长时间cAMP刺激致尿素转运蛋白A1泛素化、胞吞与降解的机制研究

项目名称： 长时间cAMP刺激致尿素转运蛋白A1泛素化、胞吞与降解的机制研究

项目编号： No.31200872

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 生理学与整合生物学

项目作者： 苏华

作者单位： 华中科技大学

项目金额： 21万元

中文摘要： 短期(＜1h)cAMP刺激可增强尿素转运蛋白A1(Urea Transporter A1,UT-A1)的功能，我们发现随着cAMP刺激时间延长(＞2h)可致UT-A1泛素化及促进其胞吞与降解。泛素化修饰类型与定位常影响蛋白的胞吞和降解，故推测长时间cAMP刺激通过影响UT-A1泛素化类型与定位启动其不同于基础状态下的胞吞和降解过程，继而下调其功能。为证实该设想我们以表达UT-A1和FLAG-Tac-UT-A1的MDCK细胞及皮下注射鞣酸加压素大鼠为研究对象，采用co-IP、胞膜与细胞微区分离、胞吞定量分析、荧光共聚焦、尿素通透性检测等方法探讨长时间cAMP刺激致UT-A1泛素化修饰类型与定位(胞膜/细胞微区)；UT-A1胞吞和降解途径及其与泛素化修饰的关系。本研究旨在探明长时间cAMP刺激对UT-A1泛素化修饰与胞吞降解途径的影响和机制，为多角度认识cAMP对UT-A1的调节机制提供依据。

中文关键词： 尿素转运蛋白；泛素化；胞吞；降解；环腺苷酸

英文摘要： Urea transporter A1 (UT-A1) is one of the most important membrane proteins to modulate urine concentration. The stimulation of cAMP in a short term (<1hr) can enhance the function of UT-A1 through the promotion of its exocytosis and phosphorylation, increasing the osmolarity of urine in turn. Our previous work have shown that the prolonged stimulation of cAMP (>2hr) can induce the ubiquitination of UT-A1 in a time and dose dependent mode and accelerate its endocytosis and degradation processes at the same time.There are many documents reported that ubiquitination can influence protein trafficking and degradation. And the different type (mono-/poly-ubiquitination; K11/K48/K63 linked poly- ubiquitination) and location(cell membrane /cytosol; lipid raft/non-lipid raft; caveolae/clathrin-coated pit) of ubiquitinated proteins exert different biological functions. We conjecture that prolonged stimulation of cAMP may influence the type and location of ubiquitinated UT-A1 and consequently promote its endocytosis and degradation through the pathway different from normal situation. By the above processes the enhanced function of UT-A1 at early stage of cAMP stimulation can be downregulated and return to the balance. In order to verify our hypothesis we are going to detect the type and location of ubiquitinated UT-A1, and

英文关键词： Urea transporter A1；ubiquitination；endocytosis；degradation；cAMP