Glutathione-dependent binding of a photoaffinity analog of agosterol A to the C-terminal half of human multidrug resistance protein

MRP1 is a 190-kDa membrane glycoprotein that confers multidrug resistance ( MDR) to tumor cells. MRP1 is characterized by an N-terminal transmembrane d omain (TMD0), which is connected to a P-glycoprotein-like core region (Delt a MRP) by a cytoplasmic Linker domain zero (L-0). It has been demonstrated that GSH plays an important role in MRP1-mediated MDR. However, the mechani sm by which GSH mediates MDR and the precise roles of TMD0 and L-0 are not known. We synthesized [I-125]11-azidophenyl agosterol A ([I-125]azidoAG-A), a photoaffinity analog of the MDR-reversing agent, agosterol A (AGA), to p hotolabel MRP1, and found that the analog photolabeled the C-proximal molec ule of MRP1 (C932-1531) in a manner that was GSH-dependent. The photolabeli ng was inhibited by anticancer agents, reversing agents and leukotriene C-4 . Based on photolabeling studies in the presence and absence of GSH using m embrane vesicles expressing various truncated, co-expressed, and mutated MR P1s, we found that L-0 is the site on MRP1 that interacts with GSH. This st udy demonstrated that GSH is required for the binding of an unconjugated ag ent to MRP1 and suggested that GSH interacts with L-0 of MRP1. The photoana log of AG-A will be useful for identifying the drug binding site within MRP 1, and the role of GSH in transporting substrates by MRP1.