Yui Naofumi, Okutsu Rie, Sohara Eisei, Rai Tatemitsu, Ohta Akihito, Noda Yumi, Sasaki Sei, Uchida Shinichi
Dept. of Nephrology, Tokyo Medical and Dental University, Japan.
Am J Physiol Cell Physiol. 2009 Dec;297(6):C1389-96. doi: 10.1152/ajpcell.00098.2009. Epub 2009 Sep 30.
FAPP2 is an adaptor protein of phosphatidylinositol-4-phosphate and is involved in the transport of some apical cargos from the trans-Golgi network (TGN). To investigate whether the regulated apical transport of aquaporin-2 (AQP2) is involved in the FAPP2-dependent apical protein-sorting machinery, we measured apical sorting of AQP2 in Madin-Darby canine kidney (MDCK) cells with or without FAPP2 knockdown. We established MDCK cell lines that stably express rat AQP2 without any tag sequence. Then, FAPP2-deficient stable cell lines were established from the AQP2-expressing cell lines by a retrovirus-mediated RNA interference system. In the established cell lines, AQP2 was detected in both apical and basolateral membranes. Forskolin increased only the apical localization of AQP2, which was not affected by basolateral treatment with 0.5% tannic acid, indicating that the forskolin-induced apical transport of AQP2 did not include the transcytotic pathway from basolateral to apical membranes but is a direct transport from TGN to the apical membranes. Using these cell lines, we tested the effect of FAPP2 knockdown on the polarized AQP2 transport to plasma membranes and found that the forskolin-induced apical transport of AQP2 was completely abolished by FAPP2 knockdown. By contrast, the basolateral localization of AQP2 was not affected by FAPP2 knockdown. AQP2 phosphorylation by forskolin was also impaired in FAPP2 knockdown MDCK cells. These results suggest that FAPP2 is necessary to generate AQP2-bearing vesicles at trans-Golgi that will undergo phosphorylation by PKA in subapical regions.
FAPP2是一种磷脂酰肌醇-4-磷酸衔接蛋白，参与从反式高尔基体网络（TGN）转运一些顶端货物。为了研究水通道蛋白2（AQP2）的调节性顶端转运是否参与FAPP2依赖性顶端蛋白分选机制，我们检测了在有或没有FAPP2敲低的Madin-Darby犬肾（MDCK）细胞中AQP2的顶端分选情况。我们建立了稳定表达无任何标签序列的大鼠AQP2的MDCK细胞系。然后，通过逆转录病毒介导的RNA干扰系统，从表达AQP2的细胞系中建立了FAPP2缺陷稳定细胞系。在建立的细胞系中，在顶端和基底外侧膜均检测到AQP2。福斯高林仅增加了AQP2的顶端定位，而0.5%单宁酸的基底外侧处理对其没有影响，这表明福斯高林诱导的AQP2顶端转运不包括从基底外侧膜到顶端膜的转胞吞途径，而是从TGN到顶端膜的直接转运。利用这些细胞系，我们测试了FAPP2敲低对AQP2向质膜的极化转运的影响，发现FAPP2敲低完全消除了福斯高林诱导的AQP2顶端转运。相比之下，AQP2的基底外侧定位不受FAPP2敲低的影响。在FAPP2敲低的MDCK细胞中，福斯高林诱导的AQP2磷酸化也受到损害。这些结果表明，FAPP2对于在反式高尔基体产生携带AQP2的囊泡是必需的，这些囊泡将在顶端下区域被蛋白激酶A磷酸化。
