14, 28, 39 Hu et al 14 demonstrated that appending tyrosine- or d

14, 28, 39 Hu et al.14 demonstrated that appending tyrosine- or dileucine-based motifs of CFTR to a Tac reporter allows for rapid internalization, indicating that the C-terminus of CFTR contains endocytic signals. However, identifying endocytic signals in a full-length polytopic protein is often difficult because creating mutations in the putative Small molecule high throughput screening sequence by alanine scanning or sequential deletion may lead to misprocessing of the full-length protein and hamper its trafficking to the plasma membrane. For example, in full-length multidrug-resistant 1 (MDR1), mutations of analogous leucine or tyrosine residues led to misprocessing and ER retention, precluding the evaluation

of its targeting function.40 find more However, we were able to successfully mutate the tyrosines in the C-terminus of full-length

BSEP and observe the same defect in endocytosis that we had demonstrated in TacCterm. To date, there are no known disease-producing point mutations of human BSEP in the identified endocytic signal region; however, there are premature stop codons that lead to deletion of the tyrosine-based motif.3 Deletion of a major portion of the C-terminus in a human disease-causing Bsep mutant in the rat (R1050X) showed proper targeting to the apical membrane of MDCK cells, indicating that a large portion of the C-terminal nucleotide-binding domain is not necessary for biosynthetic processing and apical targeting.41 However, we and others have identified a number of BSEP mutations that cause a reduction of Bsep on the cell surface through increased rate of internalization in heterologous expression systems.30, 41 This loss of Bsep protein from the canalicular membrane is characteristic of some forms of experimental cholestatic liver injury, as well as human cholestatic liver diseases. Cholestasis induced by estradiol-17β-D-glucuronide, taurolithocholic Benzatropine acid, cyclosporine A, and lipopolycharide all result in redistribution of

Bsep to the subapical cytoplasm.7, 8, 42, 43 Efforts have been made to compensate for the loss of cell surface BSEP with the administration of chemical or pharmacological agents such as MG-132 or sodium phenylbutyrate.30, 41, 44 Although the mechanisms of action are not clearly defined for these agents, one possible explanation for the increase of BSEP cell surface expression is that these compounds limit the extent of ubiquitinylation of BSEP.45 Ubiquitinylation of membrane proteins and endocytic adaptor proteins attenuates signaling of ligand-dependent activation of receptors by targeting these receptors to the endolysosomal pathway for degradation. Hayashi et al.45 showed that attaching short-chain ubiquitin to BSEP shortens the half-life of cell surface BSEP.

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