Acute regulation of aquaporin-2 phosphorylation at Ser-264 by vasopressin
Acute regulation of aquaporin-2 phosphorylation at Ser-264 by vasopressin
复制标题
DOI：
10.1073/pnas.0712338105
复制
发表时间：
2008-02-26
影响因子：
11.1
通讯作者：
Knepper, Mark A.
中科院分区：
文献类型：
--
作者：
Fenton, Robert A.;Moeller, Hanne B.;Knepper, Mark A.
通过磷酸化蛋白质组分析，我们在抗利尿激素调节的水通道aquaporin-2 （AQP2）的COOH端发现了磷酸化位点丝氨酸264 （pS264）。在本研究中，我们使用磷酸化特异性抗体（抗ps264）检测抗利尿激素对丝氨酸264位点磷酸化的AQP2 （pS264-AQP2）的调控。免疫组化分析显示，pS264-AQP2在对照小鼠的内髓集管（IMCD）上有标记，而AQP2敲除小鼠则完全没有标记。在大鼠和小鼠中，pS264-AQP2存在于整个收集管系统，从连接管到终端IMCD。免疫金电子显微镜，结合双标记共聚焦免疫荧光，细胞器特异性标记显微镜，确定大部分pS264存在于与质膜和早期内吞途径相关的室室中。在用[脱氨基- cys -1， D-Arg-8]抗利尿素（dDAVP）治疗的Brattleboro大鼠中，pS264-AQP2的丰度比对照组增加了4倍。此外，dDAVP治疗导致pS264的分布发生时间依赖性变化，从主要的细胞内囊泡到基底外侧和根尖质膜。暴露davp 60分钟后，在网格蛋白包被的囊泡、早期内体室和循环室中观察到pS264-AQP2的比例，但溶酶体中没有。总的来说，我们的结果与AVP对AQP2磷酸化和亚细胞分布的动态影响是一致的。
By phosphoproteome analysis, we identified a phosphorylation site, serine 264 (pS264), in the COOH terminus of the vasopressin-regulated water channel, aquaporin-2 (AQP2). In this study, we examined the regulation of AQP2 phosphorylated at serine 264 (pS264-AQP2) by vasopressin, using a phospho-specific antibody (anti-pS264). Immunohistochemical analysis showed pS264-AQP2 labeling of inner medullary collecting duct (IMCD) from control mice, whereas AQP2 knockout mice showed a complete absence of labeling. In rat and mouse, pS264-AQP2 was present throughout the collecting duct system, from the connecting tubule to the terminal IMCD. Immunogold electron microscopy, combined with double-labeling confocal immunofluorescence, microscopy with organelle-specific markers, determined that the majority of pS264 resides in compartments associated with the plasma membrane and early endocytic pathways. In Brattleboro rats treated with [deamino-Cys-1, D-Arg-8]vasopressin (dDAVP), the abundance of pS264-AQP2 increased 4-fold over controls. Additionally, dDAVP treatment resulted in a time-dependent change in the distribution of pS264 from predominantly intracellular vesicles, to both the basolateral and apical plasma membranes. Sixty minutes after dDAVP exposure, a proportion of pS264-AQP2 was observed in clathrin-coated vesicles, early endosomal compartments, and recycling compartments, but not lysosomes. Overall, our results are consistent with a dynamic effect of AVP on the phosphorylation and subcellular distribution of AQP2.