Liver Center, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
J Biol Chem. 2010 Jun 18;285(25):19299-307. doi: 10.1074/jbc.M109.096081. Epub 2010 Apr 19.
Multidrug resistance-associated protein 2 (Mrp2, Abcc2) is an ATP-binding cassette transporter localized at the canalicular membrane of hepatocytes that plays an important role in bile formation and detoxification. Prior in vitro studies suggest that Mrp2 can bind to Na(+)/H(+) exchanger regulatory factor 1 (NHERF-1), a PDZ protein that cross-links membrane proteins to actin filaments. However the role of NHERF-1 in the expression and functional regulation of Mrp2 remains largely unknown. Here we examine the interaction of Mrp2 and NHERF-1 and its physiological significance in HEK293 cells and NHERF-1 knock-out mice. Mrp2 co-precipitated with NHERF-1 in co-transfected HEK293 cells, an interaction that required the PDZ-binding motif of Mrp2. In NHERF-1(-/-) mouse liver, Mrp2 mRNA was unchanged but Mrp2 protein was reduced in whole cell lysates and membrane-enriched fractions to approximately 50% (p < 1 x 10(-6)) and approximately 70% (p < 0.05), respectively, compared with wild-type mice, suggesting that the down-regulation of Mrp2 expression was caused by post-transcriptional events. Mrp2 remained localized at the apical/canalicular membrane of NHERF-1(-/-) mouse hepatocytes, although its immunofluorescent labeling was noticeably weaker. Bile flow in NHERF-1(-/-) mice was reduced to approximately 70% (p < 0.001) in association with a 50% reduction in glutathione excretion (p < 0.05) and a 60% reduction in glutathione-methylfluorescein (GS-MF) excretion in isolated mouse hepatocyte (p < 0.01). Bile acid and bilirubin excretion remained unchanged compared with wild-type mice. These findings strongly suggest that NHERF-1 binds to Mrp2, and plays a critical role in the canalicular expression of Mrp2 and its function as a determinant of glutathione-dependent, bile acid-independent bile flow.
多药耐药相关蛋白 2（Mrp2，Abcc2）是一种位于肝细胞胆小管膜的 ATP 结合盒转运体，在胆汁形成和解毒中发挥重要作用。先前的体外研究表明，Mrp2 可以与 Na(+)/H(+) 交换调节因子 1（NHERF-1）结合，NHERF-1 是一种 PDZ 蛋白，可将膜蛋白交联到肌动蛋白丝上。然而，NHERF-1 在 Mrp2 的表达和功能调节中的作用在很大程度上仍是未知的。本研究在 HEK293 细胞和 NHERF-1 敲除小鼠中检测了 Mrp2 和 NHERF-1 的相互作用及其生理意义。在共转染的 HEK293 细胞中，Mrp2 与 NHERF-1 共沉淀，这种相互作用需要 Mrp2 的 PDZ 结合基序。在 NHERF-1(-/-) 小鼠肝中，Mrp2 mRNA 无变化，但 Mrp2 蛋白在全细胞裂解物和富含膜的级分中分别减少至约 50%（p < 1 x 10(-6)）和约 70%（p < 0.05），与野生型小鼠相比，表明 Mrp2 表达的下调是由转录后事件引起的。Mrp2 仍定位于 NHERF-1(-/-) 小鼠肝细胞的顶/胆小管膜，尽管其免疫荧光标记明显较弱。NHERF-1(-/-) 小鼠的胆汁流量减少至约 70%（p < 0.001），与谷胱甘肽排泄减少 50%（p < 0.05）和分离的小鼠肝细胞中谷胱甘肽-甲基荧光素（GS-MF）排泄减少 60%（p < 0.01）相关。与野生型小鼠相比，胆汁酸和胆红素排泄保持不变。这些发现强烈表明 NHERF-1 与 Mrp2 结合，并在 Mrp2 的胆小管表达及其作为谷胱甘肽依赖性、胆汁酸非依赖性胆汁流决定因素的功能中发挥关键作用。
