Pathogenesis of nephrogenic diabetes insipidus by aquaporin-2 C-terminus mutations
Pathogenesis of nephrogenic diabetes insipidus by aquaporin-2 C-terminus mutations
复制标题
DOI：
10.1046/j.1523-1755.2003.00049.x
复制
发表时间：
2003-07-01
影响因子：
19.6
通讯作者：
Sasaki, S
中科院分区：
文献类型：
--
作者：
Asai, T;Kuwahara, M;Sasaki, S
背景我们先前报道了已知引起常染色体显性肾源性尿崩症（NDI）的三种水通道蛋白-2（AQP 2）基因突变（Am J Genet 69：738，2001）。AQP 2基因C端分别为721 de 1G、763 - 772 del和812 - 818 del。野生型AQP 2是271个氨基酸的蛋白质，而这些突变基因由于移码突变导致下游180个核苷酸处产生新的终止密码子，因此预测编码330至333个氨基酸的蛋白质。非洲爪蟾卵母细胞表达研究表明，突变的AQP 2的运输受到损害。为了确定哺乳动物上皮细胞中这些NDI引起突变的细胞发病机制，Madin-Darby犬肾（MDCK）细胞用野生型AQP 2或763至772 del突变体AQP 2或两者稳定转染。细胞在膜支持物上生长，通过免疫荧光显微镜检查AQP 2蛋白的定位。共聚焦免疫荧光显微镜显示，野生型AQP 2表达在顶端区域，而突变型AQP 2显然位于基底外侧区域。此外，野生型和突变型AQP 2在共转染时共定位于基底外侧区，提示形成混合寡聚体，从而导致错误定位。由于突变体的显性负效应，野生型和763至772 del突变体AQP 2的混合寡聚体被错误定位到基底外侧膜。这一缺陷很可能解释常染色体显性NDI的发病机制。顶膜蛋白与基底外侧膜的错误定位是先天性NDI的一种新的分子发病机制。
Background. We previously reported three aquaporin-2 (AQP2) gene mutations known to cause autosomal-dominant nephrogenic diabetes insipidus (NDI) (Am J Hum Genet 69: 738, 2001). The mutations were found in the C-terminus of AQP2 (721de1G, 763 to 772del, and 812 to 818del). The wildtype AQP2 is a 271 amino acid protein, whereas these mutant genes were predicted to encode 330 to 333 amino acid proteins due to the frameshift mutations leading to the creation of a new stop codon 180 nucleotides downstream. The Xenopus oocyte expression study suggested that the trafficking of the mutant AQP2s was impaired.Methods. To determine the cellular pathogenesis of these NDI-causing mutations in mammalian epithelial cells, Madin-Darby canine kidney (MDCK) cells were stably transfected with the wild-type AQP2, or the 763 to 772del mutant AQP2, or both. Cells were grown on the membrane support to examine the localization of AQP2 proteins by immunofluorescence microscopy.Results. Confocal immunofluorescence microscopy showed that the wild-type AQP2 was expressed in the apical region, whereas the mutant AQP2 was apparently located at the basolateral region. Furthermore, the wild-type and mutant AQP2s were colocalized at the basolateral region when they were cotransfected, suggesting the formation of mixed oligomers and thereby mistargeting.Conclusion. Mixed oligomers of the wild-type and the 763 to 772del mutant AQP2s are mistargeted to the basolateral membrane due to the dominant-negative effect of the mutant. This defect is very likely to explain the pathogenesis of autosomal-dominant NDI. The mistargeting of the apical membrane protein to the basolateral membrane is a novel molecular pathogenesis of congenital NDI.