2016 Fiscal Year Research-status Report
The physiological ligand and activation mechanisms of an orphan metabotropic receptor Prrt3
| Project/Area Number |
16K18999
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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) |
2016-04-01 – 2018-03-31
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| Keywords | Receptor |
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| Outline of Annual Research Achievements |
Prrt3 is an orphan metabotropic receptor of family C GPCRs. There is no publication concerning Prrt3 and the physiological roles and functioning mechanisms of Prrt3 are totally unknown. Here we aim to clarify the physiological ligand and activation mechanisms of Prrt3. We have performed molecular biological, electrophysiological and intracellular calcium imaging experiments using Xenopus oocytes and HEK293 cells to elucidate the following questions:
1. Which classes of Gα are coupled with Prrt3? Gi/o or Gq?
2. What is the activated form of Prrt3? The cleaved form or the full-length form?
3. What is the physiological ligand of Prrt3?
We have clarified some of these questions, and the results provide us with information about the roles and activation mechanisms of Prrt3.
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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 clarify the physiological ligand and activation mechanisms of Prrt3, we have performed experiments as follows:
1. We have identified that Prrt3 couples with Gi/o and does not couple with Gq by electrophysiological recording in oocytes and intracellular calcium imaging experiment in HEK293 cells.
2. We have identified that the cleaved form of Prrt3 is not constitutively active by comparing the activity of the full-length form and the cleaved form of Prrt3 which is generated by co-expression of furin or truncated by mutagenesis.
3. We have screened over 300 of small molecules in the ligand libraries which contain over 1000 compounds. Until now, we have observed that some of cholinergic receptor agonists slightly activate Prrt3. However, these partial activators of Prrt3 are not physiological ligands.
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| Strategy for Future Research Activity |
To identify the physiological ligand of Prrt3, we plan to perform experiments as follows:
1. Screen the remaining 700 compounds in the small-molecules library.
2. If we could not obtain the ligand that activates Prrt3 in the screening of small-molecules library, there is a possibility that the physiological ligand of Prrt3 may be an unidentified molecule. We will collect the cerebrospinal fluid (CSF) from the brain of anesthetized rats and examine the effect on Prrt3 activation. If rat CSF can activate Prrt3-mediated G-protein signaling, we will identify the candidate components in CSF by LC-MS analysis.
3. Identify the ligand-binding sites by mutations of candidate amino acids in the N-terminal extracellular domain of Prrt3 using mutagenesis kit.
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| Causes of Carryover |
We originally plan to purchase small molecule library for ligand screening of Prrt3. Fortunately, some of compounds in small molecule library were kindly provided by Dr. Uesugi (Kyoto University). Therefore, we costed fewer expenses for purchasing chemicals for ligand screening of Prrt3 than the original predication.
On the other hand, there are no costs for domestic and overseas travel expenses in 2016.
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| Expenditure Plan for Carryover Budget |
We will focus on the identification of Prrt3 ligand in 2017. We plan to continue the screening of small molecule library and also investigate the effect of the composition of the rat cerebrospinal fluid. Therefore, we will use Wistar rats in the experiments in 2017.
We also plan to present our research result in 2017 that will costs domestic and overseas travel expenses.
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