| 研究概要 |
This research project focuses on elucidating physiological functions of an orphan G-protein coupled receptor (GPCR)' called proline-rich transmembrane protein 3 (Prrt3). Our previous finding using genetically modified mice has indicated that Prrt3 is involved in memory functions. This year, we achieved to develop Prrt3 specific antibodies in collaboration with Prof. Watanabe (Hokkaido University). We utilized these antibodies to explored Prrt3 expression patterns in mice. Amongst the various body parts we examined by Westem blotting (WB), Prrt3 has been fbund only in mouse brain. The predicted molecular weight of Prrt3 is 101 kDa, yet Prrt3 was found in four different sizes (140,120,80 and 70 kDa) in brain, We speculate Prrt3 may undergo post-translational modifications and/or have protein binding partners. We further examined Prrt3 expression in mouse brain using a subcellular fractionation method. Prrt3 expression has been observed in synaptosome, but not in post synaptic density fraetions. This indicates Prrt3 is expressed in the close proximity to synapses, possibly at the extrasynaptic regions or presynaptic terminals. Our collaborator, Dr Konno (University of Hokkaido) has revealed the anatomical distribution of Prrt3 using fresh frozen section of mouse brain, High expression of Prrt3 has been observed in cortex, hippocampus, thalamus and substantia nigra.
Taken together, Prrt3 is abundantly expressed in brain, and it may have roles in modulating synaptic functions as it was fbund in the synaptosome fractions.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
1: 当初の計画以上に進展している
理由
We achieved to raise Prrt3 specific antibodies, and successfully determined the expression pattern of Prrt3. However, we could not make any progress on establishing functional assays for ligand screening to deorphanize Prrt3. Transgenic (TG) mice expressing epitope-tagged Prrt3 have been successfully generated to explore Prrt3 protein binding partners using LC-MS/MS.
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| 今後の研究の推進方策 |
As we planned, we will carry out LC-MS/MS analysis at Fukata lab in NIPS, to explore binding partners for Prrt3. The presence of cleaved forms of Prrt3 (70 and 80 kDa) indicates proteases are involved in Prrt3 functions. We are now considering proteases as candidates for Prrt3 activating agents, and planning to screen various proteases on Prrt3 expressed in HEK293.
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