Characterization of the amino-terminal regions in the human multidrug resistance protein (MRP1)

The human multidrug resistance protein (MRP1) contributes to drug resistanc e in cancer cells. In addition to an MDR1-like core, MRP1 contains an N-ter minal membrane-bound (TMD0) region and a cytoplasmic linker (L-0), both cha racteristic of several members of the MRP family. In order to study the rol e of the TMD0 and L-0 regions, we constructed various truncated and mutated MRP1, and chimeric MRP1-MDR1 molecules, which were expressed in insect (Sf 9) and polarized mammalian (MDCKII) cells. The function of the various prot eins was examined in isolated membrane vesicles by measuring the transport of leukotriene C-4 and other glutathione conjugates, and by vanadate-depend ent nucleotide occlusion, Cellular localization, and glutathione-conjugate and drug transport, were also studied in MDCKII cells. We found that chimer ic proteins consisting of N-terminal fragments of MRP1 fused to the N termi nus of MDR1 preserved the transport, nucleotide occlusion and apical membra ne routing of wild-type MDR1, As shown before, MRP1 without TMD0L0 (Delta M RP1), was non-functional and localized intracellularly, so we investigated the coexpression of Delta MRP1 with the isolated L-0 region, Coexpression y ielded a functional MRPI molecule in Sf9 cells and routing to the lateral m embrane in MDCKII cells. Interestingly, the L-0 peptide was found Co be ass ociated with membranes in Sf9 cells and could only be solubilized by urea o r detergent. A 10-amino-acid deletion in a predicted amphipathic region of L-0 abolished its attachment to the membrane and eliminated MRP1 transport function, but did not affect membrane routing. Taken together, these experi ments suggest that the L-0 region forms a distinct domain within MRP1, whic h interacts with hydrophobic membrane regions and with the core region of M RP1.