Direkt zum Inhalt
Merck
  • Applications of yeast-based signaling sensor for characterization of antagonist and analysis of site-directed mutants of the human serotonin 1A receptor.

Applications of yeast-based signaling sensor for characterization of antagonist and analysis of site-directed mutants of the human serotonin 1A receptor.

Biotechnology and bioengineering (2015-04-09)
Yasuyuki Nakamura, Jun Ishii, Akihiko Kondo
ZUSAMMENFASSUNG

The monoamine neurotransmitter serotonin (5-HT) regulates a wide spectrum of human physiology through the 5-HT receptor family. One such receptor, the 5-HT1A receptor (HTR1A), is the most widely studied subtype and represents a significant molecular target in medicinal and therapeutic fields. Yeast-based fluorescent reporter systems have proven to be especially useful for GPCR assays, since detection using a fluorescent reporter considerably simplifies measurement procedures. However, previously reported systems using enhanced green fluorescent protein (EGFP) as the reporter in yeast still showed low signal-to-noise (S/N) ratios, making EGFP difficult to apply as an easily accessible tool. Therefore, we constructed a refined yeast-based GPCR biosensor employing a high-sensitivity strain that incorporated both a Gα-engineered receptor and a fluorescent reporter (ZsGreen). As we report here, the refined yeast-based fluorescent biosensor was applied successfully to antagonist characterization and analysis of site-directed mutants of the HTR1A receptor. Pindolol, a known antagonist of HTR1A, specifically inhibited agonist-induced signaling, demonstrating the ease of evaluating inhibition effects using our reporter strain. Characterization of site-specific receptor mutants confirmed the role of specific targeted residues, including the highly conserved DRY motif, in the activation of HTR1A. Thus, our refined yeast biosensor strain, which incorporates a ZsGreen reporter and an engineered Gα receptor, is expected to serve as a simple and practical sensing tool for evaluating the ligand candidates and defining residues important to the function of human GPCRs. Biotechnol.

MATERIALIEN
Produktnummer
Marke
Produktbeschreibung

Sigma-Aldrich
Natriumchlorid, for molecular biology, DNase, RNase, and protease, none detected, ≥99% (titration)
Sigma-Aldrich
Natriumchlorid -Lösung, 5 M in H2O, BioReagent, for molecular biology, suitable for cell culture
Sigma-Aldrich
Natriumchlorid -Lösung, 0.9% in water, BioXtra, suitable for cell culture
Sigma-Aldrich
Natriumchlorid, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99%
Sigma-Aldrich
Natriumchlorid, BioXtra, ≥99.5% (AT)
SAFC
Natriumchlorid -Lösung, 5 M
Sigma-Aldrich
Natriumchlorid -Lösung, BioUltra, for molecular biology, ~5 M in H2O
Sigma-Aldrich
Natriumchlorid, BioUltra, for molecular biology, ≥99.5% (AT)
Sigma-Aldrich
Natriumchlorid -Lösung, 5 M
Sigma-Aldrich
Natriumchlorid, 99.999% trace metals basis
Sigma-Aldrich
Natriumchlorid, meets analytical specification of Ph. Eur., BP, USP, 99.0-100.5%
Sigma-Aldrich
L-(−)-Glukose, ≥99%
Sigma-Aldrich
Serotonin creatinine sulfate monohydrate, powder
Sigma-Aldrich
Natriumchlorid, BioPerformance Certified, ≥99% (titration), suitable for insect cell culture, suitable for plant cell culture
Sigma-Aldrich
Natriumchlorid, AnhydroBeads, −10 mesh, 99.999% trace metals basis
Sigma-Aldrich
Natriumchlorid -Lösung, 0.85%
Sigma-Aldrich
Natriumchlorid-35Cl, 99 atom % 35Cl
Sigma-Aldrich
MOPSO, ≥99% (titration)
Sigma-Aldrich
Natriumchlorid, random crystals, optical grade, 99.9% trace metals basis
Sigma-Aldrich
Pindolol, ≥98% (TLC), powder
Sigma-Aldrich
Natriumchlorid, tablet