Receptor for activated C‐kinase 1 regulates the cellular localization and function of ABCB4

Abstract

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. 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. 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. 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.