McWilliams Ryan R, Breusegem Sophia Y, Brodsky Kelley F, Kim Eunjoon, Levi Moshe, Doctor R Brian
Department of Medicine, University of Colorado Health Sciences Center, 4200 E. 9th St., Denver, Colorado 80262, USA.
Am J Physiol Cell Physiol. 2005 Oct;289(4):C1042-51. doi: 10.1152/ajpcell.00568.2004. Epub 2005 May 25.
Proteins expressing postsynaptic density (PSD)-95/Drosophila disk 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 NaP(i) cotransporter (NaP(i)-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-NaP(i)-IIa in cotransfected OK cells showed these proteins colocalized in the apical microvilli of this PT cell model. Shank2E co-immunoprecipitated with NaP(i)-IIa from rat renal cortex tissue and HA-NaP(i)-IIa coprecipitated with Flag-Shank2E in cotransfected human embryonic kidney HEK cells. Domain analysis showed that the PDZ domain of Shank2E specifically bound NaP(i)-IIa and truncation of the COOH-terminal TRL motif from NaP(i)-IIa abolished this binding, and Far Western blotting showed that the Shank2E- NaP(i)-IIa interaction occurred directly between the two proteins. NaP(i)-IIa activity is regulated by moderating its abundance in the apical membrane. High-P(i) conditions induce NaP(i)-IIa internalization and degradation. In both rat kidney PT cells and OK cells, shifting to high-P(i) conditions 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 NaP(i)-IIa via PDZ interactions, and undergoes P(i)-induced internalization.
表达突触后致密物（PSD）-95/果蝇盘大蛋白（Dlg）/紧密连接蛋白1（ZO-1）（PDZ）结构域的蛋白质通常参与调节多种细胞类型质膜上的受体、通道和转运体活性。在肾近端小管（PT）的顶端膜上，IIa型钠-磷共转运体（NaP(i)-IIa）与特定的PDZ结构域蛋白结合。Shank2E是PDZ蛋白家族的一种剪接异构体，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，对富集的质膜组分进行免疫印迹和免疫组织学检查发现，Shank2E集中在大鼠PT细胞的刷状缘膜上。共转染的OK细胞中Flag-Shank2E和增强型绿色荧光蛋白-NaP(i)-IIa的共聚焦定位显示，这些蛋白在该PT细胞模型的顶端微绒毛中共定位。Shank2E与大鼠肾皮质组织中的NaP(i)-IIa进行共免疫沉淀，并且在共转染的人胚肾HEK细胞中，HA-NaP(i)-IIa与Flag-Shank2E共沉淀。结构域分析表明，Shank2E的PDZ结构域特异性结合NaP(i)-IIa，并且从NaP(i)-IIa的COOH末端TRL基序截断会消除这种结合，Far Western印迹显示Shank2E与NaP(i)-IIa之间的相互作用直接发生在这两种蛋白质之间。NaP(i)-IIa的活性通过调节其在顶端膜中的丰度来调控。高磷条件会诱导NaP(i)-IIa内化和降解。在大鼠肾PT细胞和OK细胞中，转变至高磷条件均会诱导Shank2E发生急性内向再分布，并且在OK细胞中会发生显著程度的降解。总之，Shank2E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NaP(i)-IIa，并经历磷诱导的内化。
