AP2 adaptor complex mediates bile salt export pump internalization and modulates its hepatocanalicular expression and transport function

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 aand l2-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 33FLAG-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 CMresident 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;55:1889-1900

Differential Roles of Ubiquitination in the Degradation Mechanism of Cell Surface–Resident Bile Salt Export Pump and Multidrug Resistance–Associated Protein 2

“…Coimmunoprecipitation and subsequent IB analysis showed that 3Â FLAG-BSEP, which is trafficked to the plasma membrane as native BSEP and shows a degradation rate from the cell surface similar to that of native BSEP (Hayashi et al, 2012a), and MRP2 were both ubiquitinated in the same way in 293 cells as in rat hepatocytes, MDCKII cells, and McA-RH7777 cells (Hayashi and Sugiyama, 2009;Hayashi et al, 2012b). Smeared bands around 190 and 210 kDa were detected by anti-Ub antibody in the immunoprecipitates from 293 cells expressing 3Â FLAG-BSEP and MRP2 with anti-FLAG and anti-MRP2 antibodies, respectively (Fig.…”

“…Dronpa-BSEP is trafficked to the CM and undergoes the same regulation for internalization as native BSEP (Hayashi et al, 2012a). In this assay, the internalization of Dronpa-BSEP contributes to the disappearance of fluorescence from the CM after photoactivation of the CM-resident Dronpa-BSEP (Fig.…”

“…EV, HA-Ub DGG , and HA-Ub DGG/I44A 293 cells were seeded onto 24-well plates coated with poly-L-lysine and poly-L-ornithine at a density of 1.5 Â 10 5 cells per well. The cells were cultured for 48 hours at 37°C in DMEM with 10% FBS and 2 mM tetracycline to induce the expression of EV, HA-Ub DGG , and HAUb DGG/I44A and subjected to a [ 125 I]Tf uptake study as described previously (Hayashi et al, 2012a). The uptake of [ …”

“…Photoactivation of Dronpa-BSEP and Dronpa-BSEP Y1311A was performed as described previously (Hayashi et al, 2012a). Fluorescence intensity in the photoactivated region at each time point was manually determined by measuring the pixel sum using LAS AF (Leica).…”

“…We have previously shown that a C-terminal tyrosine-based motif, 1311 YKL 1314 V, of BSEP plays a dominant role in its internalization through interaction with the AP2 adaptor complex (AP2) that functions in cargo selection during clathrin-mediated endocytosis (Hayashi et al, 2012a). To examine the mutual relationship between BSEP internalization mediated by ubiquitination and that mediated by clathrin-mediated endocytosis involving AP2, we studied the effect of Ub DGG on a mutated form of BSEP, BSEP Y1311A , unable to interact with AP2 (Hayashi et al, 2012a).…”