Identification and localization of three photobinding sites of iodoarylazidoprazosin in hamster P‐glycoprotein

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‐glycoprotein‐containing 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.