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

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. Bioeng. 2015;112: 1906–1915. © 2015 Wiley Periodicals, Inc.