Analysis of endosome/post-endosome system with special reference to water channel aquaporin 2

特别参考水通道水通道蛋白 2 的内体/内体后系统分析

基本信息

批准号：
18390057
负责人：
TAKATA Kuniaki
金额：
$ 10.21万
依托单位：
Gunma University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2009
项目状态：
已结题

项目摘要

Vesicular trafficking that determines the number of cell surface molecules serves as one of important regulatory mechanisms found in the control of the reabsorption of water in the kidney, maintenance of blood glucose level, etc.Intracellular vesicles involved in the endocytosis and subsequent recycling process, such as endosomes and post-endosomal vesicles are key components in such vesicular trafficking and storage. We used water channel aquaporin 2 whose trafficking is regulated by hypophysial hormone vasopressin. Glucose transporter 4(GLUT4), whose trafficking is regulated by insulin in adipocytes and muscle cells, was coexpressed with aquaporin 2 in MDCK cells, and their localization was visualized by immunofluorescence and GFP-tagging methods. Aquaporin 2 and GLUT4 exhibited distinct localization pattern each other in both the resting and stimulated states, showing that aquaporin 2 and GLUT4 are stored in distinct post-endosomal compartments each other, and that their trafficking is differentially regulated. Translocation of aquaporin 2 has been considered to be regulated by its phosphorylation. We raised antibodies that specifically detect phosphorylated aquaporin 2. By western blotting and immunofluorescence microscopy, phosphorylation of aquaporin 2 was found not only on the cell surface but also intracellular vesicles, suggesting the ubiquitous presence of phosphorylated aquaporin 2.

决定细胞表面分子数量的囊泡运输是控制肾脏水重吸收、维持血糖水平等的重要调节机制之一。细胞内囊泡参与内吞作用和随后的再循环过程，例如因为内体和内体后囊泡是这种囊泡运输和储存的关键组成部分。我们使用水通道水通道蛋白 2，其运输受垂体激素加压素调节。葡萄糖转运蛋白 4 (GLUT4) 的运输受脂肪细胞和肌肉细胞中的胰岛素调节，在 MDCK 细胞中与水通道蛋白 2 共表达，并通过免疫荧光和 GFP 标记方法可视化其定位。水通道蛋白 2 和 GLUT4 在静息和刺激状态下均表现出彼此不同的定位模式，表明水通道蛋白 2 和 GLUT4 彼此储存在不同的内体后区室中，并且它们的运输受到不同的调节。水通道蛋白2的易位被认为是通过其磷酸化来调节的。我们制备了特异性检测磷酸化水通道蛋白 2 的抗体。通过蛋白质印迹和免疫荧光显微镜，发现水通道蛋白 2 的磷酸化不仅存在于细胞表面，而且还存在于细胞内囊泡中，表明磷酸化水通道蛋白 2 无处不在。