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Research ArticleArticle

Characterization of the Dexniguldipine Binding Site in the Multidrug Resistance-Related Transport Protein P-Glycoprotein by Photoaffinity Labeling and Mass Spectrometry

Christoph Borchers, Rainer Boer, Kurt Klemm, Volker Figala, Thomas Denzinger, Wolf-Rüdiger Ulrich, Sabine Haas, Wolfgang Ise, Volker Gekeler and Michael Przybylski
Molecular Pharmacology June 2002, 61 (6) 1366-1376; DOI: https://doi.org/10.1124/mol.61.6.1366
Christoph Borchers
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Rainer Boer
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Kurt Klemm
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Volker Figala
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Thomas Denzinger
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Wolf-Rüdiger Ulrich
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Sabine Haas
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Wolfgang Ise
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Volker Gekeler
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Michael Przybylski
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Abstract

Human P-glycoprotein (P-gp), an integral membrane transport protein, is responsible for the efflux of various drugs, including cytostatics from cancer cells leading to multidrug resistance. P-gp is composed of two homologous half domains, each carrying one nucleotide binding site. The drug extrusion is ATP-dependent and can be inhibited by chemosensitizers, such as the dihydropyridine derivative dexniguldipine-HCl, through direct interaction with P-gp. To evaluate the mechanism(s) of chemosensitization and identify the binding sites of dexniguldipine-HCl, a tritium-labeled azido analog of dexniguldipine, [3H]B9209-005, was used as a photoaffinity probe. Using the multidrug resistant T-lymphoblastoid cell line CCRF-ADR5000, two proteins were specifically labeled in membranes by [3H]B9209-005. These proteins were identified by immunoprecipitation such as P-gp and its N-terminal fragment. The membranes were solubilized and the labeled P-gp proteins first isolated by lectin-chromatography and then digested with trypsin. SDS-polyacrylamide gel electrophoresisanalysis of the digest revealed a major radioactive 7-kDa fragment. The tryptic fragments were separated by high-performance liquid chromatography and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The MS results, corroborated by MALDI-MS of peptides after one step of Edman analysis, identified the radioactive 7-kDa band as the dexniguldipine-bound, tryptic P-gp peptide, 468–527. This sequence region is flanked by the Walker motifs A and B of the N-terminal ATP-binding cassette suggesting direct interaction of the chemosensitizer with the nucleotide binding site is involved in the mechanism of chemosensitization.

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Molecular Pharmacology: 61 (6)
Molecular Pharmacology
Vol. 61, Issue 6
1 Jun 2002
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Research ArticleArticle

Characterization of the Dexniguldipine Binding Site in the Multidrug Resistance-Related Transport Protein P-Glycoprotein by Photoaffinity Labeling and Mass Spectrometry

Christoph Borchers, Rainer Boer, Kurt Klemm, Volker Figala, Thomas Denzinger, Wolf-Rüdiger Ulrich, Sabine Haas, Wolfgang Ise, Volker Gekeler and Michael Przybylski
Molecular Pharmacology June 1, 2002, 61 (6) 1366-1376; DOI: https://doi.org/10.1124/mol.61.6.1366

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Research ArticleArticle

Characterization of the Dexniguldipine Binding Site in the Multidrug Resistance-Related Transport Protein P-Glycoprotein by Photoaffinity Labeling and Mass Spectrometry

Christoph Borchers, Rainer Boer, Kurt Klemm, Volker Figala, Thomas Denzinger, Wolf-Rüdiger Ulrich, Sabine Haas, Wolfgang Ise, Volker Gekeler and Michael Przybylski
Molecular Pharmacology June 1, 2002, 61 (6) 1366-1376; DOI: https://doi.org/10.1124/mol.61.6.1366
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