Dubin-Johnson syndrome (DJS) is a hereditary disease characterized by hyperbilirubinemia.We investigated the consequences of 2 missense mutations, R768W and Q1382R, of nucleotidebinding domains (NBDs) of the multidrug resistance protein 2 (MRP2; ABCC2) that were previously identified in patients with DJS. Pulse chase analysis revealed that the precursor form of the wild-type and Q1382R MRP2 were converted to the mature form, which is resistant to endoglycosidase H (Endo H) in about 60 minutes. However, the precursor form of the R768W MRP2, which is sensitive to endoglycosidase H, was degraded within 120 minutes and did not mature to the fully glycosylated form. Proteasome inhibitors inhibited the degradation of the precursor form of the R768W MRP2. Unlike the R768W MRP2, the Q1382R MRP2 was mainly localized on the apical membrane in the wild-type form. However, efflux of glutathione monochlorobimane (GS-MCLB) and ATP-dependent leukotriene C 4 (LTC 4 ) uptake into plasma membrane vesicles from cells expressing the Q1382R MRP2 were markedly reduced, suggesting that the Q1382R MRP2 on the apical membrane was nonfunctional. Vanadateinduced nucleotide trapping with 8-azido-[␣-32P]ATP in the wild-type MRP2 was stimulated by estradiol glucuronide (E 2 17␤G) in a concentration-dependent manner but that in the Q1382R MRP2 was not. In conclusion, the R768W mutation causes deficient maturation and impaired sorting, and the Q1382R mutation does not affect maturation or sorting but impairs the substrate-induced ATP hydrolysis. (HEPATOLOGY 2002;36:1236-1245

The human multidrug resistance protein MRP1 and its homolog, MRP2, are both thought to be involved in cancer drug resistance and the transport of a wide variety of organic anions, including the cysteinyl leukotriene C 4 (LTC 4 ) (K m ‫؍‬ 0.1 and 1 M). To determine which domain of these proteins is associated with substrate specificity and subcellular localization, we constructed various chimeric MRP1/MRP2 molecules and expressed them in polarized mammalian LLC-PK1 cells. We examined the kinetic properties of each chimeric protein by measuring LTC 4 and methotrexate transport in insideout membrane vesicles, sensitivity to an anticancer agent, etoposide, and subcellular localization by indirect immunofluorescence methods. The following results were determined in these studies: (i) when the NH 2 -proximal 108 amino acids of MRP2, including transmembrane (TM) helices 1-3, were exchanged with the corresponding region of MRP1, K m (LTC 4 ) values of the chimera decreased ϳ4-fold and K m (methotrexate) values increased ϳ5-fold relative to those of wild-type MRP2 and MRP1, respectively, whereas resistance to etoposide increased ϳ3-fold; (ii) when the NH 2 -proximal region up to TM9 of MRP2 was exchanged with the corresponding region of MRP1, a further increase in etoposide resistance was observed, and subcellular localization moved from the apical to the lateral membrane; (iii) when two-thirds of MRP2 at the NH 2 terminus were exchanged with the corresponding MRP1 region, the chimeric protein transported LTC 4 with an efficiency comparable with that achieved by the wild-type MRP1; and (iv) exchange of the COOH-terminal 51 amino acids between MRP1 and MRP2 did not affect the localization of either of the proteins. These results provide a strong framework for further studies aimed at determining the precise domains of MRP1 and MRP2 with affinity for LTC 4 and anticancer agents. Two representative genes for the ATP binding cassette (ABC)1 transporter superfamily proteins, P-gp/MDR1 and MRP1, mediate acquisition of a multidrug resistance phenotype through altered membrane transport of various anticancer agents in tumor cells (1, 2). MRP1 confers resistance to a number of relatively hydrophobic natural product drugs including certain anthracyclines, epipodophyllotoxins, methotrexate, and vinca alkaloids (3-7). However, unlike P-gp, MRP1 can also transport a wide range of relatively hydrophilic anionic compounds including potential physiological substrates such as LTC 4 and E 2 17␤G (8 -15). Topology studies of MRP1 have demonstrated that MRP1 and P-gp share a similar core structure consisting of two membrane-spanning domains (MSD2 and MSD3) and two nucleotide-binding domains (NBD1 and NBD2), referred to as the MDR-like core (16). The primary distinguishing characteristic of MRP1 and its related proteins, MRP2, -3, -6, and -7, is an additional NH 2 -terminal region forming a membrane-spanning domain (MSD1) with five

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