Dynamics of aquaporin-2 serine-261 phosphorylation in response to short-term vasopressin treatment in collecting duct
Dynamics of aquaporin-2 serine-261 phosphorylation in response to short-term vasopressin treatment in collecting duct
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DOI：
10.1152/ajprenal.00284.2006
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发表时间：
2007-02-01
影响因子：
4.2
通讯作者：
Knepper, Mark A.
中科院分区：
文献类型：
--
作者：
Hoffert, Jason D.;Nielsen, Jakob;Knepper, Mark A.
我们最近在加压素调节的水通道水通道蛋白 2 (AQP2) 的 COOH 末端发现了一个新的磷酸化位点丝氨酸 261 (pS261)。为了确定该位点的磷酸化是否受加压素调节，产生了兔多克隆磷酸化特异性抗体。点印迹和免疫印迹分析表明，该抗体特异性识别 pS261 处磷酸化的 AQP2，并且已知受加压素调节的位点 S256 (pS256) 的磷酸化不会干扰抗体识别。免疫组织化学分析显示，野生型小鼠的内髓集合管 (IMCD) 存在强烈的 pS261 标记，而 AQP2 敲除动物的切片则显示普遍缺乏标记。 AQP2 pS261 存在于从连接小管到末端 IMCD 的所有小鼠和大鼠远端小管段的主细胞中。用磷酸特异性抗体和总 AQP2 抗体对集合管进行联合免疫标记表明 pS261 和 pS256 具有不同的亚细胞分布。在新鲜分离的大鼠 IMCD 样品中，与 1 nM [脱氨基-Cys(1)、D-Arg(8)] 加压素孵育 30 分钟后，pS256 水平增加，而 pS261 量显着下降。同样，根据免疫组织化学标记，用[脱氨基-Cys(1)，D-Arg(8)]加压素治疗2小时的Brattleboro大鼠所有集合管段中pS261的量均减少。这项研究揭示了短期加压素暴露后 S256 和 S261 磷酸化的相互变化，表明这些残基可能在调节 AQP2 亚细胞分布和集合管水渗透性中发挥不同的作用。
We recently identified a novel phosphorylation site, serine-261 (pS261), in the COOH-terminus of the vasopressin-regulated water channel, aquaporin-2 (AQP2). To address whether phosphorylation at this site is regulated by vasopressin, a rabbit polyclonal phospho-specific antibody was generated. Dot blot and immunoblot analysis demonstrated that this antibody specifically recognizes AQP2 phosphorylated at pS261, and that phosphorylation of S256 (pS256), a site already known to be regulated by vasopressin, does not interfere with antibody recognition. Immunohistochemical analysis revealed intense pS261 labeling of inner medullary collecting duct (IMCD) from wild-type mice, while sections from AQP2 knockout animals showed a general absence of labeling. AQP2 pS261 was present in principal cells of all mouse and rat distal tubule segments from the connecting tubule to the terminal IMCD. Co-immunolabeling of collecting duct with phospho-specific and total AQP2 antibodies revealed that pS261 and pS256 have distinct subcellular distributions. Levels of pS256 increased, while the amount of pS261 significantly decreased in freshly isolated rat IMCD samples incubated with 1 nM [ deamino-Cys(1), D-Arg(8)] vasopressin for 30 min. Similarly, based on immunohistochemical labeling, the amount of pS261 was reduced in all collecting duct segments of Brattleboro rats treated with [deamino-Cys(1), D-Arg(8)] vasopressin for 2 h. This study reveals a reciprocal change in S256 and S261 phosphorylation in response to short-term vasopressin exposure, suggesting that these residues may serve distinct roles in regulation of AQP2 subcellular distribution and collecting duct water permeability.