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Development of the 5-HT2CR-Tango System Combined with an EGFP Reporter Gene
Yoshihisa Watanabe1, Atsushi Tsujimura1, Miku Aoki1,2
1Department of Basic Geriatrics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto, 602-8566, Japan.
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.
Insights
Researchers developed a novel 5-HT2CR-Tango assay to measure serotonin 2C receptor (5-HT2CR) activity. This system enables high-throughput drug screening and future in vivo brain studies for neurological and metabolic disorders.
Area of Science:
- Neuroscience
- Pharmacology
- Molecular Biology
Background:
- The serotonin 2C receptor (5-HT2CR) is a G-protein-coupled receptor crucial for emotion, feeding, reward, and cognition.
- Dysregulation of 5-HT2CR is linked to mental disorders, metabolic abnormalities, and reward system dysfunction.
- Existing methods for measuring 5-HT2CR activity are not suitable for in vivo applications.
Purpose of the Study:
- To develop a novel assay system for measuring 5-HT2CR activity.
- To create a tool applicable for both in vitro drug screening and future in vivo studies.
- To validate the new assay system using different 5-HT2CR isoforms and an antagonist.
Main Methods:
- Developed the 5-HT2CR-Tango assay based on G-protein-coupled receptor (GPCR)-arrestin interaction.
- Utilized a LexA transcriptional activation system to convert the GPCR-arrestin interaction into an EGFP reporter signal.
- Validated the assay in HEK293 cells by measuring activities of 5-HT2CR RNA-editing isoforms (INI and VGV) and the effect of SB242084.
Main Results:
- The 5-HT2CR-Tango assay successfully detected differences in basal and stimulated activity between 5-HT2CR INI and VGV isoforms.
- The INI isoform exhibited higher basal and 5-HT-stimulated activity compared to the VGV isoform.
- The assay system detected the inhibitory effect of the 5-HT2CR antagonist SB242084.
Conclusions:
- The 5-HT2CR-Tango assay is a novel and effective tool for measuring 5-HT2CR activity.
- This assay system is suitable for in vitro high-throughput drug screening targeting 5-HT2CR.
- The developed assay holds potential for future in vivo studies of brain function related to 5-HT2CRs in animal models.
