2018 Fiscal Year Research-status Report
Behavioral analyses and ligand screening toward the identification of the physiological roles of an orphan metabotropic receptor Prrt3
| Project/Area Number |
18K15020
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| Research Institution | National Institute for Physiological Sciences |
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Principal Investigator |
陳 以珊 生理学研究所, 分子細胞生理研究領域, 特任助教 (40757770)
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| Project Period (FY) |
2018-04-01 – 2020-03-31
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| Keywords | GPCR |
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| Outline of Annual Research Achievements |
Prrt3 is an orphan metabotropic receptor of family C GPCR and there is no publication concerning Prrt3. We have observed that Prrt3 is highly expressed in mouse brain. Since the physiological roles of Prrt3 are still unknown, we aim to identify the endogenous ligands of Prrt3 and elucidate the roles of Prrt3 in the central nervous system (CNS). We have performed behavioral analyses using homozygous Prrt3 KO mice, and molecular biological, electrophysiological experiments using Xenopus oocytes to elucidate the following questions:
(1) What are the physiological roles of Prrt3 in the CNS?
(2) What are the endogenous ligands of Prrt3?
(3) Where are the ligand-binding sites?
In the course of screening of Prrt3 ligands, we happened to identify some antihistamine drugs directly inhibit GIRK channel.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
In order to identify the endogenous ligands of Prrt3 and elucidate the roles of Prrt3 in the CNS, we have performed experiments as follows:
(1) We have created homozygous Prrt3 KO mice and confirm the presence and absence of Prrt3 expression in those mice. We have collected sufficient number of Prrt3 KO mice and control mice for behavioral analyses. An overall course of behavioral experiments using those mice is now in progress.
(2) We have screened some compounds from a small-molecule library provided by Prof. Uesugi (Kyoto University) through monitoring GIRK current in oocytes expressing Prrt3 and GIRK1/GIRK2 channel by two-electrode voltage clamp. The ligand screening is still in progress.
(3) We happened to find that some compounds do not affect Prrt3 but inhibit GIRK channel itself.
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| Strategy for Future Research Activity |
To reach the aim of this research, we plan to perform experiments as follows:
(1) Complete the overall course of behavioral analyses using homozygous Prrt3 KO mice and analyze the results.
(2) Continue the ligand screening. If we did not find Prrt3 ligands from the present small-molecule library, we will buy other ligand libraries and screen these compounds at one time by Prrt3 and Gα16z44 transfected HEK293 cells, and then measure the effects of ligand libraries as the Gq-mediated [Ca2+]i increase using fura-2 AM by a fluorescence plate reader.
(3) If we find Prrt3 ligand from the step 2, we will identify the ligand-binding sites by comparing the ligand-stimulated response of full-length and extracellular domain-cleaved form of Prrt3 and mutations of candidate amino acid residues.
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| Causes of Carryover |
We originally plan to purchase some small molecule libraries and a fluorescence plate reader for ligand screening. Since we made most effort in the creation, collection and behavioral analyses of Prrt3 KO mice in 2018 and only performed the ligand screening of Prrt3 from the present small-molecule library partly, we plan to purchase other compounds and also the fluorescence plate reader in the next year. Therefore, we costed fewer expenses in 2018 than the original predication.
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