NHE3 Regulation by Lipid Rafts and Hsc70

描述（由申请人提供）： 上皮刷状缘膜（BBM）Na+/H+-交换器3（NHE 3）是负责肠道和肾脏中性钠吸收的关键转运蛋白。在小肠中，NHE 3抑制是大多数小肠疾病的主要机制。因此，了解NHE 3的调控机制在胃肠病学中具有重要意义。本研究将探讨脂筏和热休克同源蛋白70（Hsc 70）在NHE 3调控中的作用。待测试的假设是：1）脂筏参与NHE 3膜分配和运输，并且是NHE 3的适当功能所需的;和2）通过与NHE 3和内吞机制的相互作用，Hsc 70通过促进NHE 3运输和/或NHE 3复合物组装/拆卸参与基础和调节的NHE 3活性。这一假设得到了强有力的初步数据的支持：（i）NHE 3存在于兔回肠BBM和培养的负鼠肾（OK）细胞的脂筏中;（ii）EGF通过增加BBM脂筏中NHE 3的量来刺激NHE 3活性;（iii）脂筏的破坏抑制回肠BBM NHE 3的内吞作用和OK细胞的NHE 3活性;（iv）NHE 3在Caco-2/E3 V（用VSVG标记的NHE 3稳定转染的Caco-2细胞）和肾近端小管细胞系OK/E3 V中作为大复合物（400-900 kDa）存在。NHE 3复合物含有多达10种不同的蛋白质，包括PS 120/E3 V细胞、Caco-2细胞和肾近端小管上皮细胞中的Hsc 70;（V），免疫共沉淀和体外结合测定显示Hsc 70与NHE 3直接相互作用。拟议的研究将导致理解两个新的机制，上皮NHE 3的调节：在脂筏和复合物形成与Hsc 70的区室化。

DESCRIPTION (provided by applicant): Epithelial brush border membrane (BBM) Na+/H+-exchanger 3 (NHE3) is a critical transporter responsible for intestinal and renal neutral Na absorption. In small intestine, NHE3 inhibition is a major mechanism for most diarrheal diseases. Therefore understanding the regulatory mechanism of NHE3 is of great significance in gastroenterology. This proposal will examine the roles of lipid rafts and heat shock cognate protein 70 (Hsc70) in the regulation of NHE3. The hypothesis to be tested is that 1), lipid rafts are involved in NHE3 membrane partitioning and trafficking and are required for the proper function of NHE3; and 2), through interaction with NHE3 and endocytic machinery Hsc70 is involved in basal and regulated NHE3 activity by facilitating NHE3 trafficking and/or NHE3 complex assembly/disassembly. This hypothesis is supported by strong preliminary data (i), NHE3 is present in the lipid rafts of both rabbit ileal BBM and cultured opossum kidney (OK) cells; (ii), EGF stimulates NHE3 activity by increasing NHE3 amount in BBM lipid rafts; (iii), Disruption of lipid rafts inhibits endocytosis of ileal BBM NHE3 and the NHE3 activity of OK cells; (iv), NHE3 exists as large complexes (400-900 kDa) in Caco-2/E3V (Caco-2 cells stable transfected with NHE3 tagged with VSVG) and kidney proximal tubule cell line OK/E3V. NHE3 complexes contain up to 10 distinct proteins, including Hsc70 in PS 120/E3V cells, Caco-2 cells and kidney proximal tubule epithelial cells; (v), Co-immunoprecipitation and in vitro binding assay show that Hsc70 interacts with NHE3 directly. The proposed studies will lead to the understanding of two novel mechanisms by which epithelial NHE3 is regulated: compartmentation in lipid rafts and complex formation with Hsc70.