title: Characterization of rhodamine 123 binding to P-glycoprotein in human multidrug-resistant cells.
abstract: The overexpression of P-glycoprotein is currently believed to be responsible for the enhanced efflux or decreased influx of cytotoxic drugs across the cell membrane in drug-resistant cells. P-glycoprotein has been proposed to mediate the efflux of a large number of structurally and functionally unrelated drugs. Although it has been suggested that P-glycoprotein binds directly to many lipophilic cations, it remains unclear whether one or more sites in P-glycoprotein mediate its broad substrate specificity. In this report, a photoactive derivative of rhodamine 123 (Rh123) [125I-azidosalicylic acid (ASA)-Rh123] was synthesized and used in a photoaffinity labeling assay to demonstrate, for the first time, direct and specific binding to P-glycoprotein. The photoaffinity labeling of P-glycoprotein by ASA-Rh123 was specifically inhibited in the presence of vinblastine and verapamil but not in the presence of colchicine. Surprisingly, ASA-Rh123 photoaffinity labeled a 6-kDa V8 peptide in P-glycoprotein that was previously shown to be photoaffinity labeled by another multidrug resistance-associated drug, [125I]iodoarylazidoprazosin. Photoaffinity labeling of mitochondria from drug-sensitive or -resistant cells with 125I-ASA-Rh123 did not reveal significant differences in the mitochondrial proteins from sensitive or resistant cells. Interestingly, however, 125I-ASA-Rh123 did photolabel a 66-kDa protein in mitochondria that was not detected in plasma membrane preparations with this assay. Taken together, our results demonstrate for the first time that Rh123 binds specifically to P-glycoprotein and that its binding site may be shared by other multidrug resistance-associated drugs.
authors.affiliation: Institute of Parasitology, McGill University, Ste. Anne de Bellevue, Canada.
authors.forename: B
authors.initials: B
authors.lastname: Nare
authors.forename: R K
authors.initials: RK
authors.lastname: Prichard
authors.forename: E
authors.initials: E
authors.lastname: Georges
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000345:Affinity Labels; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D003078:Colchicine / Q000494:pharmacology; D004351:Drug Resistance; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism*; D020112:Rhodamine 123; D012235:Rhodamines / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014700:Verapamil / Q000494:pharmacology; D014750:Vincristine / Q000494:pharmacology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000345:Affinity Labels; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D012235:Rhodamines; D020112:Rhodamine 123; D014750:Vincristine; D014700:Verapamil; D003078:Colchicine