オーファン代謝型受容体とそのスプライシングイソフォームの分泌蛋白の機能解析

今年我们的重点是鉴定富含脯氨酸的跨膜蛋白3(Prrt3)的结合蛋白，Prrt3是一种孤儿G蛋白偶联受体。在Fukata教授(NIPS)和NIPS共用转基因动物实验室的合作下，建立了表达表位标记Prrt3的转基因(TG)小鼠，以探索Prrt3结合蛋白。然而，当用LC-MS/MS对分离的Prrt3复合体进行分析时，我们观察到由于表位标记的Prrt3在TG小鼠脑中的高表达，所以我们观察到非特异的蛋白质结合。然后，我们改变了策略，使用与渡边教授(北海道大学)共同开发的Prrt3特异性抗体，从野生型(WT)小鼠脑中分离出生理性表达的Prrt3蛋白复合体。经LC-MS/MS分析，Gao蛋白和兴奋性氨基酸转运蛋白2(EAAT2)为Prrt3结合对。我们的发现也得到了蛋白质印迹实验的证实。我们先前在WT小鼠大脑中发现了Prrt3的截短形式，这表明Prrt3的N-末端结构域被从其跨膜结构域中以未知的机制切割出来。在Prrt3的N-末端区域中发现的保守氨基酸序列的点突变阻止了在HEK293细胞中表达的Furin的切割。这表明保守的位点可能在Prrt3的翻译后修饰中发挥重要作用。综上所述，我们的发现提示Prrt3可能通过与EAAT2在小鼠体内形成蛋白复合体来调节谷氨酸能信号转导。当被激活时，Prrt3最有可能传输G_&o；I/O；信令级联。这一发现在建立探索Prrt3激动剂的功能筛选试验时尤其有用。

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.