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 ...

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