Department of Pharmacology and Brain Korea 21 Project for Medical Science, Institute of Gastroenterology, Yonsei University College of Medicine, Seoul 120-752, Korea.
J Biol Chem. 2010 Mar 12;285(11):8104-13. doi: 10.1074/jbc.M109.055079. Epub 2010 Jan 14.
Na(+)/H(+) exchanger 3 (NHE3) plays an important role in neutral Na(+) transport in mammalian epithelial cells. The Rho family of small GTPases and the PDZ (PSD-95/discs large/ZO-1) domain-based adaptor Shank2 are known to regulate the membrane expression and activity of NHE3. In this study we examined the role of betaPix, a guanine nucleotide exchange factor for the Rho GTPase and a strong binding partner to Shank2, in NHE3 regulation using integrated molecular and physiological approaches. Immunoprecipitation and pulldown assays revealed that NHE3, Shank2, and betaPix form a macromolecular complex when expressed heterologously in mammalian cells as well as endogenously in rat colon, kidney, and pancreas. In addition, these proteins co-segregated at the apical surface of rat colonic epithelial cells, as detected by immunofluorescence staining. When expressed in PS120/NHE3 cells, betaPix increased membrane expression and basal activity of NHE3. Interestingly, the effects of betaPix on NHE3 were abolished by cotransfection with dominant-negative Shank2 mutants and by treatment with Clostridium difficile toxin B, a Rho GTPase inhibitor, indicating that Shank2 and Rho GTPases are involved in betaPix-mediated NHE3 regulation. Knockdown of endogenous betaPix by RNA interference decreased Shank2-induced increase of NHE3 membrane expression in HEK 293T cells. These results indicate that betaPix up-regulates NHE3 membrane expression and activity by Shank2-mediated protein-protein interaction and by activating Rho GTPases in the apical regions of epithelial cells.
钠氢交换蛋白 3（NHE3）在哺乳动物上皮细胞中发挥着重要作用，其负责中性钠离子的转运。Rho 家族小 G 蛋白和 PDZ（PSD-95/discs large/ZO-1）域基衔接蛋白 Shank2 被认为可以调节 NHE3 的膜表达和活性。本研究采用综合分子生物学和生理学方法，研究了 Rho GTPase 的鸟嘌呤核苷酸交换因子βPix 在 NHE3 调节中的作用，以及它与 Shank2 之间的关系。免疫沉淀和下拉实验表明，当 NHE3、Shank2 和βPix 在哺乳动物细胞中异源表达以及在大鼠结肠、肾脏和胰腺中内源表达时，它们会形成一个大分子复合物。此外，这些蛋白在大鼠结肠上皮细胞的顶膜表面共定位，通过免疫荧光染色可以检测到。当βPix 在 PS120/NHE3 细胞中表达时，它会增加 NHE3 的膜表达和基础活性。有趣的是，βPix 对 NHE3 的影响可以通过共转染显性失活的 Shank2 突变体和用梭状芽胞杆菌毒素 B（一种 Rho GTP 酶抑制剂）处理来消除，这表明 Shank2 和 Rho GTP 酶参与了βPix 介导的 NHE3 调节。用 RNA 干扰敲低内源性βPix 会降低 Shank2 诱导的 HEK 293T 细胞中 NHE3 膜表达的增加。这些结果表明，βPix 通过 Shank2 介导的蛋白-蛋白相互作用和激活上皮细胞顶区的 Rho GTP 酶来上调 NHE3 的膜表达和活性。
