Short-chain ubiquitination mediates the regulated endocytosis of the aquaporin-2 water channel
Short-chain ubiquitination mediates the regulated endocytosis of the aquaporin-2 water channel
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DOI：
10.1073/pnas.0604073103
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发表时间：
2006-11-28
影响因子：
11.1
通讯作者：
Deen, Peter M. T.
中科院分区：
文献类型：
--
作者：
Kamsteeg, Erik-Jan;Hendriks, Giel;Deen, Peter M. T.
为了调节哺乳动物的水平衡，精氨酸加压素(AVP)诱导磷酸化，从而使肾水通道蛋白-2(AQP2)的水通道从小泡重新分布到顶膜。反之亦然，AVP(或forsklin)的去除和激素激活PKC会导致AQP2内化，但机制尚不清楚。在这里，我们展示了在体外和体内，AQP2的一部分被两到三个泛素部分修饰。诱变实验表明，AQP2泛素化，仅在K270处有一条K63连接链。在Madin-Darby犬肾细胞中，AQP2泛素化优先发生在顶膜，随着Forskolin的去除或PKC的激活而瞬时增加，并先于其内化。野生型(Wt)和泛素化缺陷(K270R)AQP2的内化动力学分析表明，泛素化增强了AQP2的内吞作用。电子显微镜显示，AQP2与泛素的翻译融合(AQP2-Ub)仅定位于多囊泡体(MVbs)的内部小泡，而AQP2-K270R主要定位于MVB的顶膜、早期内体和界限膜。与这种分布模式一致，AQP2-Ub溶酶体降解广泛，AQP2-K270R低，wt-AQP2中等。我们的数据表明，短链泛素化参与了AQP2的调节内吞、MVB分类和降解，这可能是AVP清除和PKC激活激素减少肾水重吸收的机制。此外，由于其他几个通道也是(短链)泛素化的，我们的数据表明泛素化可能是高等真核生物中调节内吞和通道降解的一般中介。
To regulate mammalian water homeostasis, arginine-vasopressin (AVP) induces phosphorylation and thereby redistribution of renal aquaporin-2 (AQP2) water channels from vesicles to the apical membrane. Vice versa, AVP (or forskolin) removal and hormones activating PKC cause AQP2 internalization, but the mechanism is unknown. Here, we show that a fraction of AQP2 is modified with two to three ubiquitin moieties in vitro and in vivo. Mutagenesis revealed that AQP2 is ubiquitinated with one K63-linked chain at K270 only. In Madin-Darby canine kidney cells, AQP2 ubiquitination occurs preferentially when present in the apical membrane, is transiently increased with forskolin removal or PKC activation, and precedes its internalization. Internalization kinetics assays with wild type (wt) and ubiquitination-deficient (K270R) AQP2 revealed that ubiquitination enhances AQP2 endocytosis. Electron microscopy showed that a translational fusion of AQP2 with ubiquitin (AQP2-Ub) localized particularly to internal vesicles of multivesicular bodies (MVBs), whereas AQP2-K270R largely localized to the apical membrane, early endosomes, and the limiting membrane of MVBs. Consistent with this distribution pattern, lysosomal degradation was extensive for AQP2-Ub, low for AQP2-K270R, and intermediate for wt-AQP2. Our data show that short-chain ubiquitination is involved in the regulated endocytosis, MVB sorting, and degradation of AQP2 and may be the mechanism used by AVP removal and PKC-activating hormones to reduce renal water reabsorption. Moreover, because several other channels are also (short-chain) ubiquitinated, our data suggest that ubiquitination may be a general mediator for the regulated endocytosis and degradation of channels in higher eukaryotes.