TY - JOUR T1 - Characterization of the Dexniguldipine Binding Site in the Multidrug Resistance-Related Transport Protein P-Glycoprotein by Photoaffinity Labeling and Mass Spectrometry JF - Molecular Pharmacology JO - Mol Pharmacol SP - 1366 LP - 1376 DO - 10.1124/mol.61.6.1366 VL - 61 IS - 6 AU - Christoph Borchers AU - Rainer Boer AU - Kurt Klemm AU - Volker Figala AU - Thomas Denzinger AU - Wolf-Rüdiger Ulrich AU - Sabine Haas AU - Wolfgang Ise AU - Volker Gekeler AU - Michael Przybylski Y1 - 2002/06/01 UR - http://molpharm.aspetjournals.org/content/61/6/1366.abstract N2 - 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. ER -