title: Applications of yeast-based signaling sensor for characterization of antagonist and analysis of site-directed mutants of the human serotonin 1A receptor.
abstract: 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.
authors: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan.
authors: Yasuyuki
authors: Y
authors: Nakamura
authors: Organization of Advanced Science and Technology, Kobe University, Kobe, Japan.
authors: Jun
authors: J
authors: Ishii
authors: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan. akondo@kobe-u.ac.jp.
authors: Akihiko
authors: A
authors: Kondo
mesh_terms: D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D010957:Plasmids; D044282:Receptor, Serotonin, 5-HT1A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D058829:Serotonin 5-HT1 Receptor Antagonists / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D058829:Serotonin 5-HT1 Receptor Antagonists; C485184:enhanced green fluorescent protein; D044282:Receptor, Serotonin, 5-HT1A; D049452:Green Fluorescent Proteins
keywords: G-protein signaling; G-protein-coupled receptor; Saccharomyces cerevisiae; green fluorescent protein; mutagenesis; serotonin 1A receptor