title: Development of the 5-HT2CR-Tango System Combined with an EGFP Reporter Gene.
abstract: The serotonin 2C receptor (5-HT2CR) is a G-protein-coupled receptor implicated in emotion, feeding, reward, and cognition. 5-HT2CRs are pharmacological targets for mental disorders and metabolic and reward system abnormalities, as alterations in 5-HT2CR expression, RNA editing, and SNPs are involved in these disturbances. To date, 5-HT2CR activity has mainly been measured by quantifying inositol phosphate production and intracellular Ca(2+) release, but these assays are not suitable for in vivo analysis. Here, we developed a 5-HT2CR-Tango assay system, a novel analysis tool of 5-HT2CR activity based on the G-protein-coupled receptor (GPCR)-arrestin interaction. With desensitization of activated 5-HT2CR by arrestin, this system converts the 5-HT2CR-arrestin interaction into EGFP reporter gene signal via the LexA transcriptional activation system. For validation of our system, we measured activity of two 5-HT2CR RNA-editing isoforms (INI and VGV) in HEK293 cells transfected with EGFP reporter gene. The INI isoform displayed both higher basal- and 5-HT-stimulated activities than the VGV isoform. Moreover, an inhibitory effect of 5-HT2CR antagonist SB242084 was also detected by 5-HT2CR-Tango system. This novel tool is useful for in vitro high-throughput targeted 5-HT2CR drug screening and can be applied to future in vivo brain function studies associated with 5-HT2CRs in transgenic animal models.
authors.affiliation: Department of Basic Geriatrics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto, 602-8566, Japan.
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Watanabe
authors.affiliation: Department of Basic Geriatrics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto, 602-8566, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Tsujimura
authors.affiliation: Department of Basic Geriatrics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto, 602-8566, Japan.|Department of Dental Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japan.
authors.forename: Miku
authors.initials: M
authors.lastname: Aoki
authors.affiliation: Department of Basic Geriatrics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto, 602-8566, Japan.
authors.forename: Katsutoshi
authors.initials: K
authors.lastname: Taguchi
authors.affiliation: Department of Basic Geriatrics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto, 602-8566, Japan. mtanaka@koto.kpu-m.ac.jp.
authors.forename: Masaki
authors.initials: M
authors.lastname: Tanaka
mesh_terms: D000818:Animals; D019393:Arrestin / Q000235:genetics* / Q000378:metabolism; D019556:COS Cells; D002522:Chlorocebus aethiops; D055808:Drug Discovery / Q000379:methods; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D044405:Receptor, Serotonin, 5-HT2C / Q000235:genetics* / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000235:genetics* / Q000378:metabolism; D058830:Serotonin 5-HT2 Receptor Antagonists / Q000494:pharmacology*
chemical_list: D019393:Arrestin; D044405:Receptor, Serotonin, 5-HT2C; D043562:Receptors, G-Protein-Coupled; D058830:Serotonin 5-HT2 Receptor Antagonists; D049452:Green Fluorescent Proteins
keywords: 5-HT2CR; Arrestin; Drug screening; GPCR; Tango