Dept. of Gastroenterology, Hepatology and Infectious Diseases, Medical Faculty, Univ. Clinic of the Heinrich-Heine Univ. of Düsseldorf, Moorenstrasse 5, D-40225 Düsseldorf, Germany.
Am J Physiol Gastrointest Liver Physiol. 2013 Nov 15;305(10):G722-30. doi: 10.1152/ajpgi.00056.2013. Epub 2013 Sep 5.
The sodium taurocholate cotransporting polypeptide (Ntcp) is the major uptake transporter for bile salts into liver parenchymal cells, and PKC-mediated endocytosis was shown to regulate the number of Ntcp molecules at the plasma membrane. In this study, mechanisms of Ntcp internalization were analyzed by flow cytometry, immunofluorescence, and Western blot analyses in HepG2 cells. PKC activation induced endocytosis of Ntcp from the plasma membrane by ~30%. Endocytosis of Ntcp was clathrin dependent and was followed by lysosomal degradation. A dileucine motif located in the third intracellular loop of Ntcp was essential for endocytosis but also for processing and plasma membrane targeting, suggesting a dual function of this motif for intracellular trafficking of Ntcp. Mutation of two of five potential phosphorylation sites surrounding the dileucine motif (Thr225 and Ser226) inhibited PKC-mediated endocytosis. In conclusion, we could identify a motif, which is critical for Ntcp plasma membrane localization. Endocytic retrieval protects hepatocytes from elevated bile salt concentrations and is of special interest, because NTCP has been identified as a receptor for the hepatitis B and D virus.
牛磺胆酸钠共转运蛋白（Ntcp）是将胆汁盐摄取到肝实质细胞中的主要摄取转运体，已有研究表明蛋白激酶 C（PKC）介导的内吞作用可调节质膜上 Ntcp 分子的数量。本研究通过流式细胞术、免疫荧光和 Western blot 分析，在 HepG2 细胞中分析了 Ntcp 内化的机制。结果表明，PKC 的激活可诱导 Ntcp 从质膜内化约 30%。Ntcp 的内化依赖于网格蛋白，并随后被溶酶体降解。Ntcp 第三细胞内环中的亮氨酸基序对于内吞作用以及加工和质膜靶向是必需的，这表明该基序对于 Ntcp 的细胞内运输具有双重功能。亮氨酸基序周围的五个潜在磷酸化位点中的两个（Thr225 和 Ser226）的突变抑制了 PKC 介导的内吞作用。总之，我们可以确定一个对于 Ntcp 质膜定位至关重要的基序。内吞作用的回收可以保护肝细胞免受升高的胆汁盐浓度的影响，这一点特别有趣，因为 NTCP 已被鉴定为乙型肝炎和丁型肝炎病毒的受体。
