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

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 analyze the pathway of UT-A1 endocytosis(lipid raft-mediated/caveolae-mediated/ clathrin-coated pit mediated) and degradation(lysosomal mediated/proteasomal mediated) and its relationship to UT-A1 ubiquitination under the prolonged stimulation of cAMP by using gradient sucrose ultracentrifugation and other methods for subcellular domain isolation, biotinylation, endocytic internalization analysis, transepithelial urea flux assay, co-IP, immunofluorescence confocal microscopy technique, western blot in UT-A1 and FLAG-Tac-UT-A1 stably expressed MDCK cells, renal inner medullary tissue and IMCD cells from rats that are subcutaneously injected with Pitressin Tannate. The aim of our project is to clarify the mechanisms of UT-A1 ubiquitination, endocytosis and degradation which is induced by the prolonged stimulation of cAMP. This investigation could enhance the understanding of the mechanisms involved in UT-A1 regulation under cAMP stimulation.

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。

短时间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刺激时间延长其可致UT-A1泛素化及促进其胞吞与降解。而泛素化修饰类型与定位常影响膜蛋白的胞吞和降解，本研究证实cAMP激动剂呈时间依赖性促进UT-A1单泛素化，且泛素化发生部位主要位于细胞膜的脂筏区；并且cAMP激动剂呈时间依赖性促进膜蛋白UT-A1经clathrin依赖的途经内吞；UT-A1内吞后进入early endosome，最后进入溶酶体降解。另外抑制UT-A1泛素化能抑制cAMP激动剂诱导的UT-A1内吞与降解。体内动物实验也进一步证实长时间cAMP刺激促进UT-A1泛素化与降解，在糖尿病状态下UT-A1泛素化修饰增强，并且主要发生于脂筏区。故长时间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。本研究探明了长时间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。