The multidrug resistance protein is photoaffinity labeled by a quinoline-based drug at multiple sites

Tumor cells overcome cytotoxic drug pressure by the overexpression of eithe r or both transmembrane proteins, the P-glycoprotein (P-gp) and the multidr ug resistance protein (MRP). The MRP has been shown to mediate the transpor t of cytotoxic natural products, in addition to glutathione-, glucuronidate -, and sulfate-conjugated cell metabolites. However, the mechanism of MRP d rug binding and transport is at present not clear. In this study, we have u sed a photoreactive quinoline-based drug, N-(hydrocinchonidin-8'-yl)-4-azid o-2-hydroxybenzamide (IACI), to show the photoaffinity labeling of the 190 kDa protein in membranes from the drug resistant SCLC H69/AR cells. The pho toaffinity labeling of the 190 kDa protein by IACI was saturable and specif ic. The identity of the IACI-photolabeled protein as the MRP was confirmed by immunoprecipitation with the monoclonal antibody QCRL-1. Furthermore, a molar excess of leukotriene C-4, doxorubicin, colchicine, and other quinoli ne-based drugs, including MK571, inhibited the photoaffinity labeling of th e MRP. Drug transport studies showed lower IACI accumulation in MRP-express ing cells which was reversed by depleting ATP levels in H69/AR cells. Mild digestion of the purified IACI-photolabeled MRP with trypsin showed two lar ge polypeptides (similar to 111 and similar to 85 kDa). The 85 kDa polypept ide which contains the QCRL-1 and MRPm6 monoclonal antibody epitopes corres ponds to the C-terminal half of the MRP (amino acids similar to 900-1531) c ontaining the third multiple spanning domain (MSD3) and the second nucleoti de binding site. The 111 kDa polypeptide which contains the epitope sequenc e of the MRPr1 monoclonal antibody encodes the remainder of the MRP sequenc e (amino acids 1-900) containing the MSD1 and MSD2 plus the first nucleotid e binding domain. Cleveland maps of purified IACI-labeled 85 and 111 kDa po lypeptides revealed 6 kDa and similar to 6 plus 4 kDa photolabeled peptides , respectively. In addition, resolution of the exhaustively digested IACI-p hotolabeled MRP by HPLC showed two major and one minor radiolabeled peaks t hat eluted late in the gradient (60 to 72% acetonitrile). Taken together, t he results of this study show direct binding of IACI to the MRP at physiolo gically relevant sites. Moreover, IACI photolabels three small peptides whi ch localize to the N- and C-halves of the MRP. Finally, IACI provides a sen sitive and specific probe for studying MRP-drug interactions.