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(NHE3)是肠道和肾脏中性钠吸收的关键转运体。在小肠中，NHE3抑制是大多数腹泻疾病的主要机制。因此，了解NHE3的调控机制在胃肠病学中具有重要意义。这项建议将研究脂筏和热休克相关蛋白70(Hsc70)在NHE3调控中的作用。需要检验的假设是：1)，脂筏参与NHE3膜的分配和运输，是NHE3正常功能所必需的；2)通过与NHE3和内吞机制的相互作用，Hsc70通过促进NHE3的运输和/或NHE3复合体的组装/拆解，参与基础和调节NHE3的活动。这一假说得到了强有力的初步数据的支持：(I)NHE3存在于兔回肠BBM和培养的负鼠肾脏(OK)细胞的脂筏中；(Ii)EGF通过增加BBM脂筏中NHE3的数量来刺激NHE3的活性；(Iii)脂筏的破坏抑制了回肠BBM NHE3的内吞作用和OK细胞的NHE3活性；(Iv)NHE3以大分子复合体形式存在于Caco-2/E3V(稳定表达VSVG标记的NHE3的Caco-2细胞)和肾脏近端小管细胞系Caco-2/OK/E3V中。NHE3复合体含有多达10种不同的蛋白质，包括PS 120/E3V细胞、Caco-2细胞和肾脏近端小管上皮细胞中的Hsc70；(V)免疫共沉淀和体外结合实验表明Hsc70与NHE3直接相互作用。拟议的研究将有助于理解上皮细胞NHE3受调控的两个新机制：脂筏中的区隔和与Hsc70的复合体形成。

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.