• Top page
  • Timetable
  • Per session
  • Per presentation
  • How to
  • Meeting Planner



Molecular, Biochemical, and Genetic Techniques

開催日 2014/9/11
時間 16:00 - 17:00
会場 Poster / Exhibition(Event Hall B)

Development of Tango system for the monitoring of 5-HT2CR activity

  • P1-382
  • 渡邊 義久 / Yoshihisa Watanabe:1 辻村 敦 / Atsushi Tsujimura:1 青木 美空 / Miku Aoki:1,2 田口 勝敏 / Katsutoshi Taguchi:1 田中 雅樹 / Masaki Tanaka:1 
  • 1:京都府立医科大学大学院 / Kyoto Pref Univ of Med, Kyoto, Japan 2:京都府立医科大学大学院 歯科口腔科学 / Dept of Dental Med, Kyoto Pref Univ of Med, Kyoto, Japan 

5-HT2CR is a member of the seven transmembrane-spanning G-protein coupled receptors (GPCRs) and couples to Gq/11 that subsequently activates phospholipase C. It is widely distributed in the central nervous system and is involved in the regulation of anxiety, mood, and feeding. 5-HT2CR is known to undergo RNA-editing at five sites of second intracellular loop by adenosine deaminases acting on RNA (ADARs). 5-HT2CR RNA-editing leads to decrease in 5-HT potency, agonist binding affinity, constitutive activities, and G protein coupling activity. To date, the measurement of 5-HT2CR activity has been performed by G-protein dependent functional assays, such as inositol-1,4,5-trisphosphate (IP3) production assay and calcium flux assay. Although these assays are excellent methods, they are not appropriate for high-throughput drug screening (HTS) and application to in vivo live-imaging. Now, we are developing a novel method of 5-HT2CR monitoring to solve these problems. Tango system is a powerful tool for GPCRs assays based on ligand binding to a specific GPCR that triggers desensitization, a process mediated by the recruitment of arrestin to the activated receptor. For validation of 5-HT2CR Tango system, we constructed the following plasmids: pEF1_5HT2CR-TEVc-LexA-2A-arrestin-TEVp and pLexOP-EGFP. A cleavage site (TEVc) fused to the C-terminus of 5-HT2CR is cleaved by the TEV protease-tagged arrestin (arrestin-TEVp), resulting in the release of LexA transcriptional factor. Released LexA is translocated to the nucleus and induces EGFP-expression driven by the LexA promoter. These two plasmids were transfected into 293 cells, and EGFP expression was monitored with a fluorometer. We verified that 5-HT induced the expression of reporter gene in a dose-dependent manner and that its expression was inhibited by the 5-HT2CR antagonist SB242084. Moreover, this system was able to detect the difference of 5-HT2CR activities among RNA-editing isoforms (VGV and INI). These results suggest that our system will be useful for live-imaging of 5-HT2CR activity and HTS for drug discovery.

Copyright © Neuroscience2014. All Right Reserved.