Single amino acid (482) variants of the ABCG2 multidrug transporter

Major differences in transport capacity and substrate recognition

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64 Citations (Scopus)

Abstract

The human ABCG2 protein is an ATP binding cassette half-transporter, which protects our cells and tissues against various xenobiotics, while overexpression of ABCG2 in tumor cells confers multidrug resistance. It has been documented that single amino acid changes at position 482 resulted in altered drug resistance and transport capacity. In this study, we have generated nine Arg-482 mutants (G, I, M, S, T, D, N, K, Y) of ABCG2, and expressed them in insect cells. All ABCG2 variants showed cell surface expression and, in isolated membranes, an ABCG2-specific ATPase activity. When methotrexate accumulation was measured in inside-out membrane vesicles, this transport was supported only by the wild-type ABCG2. In intact cells, mitoxantrone was transported by all ABCG2 variants, except by R482K. Rhodamine 123 was extruded by most of the mutants, except by R482K, Y and by wild-type ABCG2. Hoechst 33342 was pumped out from cells expressing the wild-type and all Arg-482 variants, but not from those expressing R482K and Y. Our study demonstrates that the substrate specificity of the Arg (wild-type) form is unique and that amino acid replacements at position 482 induce major alterations in both the transport activity and substrate specificity of this protein.

Original languageEnglish
Pages (from-to)53-63
Number of pages11
JournalBBA - Biomembranes
Volume1668
Issue number1
DOIs
Publication statusPublished - Feb 1 2005

Fingerprint

Cells
Membranes
Rhodamine 123
Amino Acids
Mitoxantrone
Xenobiotics
Substrates
Methotrexate
Adenosine Triphosphatases
Tumors
Adenosine Triphosphate
Tissue
Substrate Specificity
Pharmaceutical Preparations
Transport Vesicles
Proteins
ATP-Binding Cassette Transporters
Multiple Drug Resistance
Drug Resistance
Insects

Keywords

  • Cell surface localization
  • Fluorescent dye extrusion
  • Membrane ATPase
  • Multidrug half-transporter
  • Single amino acid mutant
  • Vesicular transport

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Biophysics

Cite this

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title = "Single amino acid (482) variants of the ABCG2 multidrug transporter: Major differences in transport capacity and substrate recognition",
abstract = "The human ABCG2 protein is an ATP binding cassette half-transporter, which protects our cells and tissues against various xenobiotics, while overexpression of ABCG2 in tumor cells confers multidrug resistance. It has been documented that single amino acid changes at position 482 resulted in altered drug resistance and transport capacity. In this study, we have generated nine Arg-482 mutants (G, I, M, S, T, D, N, K, Y) of ABCG2, and expressed them in insect cells. All ABCG2 variants showed cell surface expression and, in isolated membranes, an ABCG2-specific ATPase activity. When methotrexate accumulation was measured in inside-out membrane vesicles, this transport was supported only by the wild-type ABCG2. In intact cells, mitoxantrone was transported by all ABCG2 variants, except by R482K. Rhodamine 123 was extruded by most of the mutants, except by R482K, Y and by wild-type ABCG2. Hoechst 33342 was pumped out from cells expressing the wild-type and all Arg-482 variants, but not from those expressing R482K and Y. Our study demonstrates that the substrate specificity of the Arg (wild-type) form is unique and that amino acid replacements at position 482 induce major alterations in both the transport activity and substrate specificity of this protein.",
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T1 - Single amino acid (482) variants of the ABCG2 multidrug transporter

T2 - Major differences in transport capacity and substrate recognition

AU - Özvegy-Laczka, C.

AU - Köblös, Gabriella

AU - Sarkadi, B.

AU - Váradi, A.

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N2 - The human ABCG2 protein is an ATP binding cassette half-transporter, which protects our cells and tissues against various xenobiotics, while overexpression of ABCG2 in tumor cells confers multidrug resistance. It has been documented that single amino acid changes at position 482 resulted in altered drug resistance and transport capacity. In this study, we have generated nine Arg-482 mutants (G, I, M, S, T, D, N, K, Y) of ABCG2, and expressed them in insect cells. All ABCG2 variants showed cell surface expression and, in isolated membranes, an ABCG2-specific ATPase activity. When methotrexate accumulation was measured in inside-out membrane vesicles, this transport was supported only by the wild-type ABCG2. In intact cells, mitoxantrone was transported by all ABCG2 variants, except by R482K. Rhodamine 123 was extruded by most of the mutants, except by R482K, Y and by wild-type ABCG2. Hoechst 33342 was pumped out from cells expressing the wild-type and all Arg-482 variants, but not from those expressing R482K and Y. Our study demonstrates that the substrate specificity of the Arg (wild-type) form is unique and that amino acid replacements at position 482 induce major alterations in both the transport activity and substrate specificity of this protein.

AB - The human ABCG2 protein is an ATP binding cassette half-transporter, which protects our cells and tissues against various xenobiotics, while overexpression of ABCG2 in tumor cells confers multidrug resistance. It has been documented that single amino acid changes at position 482 resulted in altered drug resistance and transport capacity. In this study, we have generated nine Arg-482 mutants (G, I, M, S, T, D, N, K, Y) of ABCG2, and expressed them in insect cells. All ABCG2 variants showed cell surface expression and, in isolated membranes, an ABCG2-specific ATPase activity. When methotrexate accumulation was measured in inside-out membrane vesicles, this transport was supported only by the wild-type ABCG2. In intact cells, mitoxantrone was transported by all ABCG2 variants, except by R482K. Rhodamine 123 was extruded by most of the mutants, except by R482K, Y and by wild-type ABCG2. Hoechst 33342 was pumped out from cells expressing the wild-type and all Arg-482 variants, but not from those expressing R482K and Y. Our study demonstrates that the substrate specificity of the Arg (wild-type) form is unique and that amino acid replacements at position 482 induce major alterations in both the transport activity and substrate specificity of this protein.

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