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Development of the 5-HT2CR-Tango System Combined with an EGFP Reporter Gene

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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 Ca2+ 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.

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Acknowledgments

We thank Dr. David J. Anderson (California Institute of Technology) for providing the Valium-TEVcs-LexA-HAtag-2A-arrestin1-TEVp plasmid vector. This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (Nos. 25290014 and 25640024 to MT).

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Authors and Affiliations

  1. Department of Basic Geriatrics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto, 602-8566, Japan

    Yoshihisa Watanabe, Atsushi Tsujimura, Miku Aoki, Katsutoshi Taguchi & Masaki Tanaka

  2. Department of Dental Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japan

    Miku Aoki

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  1. Yoshihisa Watanabe
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  2. Atsushi Tsujimura
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  3. Miku Aoki
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  4. Katsutoshi Taguchi
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  5. Masaki Tanaka
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Corresponding author

Correspondence to Masaki Tanaka.

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Fig. S1

SB242084 had no effect on cell viability and the direct inhibition of the reporter gene expression. A. HEK293 cells stably transfected with reporter plasmid were cultured with 0–20 μg/mL SB242084 for 24 h, followed by a cell viability assay. Values (%) are shown relative to SB242084-untreated cells and are represented by mean ± S.D. Statistical analysis was performed with one-way ANOVA (post hoc Tukey’s test). The experiment was repeated five times. B. The reporter gene expression of these cells was measured. Values (%) are shown relative to SB242084-untreated cells and are represented by mean ± S.D. Statistical analysis was performed with one-way ANOVA (post hoc Tukey’s test). The experiment was repeated five times. (PPTX 57 kb)

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Watanabe, Y., Tsujimura, A., Aoki, M. et al. Development of the 5-HT2CR-Tango System Combined with an EGFP Reporter Gene. J Mol Neurosci 58, 162–169 (2016). https://doi.org/10.1007/s12031-015-0650-2

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  • DOI: https://doi.org/10.1007/s12031-015-0650-2

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