Shank2E binds NaP(i) cotransporter at the apical membrane of proximal tubule cells.
Shank2E binds NaP(i) cotransporter at the apical membrane of proximal tubule cells.
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Shank2E 在近曲小管细胞的顶膜处结合 NaP(i) 协同转运蛋白。
DOI：
10.1152/ajpcell.00568.2004
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Doctor,RBrian
中科院分区：
文献类型：
--
作者：
McWilliams,RyanR;Breusegem,SophiaY;Brodsky,KelleyF;Kim,Eunjoon;Levi,Moshe;Doctor,RBrian
表达突触后密度（PSD）-95/果蝇盘大（Dlg）/闭锁小带-1（ZO-1）（PDZ）结构域的蛋白质通常参与调节多种细胞类型中质膜上的受体、通道和转运蛋白活性。在肾近端小管（PT）的顶膜处，IIa型Na Picotransporter（NaPi-IIa）结合特异性PDZ结构域蛋白。Shank 2 E是PDZ蛋白家族的剪接形式，其集中在肝脏和胰腺上皮细胞类型的顶端结构域，并在肾脏中表达。在本研究中，富集质膜组分的免疫印迹和免疫组织学发现Shank 2 E集中在大鼠PT细胞的刷状缘膜。Flag-Shank 2 E和增强型绿色荧光蛋白-NaPi-IIa在共转染的OK细胞中的共聚焦定位显示这些蛋白共定位在PT细胞模型的顶端微绒毛中。Shank 2 E与来自大鼠肾皮质组织的NaPi-IIa共免疫沉淀，HA-NaPi-IIa与共转染的人胚肾HEK细胞中的Flag-Shank 2 E共沉淀。结构域分析表明，Shank 2 E的PDZ结构域特异性结合NaPi-IIa和截断的COOH-末端TRL基序从NaPi-IIa取消这种结合，和远Western印迹表明，Shank 2 E- NaPi-IIa相互作用直接发生在两个蛋白质之间。NaPi-IIa活性通过调节其在顶膜中的丰度来调节。高浓度条件诱导NaPi-IIa内化和降解。在大鼠肾脏PT细胞和OK细胞，转移到高piconditions诱导急性内部重新分配的Shank 2 E，并在OK细胞，一个显着程度的降解。总之，Shank 2 E集中在肾PT细胞的顶端结构域，通过PDZ相互作用特异性结合NaPi-IIa，并经历Pi诱导的内化。
Proteins expressing postsynaptic density (PSD)-95/Drosophiladisk large (Dlg)/zonula occludens-1 (ZO-1) (PDZ) domains are commonly involved in moderating receptor, channel, and transporter activities at the plasma membrane in a variety of cell types. At the apical membrane of renal proximal tubules (PT), the type IIa NaPicotransporter (NaPi-IIa) binds specific PDZ domain proteins. Shank2E is a spliceoform of a family of PDZ proteins that is concentrated at the apical domain of liver and pancreatic epithelial cell types and is expressed in kidney. In the present study, immunoblotting of enriched plasma membrane fractions and immunohistology found Shank2E concentrated at the brush border membrane of rat PT cells. Confocal localization of Flag-Shank2E and enhanced green fluorescent protein-NaPi-IIa in cotransfected OK cells showed these proteins colocalized in the apical microvilli of this PT cell model. Shank2E coimmunoprecipitated with NaPi-IIa from rat renal cortex tissue and HA-NaPi-IIa coprecipitated with Flag-Shank2E in cotransfected human embryonic kidney HEK cells. Domain analysis showed that the PDZ domain of Shank2E specifically bound NaPi-IIa and truncation of the COOH-terminal TRL motif from NaPi-IIa abolished this binding, and Far Western blotting showed that the Shank2E- NaPi-IIa interaction occurred directly between the two proteins. NaPi-IIa activity is regulated by moderating its abundance in the apical membrane. High-Piconditions induce NaPi-IIa internalization and degradation. In both rat kidney PT cells and OK cells, shifting to high-Piconditions induced an acute internal redistribution of Shank2E and, in OK cells, a significant degree of degradation. In sum, Shank2E is concentrated in the apical domain of renal PT cells, specifically binds NaPi-IIa via PDZ interactions, and undergoes Pi-induced internalization.