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

水通道蛋白-11（AQP 11）最近被鉴定并显示在肾脏近端小管表达。AQP 11-null小鼠不能存活超过新生期，推测是由于与肾囊肿相关的肾衰竭。AQP 11具有几种独特的氨基酸序列模式，包括一个天冬酰胺-脯氨酸-半胱氨酸（NPC）基序，其对应于常规AQPs的N-末端天冬酰胺-脯氨酸-丙氨酸（NPA）基序，一个AQPs的特征基序，以及一个C-末端二赖氨酸基序（NKKE），其类似于KKXX ER-保留信号。在这项研究中，我们评估了NPC和NKKE基序在AQP 11亚细胞定位和寡聚化中的作用。蛋白质印迹法从差速离心全肾匀浆产生的蛋白质组分揭示了AQP 11的细胞内定位。这两个共聚焦显微镜研究和细胞表面的生物素化实验与细胞瞬时表达AQP 11也支持细胞内，特别是ER定位AQP 11。免疫共沉淀和化学交联实验显示了AQP 11的寡聚体结构。在NPC或NKKE基序的定点突变不直接AQP 11质膜。NKKE基序的突变对寡聚化没有显著影响，而NPC基序中的C101 A突变减少AQP 11的寡聚化。虽然已知NPA基序对于水渗透孔的形成是重要的，但我们的观察表明，AQP 11的相应NPC基序在其3-D蛋白结构中也具有作用。

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.