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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Phosphorylation of aquaporin-2 regulates its endocytosis and protein-protein interactions
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Phosphorylation of aquaporin-2 regulates its endocytosis and protein-protein interactions

机译：Aquaporin-2的磷酸化调节其内吞作用和蛋白质-蛋白质相互作用

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摘要

The water channel aquaporin-2 (AQP2) is essential for urine concentration. Vasopressin regulates phosphorylation of AQP2 at four conserved serine residues at the COOH-terminal tail (S256, S261, S264, and S269). We used numerous stably transfected Madin-Darby canine kidney cell models, replacing serine residues with either alanine (A), which prevents phosphorylation, or as-partic acid (D), which mimics the charged state of phosphorylated AQP2, to address whether phosphorylation is involved in regulation of (ⅰ) apical plasma membrane abundance of AQP2, (ⅱ) inter-nalization of AQP2, (ⅲ) AQP2 protein-protein interactions, and (ⅳ) degradation of AQP2. Under control conditions, S256D- and 269D-AQP2 mutants had significantly greater apical plasma membrane abundance compared to wild type (WT)-AQP2. Activation of ad-enylate cyclase significantly increased the apical plasma membrane abundance of all S-A or S-D AQP2 mutants with the exception of 256D-AQP2, although 256A-, 261A-, and 269A-AQP2 mutants increased to a lesser extent than WT-AQP2. Biotin inter-nalization assays and confocal microscopy demonstrated that the internalization of 256D- and 269D-AQP2 from the plasma membrane was slower than WT-AQP2. The slower internalization corresponded with reduced interaction of S256D- and 269D-AQP2 with several proteins involved in endocytosis, including Hsp70, Hsc70, dynamin, and clathrin heavy chain. The mutants with the slowest rate of internalization, 256D- and 269D-AQP2, had a greater protein half-life (t_(1/2) = 5.1 h and t_(1/2) = 4.4 h, respectively) compared to WT-AQP2 (t_(1/2) = 2.9 h). Our results suggest that vaso-pressin-mediated membrane accumulation of AQP2 can be controlled via regulated exocytosis and endocytosis in a process that is dependent on COOH terminal phosphorylation and subsequent protein-protein interactions.
机译：水通道aquaporin-2（AQP2）对于尿液浓缩至关重要。加压素调节COQ末端尾部的四个保守丝氨酸残基处的AQP2磷酸化（S256，S261，S264和S269）。我们使用了许多稳定转染的Madin-Darby犬肾细胞模型，用可以防止磷酸化的丙氨酸（A）或模仿磷酸化AQP2带电状态的天冬氨酸（D）代替丝氨酸残基，以解决是否磷酸化是参与调节（ⅰ）AQP2的顶质膜丰度，（ⅱ）AQP2的内在化，（ⅲ）AQP2蛋白质-蛋白质相互作用和（ⅳ）AQP2的降解。在对照条件下，与野生型（WT）-AQP2相比，S256D-和269D-AQP2突变体的顶端质膜丰度明显更高。腺苷酸环化酶的激活显着增加了所有S-A或S-D AQP2突变体的顶质膜丰度，但256D-AQP2除外，尽管256A-，261A-和269A-AQP2突变体的增幅低于WT-AQP2。生物素内在化分析和共聚焦显微镜检查表明，从质膜中获得的256D-和269D-AQP2的内在作用比WT-AQP2慢。较慢的内在化与S256D-和269D-AQP2与参与内吞作用的几种蛋白质（包括Hsp70，Hsc70，动力蛋白和网格蛋白重链）的相互作用降低有关。与WT-相比，内化速率最低的突变体256D-和269D-AQP2具有更长的蛋白质半衰期（分别为t_（1/2）= 5.1 h和t_（1/2）= 4.4 h）。 AQP2（t_（1/2）= 2.9 h）。我们的研究结果表明，血管加压素介导的AQP2膜积聚可以通过调节的胞吐作用和内吞作用来控制，该过程依赖于COOH末端磷酸化和随后的蛋白质-蛋白质相互作用。

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