1.18) are enzymes that participate in cellular detoxification of endogenous as well as foreign electrophilic compounds, function in the cellular detoxification systems and are evolved to protect cells against reactive oxygen metabolites by conjugating the reactive molecules to the nucleophile scavenging tripeptide glutathione (GSH, ?-glu-cys-gly). The GSTs are found in all eukaryotes and prokaryotic systems, in the cytoplasm, on the microsomes, and in the mitoch...
Interactions of cholesterol reducing agent simvastatin with phospholipid model membranes Koçak, Mustafa; Severcan, Feride; Department of Biochemistry (2007) Interactions of simvastatin with zwitterionic dipalmitoyl phosphotidylcholine (DPPC) multilamellar liposomes were investigated as a function of temperature and simvastatin concentration. And acyl chain length effect on the simvastatin-model membrane interactions was monitored with DPPC and dimyristoyl phosphotidylcholine (DMPC) lipids. All studies were carried out by two non-invasive techniques, namely Fourier transform infrared (FTIR) spectroscopy, and differential scanning calorimetry (DSC). The results s...
Investigation for natural extract inhibitors of bovine lens aldose reductase responsible for the formation of diabetis dependent cataract Onay, Melih; Çoruh, Nursen; Department of Biochemistry (2008) In the polyol pathway, Aldose reductase (AR) is an important enzyme in reduction of aldehydes and aldosugars to their suitable alcohols. AR, using NADPH as a coenzyme, has a molecular weight of 37 000 dalton. AR in its activated form, known to increase the sorbitol accumulation in lens, is responsible for the cataract formation in diabetis diseases. Therefore, the inhibition of aldose reductase is important to prevent the incedence of cataract formation in diabetus mellitus. In the treatment of diabetis dep...

Citation Formats

G. E. Umanah, Ç. D. Son, F. Ding, F. Naider, and J. M. Becker, “Cross-Linking of a DOPA-Containing Peptide Ligand into its G Protein-Coupled Receptor,” FASEB JOURNAL, pp. 0–0, 2009, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/34294.