Department of Physiology (286), RUNMC Nijmegen, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands.
Nephrol Dial Transplant. 2012 Dec;27(12):4389-97. doi: 10.1093/ndt/gfs292. Epub 2012 Jul 9.
Mutations in the aquaporin-2 (AQP2) gene cause nephrogenic diabetes insipidus (NDI), a renal disorder characterized by polyuria due to a lacking antidiuretic response to vasopressin. While most AQP2 mutants in recessive NDI are misfolded and retained in the endoplasmic reticulum, AQP2-P262L in NDI was impaired in its vasopressin-dependent translocation from vesicles to the plasma membrane.
Vasopressin-induced translocation of AQP2 coincides with AQP2 phosphorylation at S256, S264 and T269 and dephosphorylation at S261. Since P262 lies adjacent to S261, we tested whether a changed phosphorylation could underlie AQP-P262L missorting in NDI.
In polarized cells, AQP2-P262L expressed as a double 29/30 kDa band, whereas wt-AQP2 expressed only as a 29 kDa band. Phosphatase treatment revealed that the 30 kDa AQP2-P262L band was due to changed phosphorylation. The use of newly developed phospho-specific antibodies showed that forskolin not only increased pS256 and pT269, but, in contrast to wt-AQP2, also pS261 in AQP2-P262L. The expression of AQP2-P262L proteins in which S261 phosphorylation was prevented (S261A), however, was still missorted to vesicles/basolateral membrane, despite the absence of the 30 kDa band.
Together, our data reveal that vasopressin induces instead of reduces the phosphorylation of S261 in AQP2-P262L, but it remains to be established whether the changed phosphorylation causes its missorting in NDI.
水通道蛋白-2（AQP2）基因的突变导致肾性尿崩症（NDI），这是一种肾脏疾病，其特征是多尿，因为缺乏抗利尿激素对血管加压素的反应。虽然隐性 NDI 中的大多数 AQP2 突变体是错误折叠并保留在内质网中，但 NDI 中的 AQP2-P262L 在其依赖血管加压素的从囊泡到质膜的易位中受损。
血管加压素诱导的 AQP2 易位与 S256、S264 和 T269 的 AQP2 磷酸化和 S261 的去磷酸化同时发生。由于 P262 位于 S261 附近，我们测试了改变的磷酸化是否是 NDI 中 AQP-P262L 错配的基础。
在极化细胞中，AQP2-P262L 表达为双 29/30 kDa 带，而 wt-AQP2 仅表达为 29 kDa 带。磷酸酶处理表明 30 kDa AQP2-P262L 带是由于磷酸化改变。使用新开发的磷酸特异性抗体表明，福司可林不仅增加了 pS256 和 pT269，而且与 wt-AQP2 相反，还增加了 AQP2-P262L 中的 pS261。然而，AQP2-P262L 蛋白的表达，其中 S261 磷酸化被阻止（S261A），尽管没有 30 kDa 带，但仍然错分到囊泡/基底外侧膜。
总的来说，我们的数据表明，血管加压素诱导而不是降低 AQP2-P262L 中 S261 的磷酸化，但尚需确定改变的磷酸化是否导致其在 NDI 中的错配。
