[Show abstract][Hide abstract]ABSTRACT: We previously showed that ubiquitination, a reversible post-translational modification, facilitates degradation of cell surface-resident bile salt export pump (BSEP) and multidrug resistance-associated protein 2 (MRP2), ABC transporters that are expressed at the canalicular membrane (CM) of hepatocytes. In the current study, its underlying mechanism was investigated by evaluating the role of ubiquitination in the processes of internalization and subsequent degradation of cell surface-resident BSEP and MRP2. Cell surface biotinylation analysis using Flp-In T-REx 293 (293) cells showed that ectopic expression of Ub(ΔGG), which is ubiquitin (Ub) lacking the two C-terminal glycines essential for the Ub conjugation reaction, inhibited the internalization of 3×FLAG-BSEP, but not of MRP2, and the degradation of the internalized MRP2, but not of the internalized 3×FLAG-BSEP. Its inhibitory effect on BSEP internalization was also indicated by a time-lapse imaging analysis using the rat hepatoma cell line McA-RH7777 in which Ub(ΔGG) delayed the loss of fluorescence from photoactivated Dronpa-BSEP on the CM. The effect of Ub(ΔGG) on BSEP internalization in these experiments was abrogated by treatment with chlorpromazine, an inhibitor of clathrin-mediated endocytosis, and the introduction of a Y1311A mutation into BSEP. This mutation eliminates the ability of BSEP to interact with the AP2 adaptor complex, an adaptor protein required for cargo selection in clathrin-mediated endocytosis. In conclusion, our data suggests that ubiquitination facilitates clathrin-mediated endocytosis of BSEP and the degradation of internalized MRP2, leading to the degradation of the cell surface-resident form of both transporters.
Preview · Article · Dec 2013 · Molecular pharmacology
[Show abstract][Hide abstract]ABSTRACT: 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 α- and μ2-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 3×FLAG-human BSEP-expressing HeLa cells and human sandwich-culture hepatocytes indicates that the impairment of AP2 function by RNA interference targeting of α-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.
[Show abstract][Hide abstract]ABSTRACT: Bile salt export pump (BSEP/ABCB11) mediates the biliary excretion of bile acids in an ATP-dependent manner. Reduced BSEP expression at the canalicular membrane caused by various factors like pregnancy and sepsis can lead to dysfunction of BSEP, ultimately inducing or deteriorating intrahepatic cholestasis. Although the rapid internalization and subsequent degradation of BSEP has been suggested to be responsible for the reduced BSEP expression under cholestatic conditions, the underlying molecular mechanism has not yet been clarified. Our current study explored the molecular mechanism of BSEP internalization. We specifically focused on clathrin adaptor protein complex 2 (AP2), which helps clathrin-mediated internalization by recruiting targeted protein into clathrin coated pits, since BSEP has several potential recognition sites for AP2 in its cytosolic region. Studies using immunostaining and co-immunoprecipitation showed the colocalization of BSEP with α-subunit of AP2 (AP2α) on the plasma membrane in 3×FLAG BSEP-expressing HeLa cells and the canalicular membrane in rat hepatocytes as well as interaction between BSEP and AP2α, suggesting the plasma membrane as a potential site of interaction. Biotinylation study demonstrated that 3×FLAG-BSEP expression at the cell surface was markedly enhanced in clathrin heavy chain knockdown HeLa cells and AP2 function-deficient HeLa cells, which were constructed by siRNA-mediated depletion of AP2α. In addition, the internalization rate of 3×FLAG-BSEP was significantly slower in AP2 function-deficient HeLa cells than in control cells by almost 85%. In conclusion, AP2 mediates clathrin-mediated internalization of BSEP through the direct interaction with BSEP, and thereby negatively modulates BSEP expression at the cell surface.