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

水通道蛋白-11(Aquaporin-11，AQP11)最近被发现并在肾脏近端小管表达。AQP11缺失的小鼠在新生期后无法存活，可能是由于与肾囊肿相关的肾功能衰竭。AQP11有几种独特的氨基酸序列模式，包括与传统AQP的N端天冬酰胺-Pro-丙氨酸(NPA)基序相对应的天冬酰胺-脯氨酸-半胱氨酸(NPC)基序、AQP的特征基序以及类似于KKXX ER保留信号的C-末端二赖氨酸基序(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。全肾匀浆差速离心法蛋白质组分的Western blotting结果显示AQP11在细胞内定位。瞬时表达AQP11的细胞的共聚焦显微镜研究和细胞表面生物素化实验也支持细胞内的AQP11，特别是内质网定位的AQP11。免疫共沉淀和化学交联实验表明，AQP11具有低聚结构。针对NPC或NKKE基序的定点突变不会将AQPll定向到质膜上。NKKE基序的突变对AQPll的寡聚化无显著影响，而NPC基序的C101A突变降低了AQPll的寡聚化。虽然已知NPA基序对于透水性孔的形成是重要的，但我们的观察表明，对应的AQP11的NPC基序也在其三维蛋白质结构中发挥作用。

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.