Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Am J Physiol Renal Physiol. 2020 Oct 1;319(4):F592-F602. doi: 10.1152/ajprenal.00205.2020. Epub 2020 Aug 17.
Aquaporin-2 (AQP2) is a vasopressin-regulated water channel protein responsible for water reabsorption by the kidney collecting ducts. Under control conditions, most AQP2 resides in the recycling endosomes of principal cells, where it answers to vasopressin with trafficking to the apical plasma membrane to increase water reabsorption. Upon vasopressin withdrawal, apical AQP2 retreats to the early endosomes before joining the recycling endosomes for the next vasopressin stimulation. Prior studies have demonstrated a role of AQP2 S269 phosphorylation in reducing AQP2 endocytosis, thereby prolonging apical AQP2 retention. Here, we studied where in the cells S269 was phosphorylated and dephosphorylated in response to vasopressin versus withdrawal. In mpkCCD collecting cells, vacuolar protein sorting 35 knockdown slowed vasopressin-induced apical AQP2 trafficking, resulting in AQP2 accumulation in the recycling endosomes where S269 was phosphorylated. Rab7 knockdown, which impaired AQP2 trafficking from the early to recycling endosomes, reduced vasopressin-induced S269 phosphorylation. Rab5 knockdown, which impaired AQP2 endocytosis, did not affect vasopressin-induced S269 phosphorylation. Upon vasopressin withdrawal, S269 was not dephosphorylated in Rab5 knockdown cells. In contrast, S269 dephosphorylation upon vasopressin withdrawal was completed in Rab7 or vacuolar protein sorting 35 knockdown cells. We conclude that S269 is dephosphorylated during Rab5-mediated AQP2 endocytosis before AQP2 joins the recycling endosomes upon vasopressin withdrawal. While in the recycling endosomes, AQP2 can be phosphorylated at S269 in response to vasopressin before apical trafficking.
水通道蛋白 2（AQP2）是一种加压素调节的水通道蛋白，负责肾脏集合管的水重吸收。在控制条件下，大多数 AQP2 位于主细胞的再循环内体中，在那里它对加压素做出反应，通过运输到顶端质膜来增加水重吸收。在加压素撤出后，顶端 AQP2 在加入再循环内体之前退回到早期内体，以备下一次加压素刺激。先前的研究表明 AQP2 S269 磷酸化在减少 AQP2 内吞作用方面起作用，从而延长顶端 AQP2 的保留时间。在这里，我们研究了在细胞中 S269 在响应加压素与撤出时在何处被磷酸化和去磷酸化。在 mpkCCD 收集细胞中，液泡蛋白分选 35 敲低减缓了加压素诱导的顶端 AQP2 转运，导致 AQP2 在再循环内体中积累，S269 在此处被磷酸化。Rab7 敲低，其损害了 AQP2 从早期到再循环内体的转运，减少了加压素诱导的 S269 磷酸化。Rab5 敲低，其损害了 AQP2 的内吞作用，不影响加压素诱导的 S269 磷酸化。在加压素撤出后，Rab5 敲低细胞中 S269 未去磷酸化。相比之下，在 Rab7 或液泡蛋白分选 35 敲低细胞中，加压素撤出后 S269 的去磷酸化完成。我们得出结论，在 Rab5 介导的 AQP2 内吞作用过程中，S269 在 AQP2 加入再循环内体之前被去磷酸化，在加压素撤出后，AQP2 可以在再循环内体中被 S269 磷酸化，然后进行顶端转运。
