Abstract
P-glycoprotein, the overexpression of which is a major cause for the failure of cancer chemotherapy in man, recognizes and transports a broad range of structurally unrelated amphiphilic compounds. This study reports on the localization of the binding site of P-glycoprotein for iodomycin, the Bolton-Hunter derivative of the anthracycline daunomycin. Plasma membrane vesicles isolated from multidrug-resistant Chinese hamster ovary B30 cells were photolabeled with [125I]iodomycin. After chemical cleavage behind the tryptophan residues, 125I-labeled peptides were separated by electrophoresis and high performance liquid chromatography. Edman sequencing revealed that [125I]iodomycin had been predominantly incorporated into the fragment 230-312 of isoform I of hamster P-glycoprotein. According to models based on hydropathy plots, the amino acid sequence 230-312 forms the distal part of transmembrane segment 4, the second cytoplasmic loop, and the proximal part of transmembrane segment 5 in the N-terminal half of P-glycoprotein. The binding site for iodomycin is recognized with high affinity by vinblastine and cyclosporin A.
Highlights
P-glycoprotein, the overexpression of which is a major cause for the failure of cancer chemotherapy in man, recognizes and transports a broad range of structurally unrelated amphiphilic compounds
This study reports on the localization of the binding site of P-glycoprotein for iodomycin, the Bolton-Hunter derivative of the anthracycline daunomycin
This study reports on the localization of the binding site for iodomycin in hamster P-glycoprotein
Summary
P-glycoprotein, the overexpression of which is a major cause for the failure of cancer chemotherapy in man, recognizes and transports a broad range of structurally unrelated amphiphilic compounds. Mutants that arose spontaneously during drug selection of cells and thereby changed their resistance profile were detected in the first cytoplasmic loop of human MDR1 protein (Gly185 Val) [9] and in transmembrane segment (TM) 6 of hamster P-glycoprotein (Gly338 Ala/Ala339 Pro) [10]. The exchange of amino acids in the first cytoplasmic loop [11], TM5, TM6, TM10, TM12 [13,14,15,16], and the cytosolic linker peptide [17] resulted in all in an altered multidrug resistance phenotype, suggesting that during binding and transport the substrate is recognized by multiple residues located either in the cytosolic, membraneous, or ectoplasmic domains. The topology of P-glycoprotein in vivo, may be more variable than predicted, e.g. under certain experimental conditions, TM segments were detected outside the membrane and loops postulated to face the cytosol were found on the extracellular side [21,22,23,24,25]
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