Characterization of the dexniguldipine binding site in the multidrug resistance-related transport protein P-glycoprotein by photoaffinity labeling and mass spectrometry.
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, [(3)H]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 [(3)H]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.
Borchers
Christoph
C
Department of Chemistry, Analytical Chemistry, University of Konstanz Konstanz, Germany.
Boer
Rainer
R
Klemm
Kurt
K
Figala
Volker
V
Denzinger
Thomas
T
Ulrich
Wolf-Rüdiger
WR
Haas
Sabine
S
Ise
Wolfgang
W
Gekeler
Volker
V
Przybylski
Michael
M
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D001386:Azides / Q000737:chemistry; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004095:Dihydropyridines / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000302:isolation & purification; D019920:Photoaffinity Labels / Q000737:chemistry; D014316:Tritium; D014357:Trypsin / Q000378:metabolism; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D001386:Azides; D004095:Dihydropyridines; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D014316:Tritium; C041130:azidopine; D014357:Trypsin; C054074:niguldipine