Receptor for activated C-kinase 1 regulates the cellular localization and function of ABCB4
Receptor for activated C-kinase 1 regulates the cellular localization and function of ABCB4
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DOI：
10.1111/j.1872-034x.2009.00544.x
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发表时间：
2009-11-01
影响因子：
4.2
通讯作者：
Suzuki, Hiroshi
中科院分区：
文献类型：
--
作者：
Ikebuchi, Yuki;Takada, Tappei;Suzuki, Hiroshi
目的：多药耐药蛋白3（multidrug resistance protein 3，MDR 3/ABCB 4）是位于肝细胞胆小管膜上的一种蛋白，负责磷脂酰胆碱的跨膜转运，其遗传缺陷可导致肝脏疾病，如进行性家族性肝内胆汁淤积症3型。方法：利用酵母双杂交技术，以人ABCB 4胞浆连接区为模板，从人肝脏cDNA文库中筛选ABCB 4的表面表达蛋白。该筛选使我们能够鉴定活化C激酶1（RACK 1）的受体作为ABCB 4的新型结合伴侣。结果：siRNA下调HeLa细胞内源性RACK 1表达后，ABCB 4定位于胞浆区，蛋白质表达减少，但内源性RACK 1的抑制并不影响ABCB 4的mRNA表达和蛋白质稳定性。通过抑制HepG 2细胞中内源性RACK 1表达，也发现ABCB 4细胞定位的类似改变。因此，ABCB 4介导的磷脂酰胆碱易位活性显着降低时，内源性RACK 1表达在HeLa细胞中被抑制。与此相反，ABCB 1的膜表面定位和蛋白表达不受内源性RACK 1表达的抑制。结论：这些结果表明，RACK 1可能具有功能意义的ABCB 4的调节辅因子，是必不可少的质膜定位和转运功能的ABCB 4。
Aim:Multidrug resistance protein 3 (MDR3/ABCB4), located on the bile canalicular membrane of hepatocytes, is responsible for the translocation of phosphatidylcholine across the plasma membrane, and its hereditary defect causes liver disorders, such as progressive familial intrahepatic cholestasis type 3. We aimed to identify the proteins responsible for the surface expression of human ABCB4.Methods:We performed yeast two-hybrid screening with the cytoplasmic linker region of ABCB4 against a human liver cDNA library. This screening allowed us to identify the receptor for activated C-kinase 1 (RACK1) as a novel binding partner of ABCB4. The association of RACK1 with the linker region of ABCB4 was further confirmed by GST-pulldown assay, although we could not find out the interaction of full length of ABCB4 and RACK1 in co-immunoprecipitation assay in HeLa cells.Results:Down-regulation of endogenous RACK1 expression by siRNA in HeLa cells resulted in the localization of ABCB4 in the cytosolic compartment as well as reduced protein expression of ABCB4, although mRNA expression and the protein stability of ABCB4 were not affected by the suppression of endogenous RACK1. Similar alterations in cellular localization of ABCB4 were also found by suppressing endogenous RACK1 expression in HepG2 cells. Consequently, ABCB4-mediated phosphatidylcholine translocation activity was significantly reduced when endogenous RACK1 expression was suppressed in HeLa cells. In contrast, the membrane surface localization and the protein expression of ABCB1 were not affected by the suppression of endogenous RACK1 expression.Conclusion:These results suggest that RACK1 may have a functional significance as a regulatory cofactor of ABCB4 and is indispensable for the plasma membrane localization and translocation function of ABCB4.