AP2 adaptor complex mediates bile salt export pump internalization and modulates its hepatocanalicular expression and transport function
AP2 adaptor complex mediates bile salt export pump internalization and modulates its hepatocanalicular expression and transport function
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DOI：
10.1002/hep.25591
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发表时间：
2012-06-01
期刊：
影响因子：
13.5
通讯作者：
Sugiyama, Yuichi
中科院分区：
文献类型：
--
作者：
Hayashi, Hisamitsu;Inamura, Kaori;Sugiyama, Yuichi
胆盐输出泵（BSEP）介导胆盐的胆汁排泄，其功能障碍引起肝内胆汁淤积。BSEP内化促进其小管表达减少是导致这种疾病状态的原因之一。然而，BSEP从管膜内化的分子机制尚不清楚。我们之前已经证明，4-苯基丁酸盐（4PBA），一种用于鸟氨酸转氨基甲酰基酶缺乏症（OTCD）的药物，可以抑制细胞表面BSEP的内化和随后的降解。目前的研究发现，在大鼠和OTCD患者的肝脏标本中，4PBA处理显著降低了a-和mu 2-适应蛋白的表达，这两种蛋白都是AP2适应蛋白复合物（AP2）的亚基，介导网格蛋白依赖的内吞作用，并且BSEP在其细胞质区域具有潜在的AP2识别基序。在此基础上，进一步探讨AP2在BSEP内化中的作用。体外对3xflag -人BSEP表达的HeLa细胞和人三明治培养肝细胞的分析表明，RNA干扰靶向a-adaptin对AP2功能的损害抑制了BSEP从质膜的内化，增加了其在细胞表面的表达和转运功能。免疫染色、共免疫沉淀、谷胱甘肽s -转移酶下拉试验和延时成像研究表明，AP2通过BSEP羧基端的酪氨酸基序与CM处的BSEP相互作用，并介导BSEP从肝细胞CM内化。结论：AP2通过与BSEP的直接相互作用介导cm驻留BSEP的内化和随后的降解，从而调节BSEP的小管表达和转运功能。这一信息将有助于了解与肝内胆汁淤积相关的严重肝脏疾病的发病机制。(肝脏病学2012;55:18891900)
The bile salt export pump (BSEP) mediates the biliary excretion of bile salts and its dysfunction induces intrahepatic cholestasis. Reduced canalicular expression of BSEP resulting from the promotion of its internalization is one of the causes of this disease state. However, the molecular mechanism underlying BSEP internalization from the canalicular membrane (CM) remains unknown. We have shown previously that 4-phenylbutyrate (4PBA), a drug used for ornithine transcarbamylase deficiency (OTCD), inhibited internalization and subsequent degradation of cell-surface-resident BSEP. The current study found that 4PBA treatment decreased significantly the expression of a- and mu 2-adaptin, both of which are subunits of the AP2 adaptor complex (AP2) that mediates clathrin-dependent endocytosis, in liver specimens from rats and patients with OTCD, and that BSEP has potential AP2 recognition motifs in its cytosolic region. Based on this, the role of AP2 in BSEP internalization was explored further. In vitro analysis with 3xFLAG-human BSEP-expressing HeLa cells and human sandwich-culture hepatocytes indicates that the impairment of AP2 function by RNA interference targeting of a-adaptin inhibits BSEP internalization from the plasma membrane and increases its cell-surface expression and transport function. Studies using immunostaining, coimmunoprecipitation, glutathione S-transferase pulldown assay, and time-lapse imaging show that AP2 interacts with BSEP at the CM through a tyrosine motif at the carboxyl terminus of BSEP and mediates BSEP internalization from the CM of hepatocytes. Conclusion: AP2 mediates the internalization and subsequent degradation of CM-resident BSEP through direct interaction with BSEP and thereby modulates the canalicular expression and transport function of BSEP. This information should be useful for understanding the pathogenesis of severe liver diseases associated with intrahepatic cholestasis. (HEPATOLOGY 2012;55:18891900)