| 研究実績の概要 |
This year we focused on identifying the binding proteins for proline rich transmembrane protein 3 (Prrt3), which is an orphan G-protein coupled receptor. In collaboration with Prof. Fukata (NIPS) and the share-use transgenic animal facility in NIPS, transgenic (TG) mice expressing epitope-tagged Prrt3 were generated to explore the Prrt3 binding proteins. However, when the isolated Prrt3 complexes were analyzed using the liquid-chromatography tandem mass spectrometry (LC-MS/MS), we observed non-specific protein bindings due to the high expression of epitope-tagged Prrt3 in the TG mouse brain. We then changed our strategy and used our Prrt3 specific antibodies developed with Prof. Watanabe (Hokkaido University) to isolate physiologically expressed Prrt3 protein complexes from wild-type (WT) mouse brain. The isolated protein complexes were analyzed by the LC-MS/MS, and Gao protein and excitatory amino acid transporter 2 (EAAT2) were identified as Prrt3 binding partners. Our finding was also confirmed by western blot experiment. We previously identified truncated forms of Prrt3 in WT mouse brain, which suggested that the N-terminal domain of Prrt3 was cleaved from its transmembrane domain by unknown mechanism. Point mutations at the conserved amino acid sequence found in the N-terminal domain of Prrt3 prevented cleavage by furin when expressed in HEK293 cells. This suggests the conserved site may play an important role in the post-translational modification of Prrt3. In summary, our findings suggest Prrt3 may have a possible role in regulating glutamatergic signaling by forming a protein complex with EAAT2 in mouse. Prrt3 is most likely to transmit G_<i/o> signaling cascade when activated. This finding is useful especially when establishing a functional screening assay to explore Prrt3 agonist.
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| 今後の研究の推進方策 |
The identification of G-protein subtype which couples with Prrt3 is important and necessary information when establishing functional screening assay to deorphanize Prrt3. Activation of GPCRs which couple to Gao protein stimulates dissociation of βγ dimer which activates G-protein gated inwardly rectifying K^+ (GIRK) channels. For the ligand screening assay, Prrt3 will be expressed with GIRK1/2 in Xenopus oocyte and Prrt3 activation will be monitored by measuring K^+ current using the two-electrode voltage clamp. Various physiologically relevant small molecules (e.g. amino-acids etc...), and molecules which are known to regulate glutamatergic neurons in mouse thalamus and hippocampus are Prrt3 agonist candidates.
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