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Molecular Pharmacology

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Research ArticleArticle

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

Kensuke Aida, Hisamitsu Hayashi, Kaori Inamura, Tadahaya Mizuno and Yuichi Sugiyama
Molecular Pharmacology March 2014, 85 (3) 482-491; DOI: https://doi.org/10.1124/mol.113.091090
Kensuke Aida
Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan (K.A., H.H., K.I., T.M.); and Sugiyama Laboratory, RIKEN Innovation Center, Research Cluster for Innovation, RIKEN, Yokohama, Japan (Y.S.)
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Hisamitsu Hayashi
Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan (K.A., H.H., K.I., T.M.); and Sugiyama Laboratory, RIKEN Innovation Center, Research Cluster for Innovation, RIKEN, Yokohama, Japan (Y.S.)
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Kaori Inamura
Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan (K.A., H.H., K.I., T.M.); and Sugiyama Laboratory, RIKEN Innovation Center, Research Cluster for Innovation, RIKEN, Yokohama, Japan (Y.S.)
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Tadahaya Mizuno
Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan (K.A., H.H., K.I., T.M.); and Sugiyama Laboratory, RIKEN Innovation Center, Research Cluster for Innovation, RIKEN, Yokohama, Japan (Y.S.)
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Yuichi Sugiyama
Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan (K.A., H.H., K.I., T.M.); and Sugiyama Laboratory, RIKEN Innovation Center, Research Cluster for Innovation, RIKEN, Yokohama, Japan (Y.S.)
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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 cells showed that ectopic expression of UbΔGG, 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 UbΔGG delayed the loss of fluorescence from photoactivated Dronpa-BSEP on the CM. The effect of UbΔGG 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 suggest 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.

Footnotes

    • Received December 6, 2013.
    • Accepted December 17, 2013.
  • K.A. and H.H. contributed equally to this work.

  • This work was supported by the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation to Y.S.; and a Grant-in-Aid for Young Scientists (B) [23790175] and 5th Mandom International Research Grants on Alternative to Animal Experiments (to H.H.).

  • dx.doi.org/10.1124/mol.113.091090.

  • Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 85 (3)
Molecular Pharmacology
Vol. 85, Issue 3
1 Mar 2014
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Research ArticleArticle

Intracellular Transport of BSEP and MRP2 via Ubiquitination

Kensuke Aida, Hisamitsu Hayashi, Kaori Inamura, Tadahaya Mizuno and Yuichi Sugiyama
Molecular Pharmacology March 1, 2014, 85 (3) 482-491; DOI: https://doi.org/10.1124/mol.113.091090

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Research ArticleArticle

Intracellular Transport of BSEP and MRP2 via Ubiquitination

Kensuke Aida, Hisamitsu Hayashi, Kaori Inamura, Tadahaya Mizuno and Yuichi Sugiyama
Molecular Pharmacology March 1, 2014, 85 (3) 482-491; DOI: https://doi.org/10.1124/mol.113.091090
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