Identification of Domains Participating in the Substrate Specificity and Subcellular Localization of the Multidrug Resistance Proteins MRP1 and MRP2

Abstract

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

Role of the NH2-terminal Membrane Spanning Domain of Multidrug Resistance Protein 1/ABCC1 in Protein Processing and Trafficking

“…Thus, the COOH-terminal region of MRP2 can indeed substitute for the native region of MRP1, even in the absence of MSD0. It also has been reported that the apical location of MRP2 is unaffected by exchange of MSD0 and part of CL3 for the analogous region from MRP1 (Konno et al, 2003). Consistent with this finding, we confirmed that MRP1 1-203 / MRP2 189 -1545 localized to apical membranes in the polarized MDCK-I cells used for this study (Supplementary Data Figure 3B).…”

“…Processing and Trafficking of MRP1 Hybrid Proteins Recently, it has been shown that the COOH-terminal region following the Walker B motif in NBD2 of MRP1 can be exchanged for the analogous domain from MRP2 without affecting activity or plasma membrane localization (Konno et al, 2003;Westlake et al, 2004). Consequently, we determined whether this was also true in the absence of MSD0.…”

“…Thus, although the integrity of the cytoplasmic loop immediately after MSD0 is essential for protein processing and activity, it has been concluded that MSD0 is dispensable, at least with respect to trafficking and transport of some substrates. Nevertheless, certain point mutations or deletions in MSD0 decrease or eliminate MRP1 transport activity (Gao et al, 1998;Leslie et al, 2003;Ito et al, 2003) and a recent study of hybrid proteins in which TM1-3 of MSD0 were switched between MRP1 and MRP2 concluded that this NH 2 -terminal region influenced the kinetics of leukotriene C 4 (LTC 4 ) and methotrexate transport by the two homologues (Konno et al, 2003).…”

“…Thus, although the integrity of the cytoplasmic loop immediately after MSD0 is essential for protein processing and activity, it has been concluded that MSD0 is dispensable, at least with respect to trafficking and transport of some substrates. Nevertheless, certain point mutations or deletions in MSD0 decrease or eliminate MRP1 transport activity (Gao et al, 1998;Leslie et al, 2003;Ito et al, 2003) and a recent study of hybrid proteins in which TM1-3 of MSD0 were switched between MRP1 and MRP2 concluded that this NH 2 -terminal region influenced the kinetics of leukotriene C 4 (LTC 4 ) and methotrexate transport by the two homologues (Konno et al, 2003).To further define the role of MSD0, we have used several approaches to examine its influence on the subcellular localization of MRP1 in both polarized and nonpolarized cells. These studies revealed that although MSD0-less MRP1 was able to traffic to the plasma membrane, the proportion of the protein in intracellular membrane compartments increased so that in transfected cells, ϳ55% of MSD0-less MRP1 was not in the plasma membrane.…”

The apical conjugate efflux pump ABCC2 (MRP2)

“…Whereas one report proposed that the PDZ-binding motif is the apical targeting signal of ABCC2 [46], this was not confirmed by all other studies [34, 93,124,183]. These latter studies indicate that the apical targeting motif of ABCC2 is not a distinct, narrowly defined amino acid sequence, but rather appears to be composed of several motifs within different parts of ABCC2 that come together only in the intact protein.…”