Christensen B M, Zelenina M, Aperia A, Nielsen S
Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus, Denmark.
Am J Physiol Renal Physiol. 2000 Jan;278(1):F29-42. doi: 10.1152/ajprenal.2000.278.1.F29.
Phosphorylation of Ser(256), in a PKA consensus site, in AQP2 (p-AQP2) appears to be critically involved in the vasopressin-induced trafficking of AQP2. In the present study, affinity-purified antibodies that selectively recognize AQP2 phosphorylated at Ser(256) were developed. These antibodies were used to determine 1) the subcellular localization of p-AQP2 in rat kidney and 2) changes in distribution and/or levels of p-AQP2 in response to [desamino-Cys(1),D-Arg(8)]vasopressin (DDAVP) treatment or V(2)-receptor blockade. Immunoelectron microscopy revealed that p-AQP2 was localized in both the apical plasma membrane and in intracellular vesicles of collecting duct principal cells. Treatment of rats with V(2)-receptor antagonist for 30 min resulted in almost complete disappearance of p-AQP2 labeling of the apical plasma membrane with only marginal labeling of intracellular vesicles remaining. Immunoblotting confirmed a marked decrease in p-AQP2 levels. In control Brattleboro rats (BB), lacking vasopressin secretion, p-AQP2 labeling was almost exclusively present in intracellular vesicles. Treatment of BB rats with DDAVP for 2 h induced a 10-fold increase in p-AQP2 labeling of the apical plasma membrane. The overall abundance of p-AQP2, however, was not increased, as determined both by immunoelectron microscopy and immunoblotting. Consistent with this, 2 h of DDAVP treatment of normal rats also resulted in unchanged p-AQP2 levels. Thus the results demonstrate that AQP2 phosphorylated in Ser(256) is present in the apical plasma membrane and in intracellular vesicles and that both the intracellular distribution/trafficking, as well as the abundance of p-AQP2, are regulated via V(2) receptors by altering phosphorylation and/or dephosphorylation of Ser(256) in AQP2.
水通道蛋白2（p-AQP2）中蛋白激酶A（PKA）共有序列位点的丝氨酸（Ser）256磷酸化似乎在血管加压素诱导的AQP2转运中起关键作用。在本研究中，开发了选择性识别Ser256磷酸化的AQP2的亲和纯化抗体。这些抗体用于确定：1）大鼠肾脏中p-AQP2的亚细胞定位；2）响应[去氨基半胱氨酸（1），D-精氨酸（8）]血管加压素（DDAVP）处理或V2受体阻断时p-AQP2的分布和/或水平变化。免疫电子显微镜显示，p-AQP2定位于集合管主细胞的顶端质膜和细胞内囊泡中。用V2受体拮抗剂处理大鼠30分钟导致顶端质膜的p-AQP2标记几乎完全消失，仅残留细胞内囊泡的少量标记。免疫印迹证实p-AQP2水平显著降低。在缺乏血管加压素分泌的对照布拉特洛维大鼠（BB）中，p-AQP2标记几乎仅存在于细胞内囊泡中。用DDAVP处理BB大鼠2小时导致顶端质膜的p-AQP2标记增加10倍。然而，通过免疫电子显微镜和免疫印迹测定，p-AQP2的总体丰度并未增加。与此一致，用DDAVP处理正常大鼠2小时也导致p-AQP2水平不变。因此，结果表明Ser256磷酸化的AQP2存在于顶端质膜和细胞内囊泡中，并且细胞内分布/转运以及p-AQP2的丰度均通过V2受体调节，通过改变AQP2中Ser256的磷酸化和/或去磷酸化来实现。
