Expression and Functional Characterization of Human ABC Transporter ABCG2 Variants in Insect Cells

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Author(s)

    • ISHIKAWA Toshihisa
    • Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology
    • KASAMATSU Shiho
    • Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology
    • HAGIWARA Yuko
    • Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology
    • MITOMO Hideyuki
    • Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology
    • KATO Ryo
    • Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology
    • SUMINO Yasuhiro
    • Strategic Product Planning Department, Takeda Chemical Industries, LTD.

Abstract

Hitherto three variant forms of ABCG2 have been documented on the basis of their amino acid moieties (i.e., Arg, Gly, and Thr) at the position 482. In the present study, we have generated those variants of ABCG2 by site-directed mutagenesis and expressed them in Sf9 insect cells. The apparent molecular weight of the expressed ABCG2 variants was 130, 000 under non-reductive conditions, whereas it was reduced to 65, 000 by treatment with mercaptoethanol. It is suggested that ABCG2 exists in the plasma membrane of Sf9 cells as a homodimer bound through cysteinyl disulfide bond(s). Both ATPase activity and drug transport of ABCG2 variants were examined by using plasma membrane fractions prepared from ABCG2-overexpressing Sf9 cells. The ATPase activity of the plasma membrane expressing ABCG2 (Gly-482) was significantly enhanced by prazosin. In contrast, ABCG2 (Arg-482) transports [<SUP>3</SUP>H]methotrexate in an ATP-dependent manner; however, no transport activity was observed with the other variants (Gly-482 and Thr-482). It is strongly suggested that the amino acid moiety at the position of 482 is critical for the substrate specificity of ABCG2.

Journal

  • Drug Metabolism and Pharmacokinetics

    Drug Metabolism and Pharmacokinetics 18(3), 194-202, 2003-08-20

    The Japanese Society for the Study of Xenobiotics

References:  39

Cited by:  4

Codes

  • NII Article ID (NAID)
    10011426121
  • NII NACSIS-CAT ID (NCID)
    AA1162652X
  • Text Lang
    ENG
  • Article Type
    Journal Article
  • ISSN
    13474367
  • Data Source
    CJP  CJPref  J-STAGE 
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