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

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Abstract

The human multidrug resistance protein (MRP1) contributes to drug resistance in cancer cells. In addition to an MDR1-like core, MRP1 contains an N-terminal membrane-bound (TMD0) region and a cytoplasmic linker (L0), both characteristic of several members of the MRP family. In order to study the role of the TMD0 and L0 regions, we constructed various truncated and mutated MRP1, and chimeric MRP1-MDR1 molecules, which were expressed in insect (Sf9) and polarized mammalian (MDCKII) cells. The function of the various proteins was examined in isolated membrane vesicles by measuring the transport of leukotriene C4 and other glutathione conjugates, and by vanadate-dependent nucleotide occlusion. Cellular localization, and glutathione-conjugate and drug transport, were also studied in MDCKII cells. We found that chimeric proteins consisting of N-terminal fragments of MRP1 fused to the N terminus of MDR1 preserved the transport, nucleotide occlusion and apical membrane routing of wild-type MDR1. As shown before, MRP1 without TMD0L0 (ΔMRP1), was non-functional and localized intracellularly, so we investigated the coexpression of ΔMRP1 with the isolated L0 region. Coexpression yielded a functional MRP1 molecule in Sf9 cells and routing to the lateral membrane in MDCKII cells. Interestingly, the L0 peptide was found to be associated with membranes in Sf9 cells and could only be solubilized by urea or detergent. A 10-amino-acid deletion in a predicted amphipathic region of L0 abolished its attachment to the membrane and eliminated MRP1 transport function, but did not affect membrane routing. Taken together, these experiments suggest that the L0 region forms a distinct domain within MRP1, which interacts with hydrophobic membrane regions and with the core region of MRP1.

Original languageEnglish
Pages (from-to)4451-4461
Number of pages11
JournalJournal of cell science
Volume113
Issue number24
Publication statusPublished - dec. 1 2000

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ASJC Scopus subject areas

  • Cell Biology

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