We define this endocytic motif by site-directed mutagenesis as a canonical tyrosine-based motif 1310YYKLV1314 (YxxØ). When expressed in HEK293T cells, TacCterm is constitutively internalized via a dynamin- and clathrin-dependent pathway. Mutation of the Y1310Y1311 amino acids in TacCterm and in full-length human BSEP blocks the internalization. Subsequent sequence analysis reveals this motif to be highly conserved between the closely related ABCB subfamily members that

mediate ATP-dependent transport of broad substrate specificity. Conclusion: Ibrutinib in vivo Our results indicate that constitutive internalization of BSEP is clathrin-mediated and dependent on RG-7388 research buy the tyrosine-based endocytic motif at the C-terminal end of BSEP. (HEPATOLOGY 2012;55:1901–1911) The bile salt export pump (BSEP, ABCB11) is an adenosine triphosphate (ATP)-dependent

bile salt pump that functions at the liver canalicular membrane. Mutations in BSEP that result in defective trafficking can cause cholestatic disorders including progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis, and cholestasis of pregnancy.1-3 The amount of BSEP on the canalicular membrane is regulated by postprandial demand for the enterohepatic circulation of bile salts.4 Pulse-chase experiments revealed a large intracellular pool of Bsep in rat liver that is

mobilized for targeting and recycling of Bsep to and from the canalicular membrane.5 Furthermore, Bsep constitutively recycles between the canalicular membrane and Rab11a-positive 上海皓元医药股份有限公司 endosomes in WIF-B9 cells.6 Thus, the maintenance and retrieval of BSEP on the apical membrane is crucial for its function.4, 6 The retrieval of Bsep also plays an important role in the pathophysiology of rat cholestatic models. Bsep protein is internalized in isolated perfused rat liver and rat hepatocyte couplets after estradiol 17β-D-glucuronide administration, a process blocked by protein kinase inhibitors or dibutyrl cyclic adenosine monophosphate (cAMP).7, 8 Cholestasis due to lipopolysaccharide administration, as well as oxidative stress in rats, results in internalization of Bsep.9 However, the mechanism by which Bsep is retrieved from the canalicular membrane remains largely unknown. In Madin-Darby canine kidney (MDCK) cells, dominant-negative expression of Eps15 increases the apical membrane expression of rat Bsep, suggesting that a clathrin-dependent mechanism may play a role in regulating cell surface Bsep expression.10 Clathrin has been shown to be involved in apical endocytosis in rat hepatocytes.11 Targeting and trafficking of membrane proteins depends on sequence motifs and protein–protein interactions with various forms of trafficking machinery.