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. 125 I]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 Nterminal half of P-glycoprotein. The binding site for iodomycin is recognized with high affinity by vinblastine and cyclosporin A. P-glycoprotein, which belongs to the large family of ABC 1 transporters (1), binds and transports a broad range of structurally unrelated compounds (2). Its overexpression may cause the phenomenon of multidrug resistance (MDR) during cancer chemotherapy, whereby the tumor cells become resistant to a variety of antineoplastic agents due to a reduced intracellular accumulation of drugs (2-4). The MDR phenotype can be overcome by modulators, i.e. substances that are bound by P-glycoprotein and inhibit its drug excluding function (3). The medically important substrates of P-glycoprotein comprise anticancer drugs such as Vinca alkaloids and anthracyclines (2-4) and approved drugs that turned out to be potent modulators such as verapamil (5, 6), calcium antagonists (7), and cyclosporins (8).The major issue of how P-glycoprotein can handle so many substrates has been mainly approached by photoaffinity labeling and mutagenesis studies. 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 (Gly 185 3 Val) (9) and in transmembrane segment (TM) 6 of hamster P-glycoprotein (Gly 338 3 Ala/Ala 339 3 Pro) (10). Active mutagenesis identified further motifs and positions in the N-and C-terminal half of P-glycoprotein which influence substrate specificity (11, 12). For example, the exchange of amino acids in the first cytoplasmic loop (11), TM5, TM6, TM10, TM12 (13-16), 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.Hydropathy plots deduced from the primary sequence predict that P-glycoprotein consists principally of two symmetrical halves, each of which contain a membrane domain with six membrane-spanning segments and a subsequent cytosolic nucleotide binding fold (18 -20). The topology of P-glycoprotein in vivo, however, 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 foun...

P-glycoprotein is an ATP-dependent drug-efflux pump which can transport a diverse range of structurally and functionally unrelated substrates across the plasma membrane. Overexpression of this protein may result in multidrug resistance and is a major cause of the failure of cancer chemotherapy. The most commonly used photoreactive substrate is iodoarylazidoprazosin. Its binding domains within the P-glycoprotein have so far been inferred from indirect methods such as epitope mapping. In this study, the binding sites were refined and relocalized by direct analysis of photolabeled peptides. P-glycoproteincontaining plasma membrane vesicles of Chinese hamster ovary B30 cells were photoaffinity-labeled with iodoarylazidoprazosin. After chemical cleavage behind tryptophan residues or enzymatic cleavage behind lysine residues, the resulting 125 I-labeled peptides were separated by tricine/ PAGE and HPLC and subjected to Edman sequencing. The major photoaffinity binding sites of iodoarylazidoprazosin were localized in the amino-acid regions 248±312 [transmembrane segment (TM)4 to TM5], 758±800 (beyond TM7 to beyond TM8) and 1160±1218 (after the Walker A motif of the second nucleotide-binding domain). Therefore the binding pocket of iodoarylazidoprazosin is made up of at least three binding epitopes.Keywords: ABC transporter; multidrug resistance; P-glycoprotein; photoaffinity labeling.Chemotherapy is one of the three major options for the treatment of cancer. However, after the initial treatment with cytostatic drugs, tumor cells can become resistant to a broad range of structurally different drugs. Such resistance during chemotherapy is known as multidrug resistance (MDR). MDR is mainly based on overexpression of P-glycoprotein, an ATP-binding cassette (ABC) transporter. P-glycoprotein actively transports many amphiphilic cytostatic drugs out of cancer cells (causing resistance against these drugs) [1±4].The large 1276 amino-acid plasma membrane protein P-glycoprotein is made up of 12 transmembrane segments (TMs) and two nucleotide-binding domains (NBDs) [3]. The major issue of how P-glycoprotein can handle so many substances has been mainly approached by photoaffinity labeling and mutagenesis studies [5,6]. Active mutagenesis identified numerous motifs and positions in the transmembrane domains and both nucleotide-binding folds which are directly or indirectly involved in drug binding [7]. These data demonstrate the complexity of the 3D structure of the substrate-binding pocket of P-glycoprotein. Moreover, binding and transport of the drug were found to be coupled to ATP hydrolysis [8] Epitope mapping revealed two major photobinding sites for the prazosin derivative [ 125 I]iodoarylazidoprazosin in each half of the P-glycoprotein (TM6 and TM12) and one minor photobinding site from TM4 up to, but not including, TM6 [15]. The minor photolabeling site overlaps that found for its photoreactive P-glycoprotein substrates iodipine and iodomycin [10,11], the second cytoplasmic loop between TM4 and TM5. Drug-transport studies ...

Both the overexpression of P-glycoprotein and the broad range of substrates of this ATP-binding cassette (ABC) transporter induce the phenomenon of multidrug resistance, one major cause of the failure of cancer chemotherapy in humans. This study reports that [ 125 I]iodipine, a structural analogue of the 1,4-dihydropyridine azidopine, shares a common binding site with iodomycin, a Bolton±Hunter derivative of the anthracycline daunomycin. This binding site is different from that described for iodoarylazidoprazosin, which is presumed to share a common binding site with azidopine. Edman sequencing revealed that [ 125 I]iodipine had photolabelled the same peptide as iodomycin and spans the primary sequence of hamster isoform pgp1 from amino acid 230 to amino acid 312.Keywords: ABC transporter; P-glycoprotein; photoaffinity labeling.The overexpression of P-glycoprotein is thought to be one reason for the failure of human cancer chemotherapy. P-glycoprotein confers resistance to a broad spectrum of structurally and chemically unrelated compounds by reducing the intracellular concentration of drugs [1]. This phenomenon of multidrug resistance (MDR) can be overcome by so-called modulators such as verapamil, cyclosporins and dihydropyridine calcium channel blockers, which resensitize the tumour cells by blocking the drug exclusion function of P-glycoprotein [2]. P-glycoprotein belongs to the protein superfamily of ATP-binding cassette (ABC) transporters. Predicted by hydropathy plot analysis, the 170-kDa large plasma membrane protein is made up of 12 transmembrane domains and two cytosolic nucleotide-binding folds [3]. A central issue of P-glycoprotein research is the question of how this protein is able to recognize and transport so many unrelated compounds. [14]. Azidopine photolabels two sites on P-glycoprotein, one in the N-terminal half between amino acid residues 198 and 440 and the other in the C-terminal half [10]. The binding site for azidopine was not analysed further (Fig. 5), but it is presumed that azidopine and [ 125 I]iodoarylazidoprazosin share a common binding site on P-glycoprotein [15].Another photoreactive substrate of P-glycoprotein is iodomycin, a photoreactive Bolton±Hunter derivative of the anthracycline daunomycin [16]. Recently we were able to localize by direct amino acid sequence analysis the [ 125 I]iodomycin-binding site of hamster isoform pgp1 [13] N-terminal in a segment of the primary sequence between amino acid 230 (TM4) and amino acid 312 (TM5).The 1,4-dihydropyridine derivative iodipine, known to be a modulator of MDR [17], was used at an early stage to characterize L-type Ca 2+ channels [18]. The photobinding of the inherently photoreactive iodipine to P-glycoprotein from Chinese hamster ovary (CHO) B30 plasma-membrane vesicles was demonstrated by . Iodipine shares the Bolton±Hunter group with iodomycin but otherwise has a structure very similar to that of azidopine (Fig. 1), for which reason the latter has been extensively employed as a reference compound in studies of drug binding ...