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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

孤儿代谢受体分泌蛋白及其剪接亚型的功能分析

基本信息

批准号：
13J00388
负责人：
山本泉
金额：
$ 1.77万
依托单位：
National Institute for Physiological Sciences
依托单位国家：
日本
项目类别：
Grant-in-Aid for JSPS Fellows
财政年份：
2013
资助国家：
日本
起止时间：
2013 至 2014
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13J00388/
关键词：
Orphan GPCR Prrt3 Protease LC-MS/MS EAAT2

项目摘要

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.

今年，我们重点鉴定富含脯氨酸的跨膜蛋白 3 (Prrt3) 的结合蛋白，这是一种孤儿 G 蛋白偶联受体。与 Fukata 教授 (NIPS) 和 NIPS 中的共享转基因动物设施合作，产生了表达表位标记的 Prrt3 的转基因 (TG) 小鼠，以探索 Prrt3 结合蛋白。然而，当使用液相色谱串联质谱 (LC-MS/MS) 分析分离的 Prrt3 复合物时，我们观察到由于 TG 小鼠大脑中表位标记的 Prrt3 高表达而导致非特异性蛋白质结合。然后，我们改变了策略，使用与 Watanabe 教授（北海道大学）合作开发的 Prrt3 特异性抗体，从野生型 (WT) 小鼠大脑中分离出生理表达的 Prrt3 蛋白复合物。通过 LC-MS/MS 分析分离的蛋白质复合物，Gao 蛋白和兴奋性氨基酸转运蛋白 2 (EAAT2) 被鉴定为 Prrt3 结合伴侣。我们的发现也得到了蛋白质印迹实验的证实。我们之前在 WT 小鼠大脑中发现了 Prrt3 的截短形式，这表明 Prrt3 的 N 末端结构域通过未知机制从其跨膜结构域中裂解出来。 Prrt3 N 端结构域中保守氨基酸序列的点突变可防止在 HEK293 细胞中表达时被弗林蛋白酶切割。这表明保守位点可能在 Prrt3 的翻译后修饰中发挥重要作用。总之，我们的研究结果表明 Prrt3 可能通过与小鼠中的 EAAT2 形成蛋白复合物来调节谷氨酸信号传导。 Prrt3 在激活时最有可能传输 G_<i/o> 信令级联。这一发现在建立功能筛选试验来探索 Prrt3 激动剂时尤其有用。