Elucidation of the role of AQPll, a novel aquaporin, on ER stress

水通道蛋白-11 （AQP11）最近被发现并显示在肾近端小管表达。aqp11缺失小鼠不能存活过新生儿期，可能是由于肾囊肿相关的肾功能衰竭。AQP11具有几个独特的氨基酸序列模式，包括一个与传统AQPs的n端天冬氨酸-脯氨酸-丙氨酸（NPA）基序对应的天冬氨酸-脯氨酸-半胱氨酸（NPC）基序，一个AQPs的特征基序，以及一个类似于KKXX er保留信号的c端二赖氨酸基序（NKKE）。在这项研究中，我们评估了NPC和NKKE基序在AQP11亚细胞定位和寡聚化中的作用。对全肾匀浆差速离心后的蛋白进行Western blotting，发现AQP11在细胞内的定位。共聚焦显微镜研究和瞬时表达AQP11的细胞表面生物素化实验也支持细胞内，特别是位于er的AQP11。共免疫沉淀和化学交联实验显示AQP11的低聚结构。NPC或NKKE基序的定点突变不会将AQPll导向质膜。NKKE基序的突变对AQPll的寡聚化无显著影响，而NPC基序的C101A突变则降低了AQPll的寡聚化。虽然已知NPA基序对可透水孔隙的形成很重要，但我们的观察表明，AQP11相应的NPC基序也在其3-D蛋白结构中发挥作用。

Aquaporin-11 (AQP11) was recently identified and shown to be expressed at the proximal tubule in the kidney. AQP11-null mice do not survive past the neonatal period, presumably due to renal failure associated with renal cysts. AQP11 has several unique amino acid sequence patterns including an asparagine-proline-cysteine (NPC) motif that corresponds to the N-terminal asparagine-proline-alanine (NPA) motif of conventional AQPs, a signature motif of the AQPs, as well as a C-terminal di-lysine motif (NKKE) which resembles the KKXX ER-retention signal. In this study we evaluated the roles of NPC and NKKE motifs in the subcellular localization and oligomerization of AQP11. Western blotting of protein fractions resulting from differential centrifugation of the whole kidney homogenate revealed the intracellular localization of AQP11. Both confocal microscopic studies and cell surface biotinylation experiments with cells transiently expressing AQP11 also supported the intracellular, especially ER-located AQP11. Co-immunoprecipitation and chemical cross-linking experiments showed the oligomeric structure of AQP11. Site-directed mutation at the NPC or the NKKE motif did not direct AQPll to the plasma membrane. Mutation at the NKKE motif had no significant effect on oligomerization, while the C101A mutation in the NPC motif reduced oligomerization of AQPll. Although the NPA motif is known to be important for a water-permeable pore formation, our observations suggest that the corresponding NPC motif of AQP11 also has a role in its 3-D protein structure.