Characterization of the Amino-Terminal Regions in the Human Multidrug Resistance Protein (MRPI). J. Cell. Sci., 113, 4451-4461
National Institute of Haematology and Immunology, Research Group of the Hungarian Academy of Sciences, H-1113 Budapest, Hungary. Journal of Cell Science
(Impact Factor: 5.43).
01/2001; 113 Pt 24(24):4451-61.
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 (TMD(0)) region and a cytoplasmic linker (L(0)), both characteristic of several members of the MRP family. In order to study the role of the TMD(0) and L(0) 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 C(4) 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 TMD(0)L(0) (Delta MRP1), was non-functional and localized intracellularly, so we investigated the coexpression of Delta MRP1 with the isolated L(0) region. Coexpression yielded a functional MRP1 molecule in Sf9 cells and routing to the lateral membrane in MDCKII cells. Interestingly, the L(0) 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 L(0) abolished its attachment to the membrane and eliminated MRP1 transport function, but did not affect membrane routing. Taken together, these experiments suggest that the L(0) region forms a distinct domain within MRP1, which interacts with hydrophobic membrane regions and with the core region of MRP1.
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- "In addition, the retention and recycling mechanisms , which determine the further intracellular trafficking of delivered proteins, are also unconfirmed. Several attempts to identify the distinct protein regions required for basolateral targeting of ABCC1, using mutagenesis or analysis of chimeras formed between ABCC1 and ABCC2, have shown that the CLD1   , as well as a cooperation between the C-terminus and MSD0 , are crucial for proper trafficking of ABCC1. However, the specific targeting signals that direct ABCC1 to the basolateral plasma membrane are currently unknown. "
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ABSTRACT: Localization of ATP-binding cassette transporter isoform C1 (ABCC1) to the basolateral membrane of polarized cells is crucial for export of a variety of cellular metabolites; however, the mechanism regulating basolateral targeting of the transporter is poorly understood. Here we describe identification of a basolateral targeting signal in the first cytoplasmic loop domain (CLD1) of human ABCC1. Comparison of the CLD1 amino acid sequences from ABCC1 and ABCC2 revealed that ABCC1 possesses a characteristic sequence, E(295)EVEALI(301), which is comprised of a cluster of acidic glutamate residues followed by a di-leucine motif. This characteristic sequence is highly conserved among vertebrate ABCC1 orthologs and is positioned at a site that is structurally equivalent to the apical targeting signal previously described in ABCC2. Alanine scanning mutagenesis of this sequence in full-length human ABCC1 showed that both L(300) and I(301) residues were required for basolateral targeting of ABCC1 in polarized HepG2 and MDCK cells. Conversely, E(295), E(296), and E(298) residues were not required for basolateral localization of the transporter. Therefore, a di-leucine motif within the CLD1 is a basolateral targeting determinant of ABCC1.
Available from: dmd.aspetjournals.org
- "However, in the heterogeneous cell populations, the mutant proteins appeared properly routed to the plasma membrane. Cys 43 and Ser 92 are both located NH 2 -proximal to the core four-domain structure of MRP1 in MSD0; and, although a role in dimerization has been proposed, MSD0 appears dispensable for organic anion transport activity (Bakos et al., 2000;Deeley et al., 2006;Yang et al., 2010). However, most in silico prediction programs based on chemical properties, amino acid conservation, and structural data classify the C43S and S92F nsSNPs as very likely to be deleterious to MRP1 function. "
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ABSTRACT: In this study we compared the in silico predictions of the effect of ABCC1 non-synonymous single nucleotide polymorphisms (nsSNPs) with experimental data on MRP1 transport function and response to chemotherapeutics and MRP1 inhibitors. Vectors encoding seven ABCC1 nsSNPs were stably expressed in human embryonic kidney (HEK) cells, and levels and localization of the mutant MRP1 proteins were determined by confocal microscopy and immunoblotting. The function of five of the mutant proteins was determined using cell-based drug and inhibitor sensitivity and efflux assays, and membrane-based organic anion transport assays. Predicted consequences of the mutations were determined by multiple bioinformatic methods. Mutants C43S and S92F were correctly routed to the HEK cell plasma membrane but levels were too low to permit functional characterization. In contrast, levels and membrane trafficking of R633Q, G671V, R723Q, A989T and C1047S were similar to wild-type MRP1. In cell-based assays, all five mutants were equally effective at effluxing calcein while only two exhibited reduced resistance to etoposide (C1047S) and vincristine (A989T; C1047S). The GSH-dependent inhibitor LY465803 was less effective at blocking calcein efflux by A989T but in a membrane-based assay, organic anion transport by A989T and C1047S was inhibited by MRP1 modulators as well as wild-type MRP1. GSH accumulation assays suggest cellular GSH efflux by A989T and C1047S may be impaired. In conclusion, although six in silico analyses consistently predict deleterious consequences of ABCC1 nsSNPs G671V, changes in drug resistance and inhibitor sensitivity were only observed for A989T and C1047S which may relate to GSH transport differences.
Available from: ncbi.nlm.nih.gov
- "ABCC1 expression can also affect the response of nonsmall
cell lung cancer , breast cancer [8–10], and ovarian
cancer  to chemotherapy. ABCC1 is a 190-kDa glycoprotein
consisting of 2 nucleotide-binding domains (NBDs) and 3
transmembrane domains [12–14]. ABCC1-mediated substrate
translocation to the cell exterior is energised by ATP binding
and hydrolysis at the NBDs [15,
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ABCC1 is a member of the ATP-binding Cassette super family of transporters, actively effluxes xenobiotics from cells. Clinically, ABCC1 expression is linked to cancer multidrug resistance. Substrate efflux is energised by ATP binding and hydrolysis at the nucleotide-binding domains (NBDs) and inhibition of these events may help combat drug resistance. The aim of this study is to identify potential inhibitors of ABCC1 through virtual screening of National Cancer Institute (NCI) compounds. A threedimensional model of ABCC1 NBD2 was generated using MODELLER whilst the X-ray crystal structure of ABCC1 NBD1 was retrieved from the Protein Data Bank. A pharmacophore hypothesis was generated based on flavonoids known to bind at the NBDs using PHASE, and used to screen the NCI database. GLIDE was employed in molecular docking studies for all hit compounds identified by pharmacophore screening. The best potential inhibitors were identified as compounds possessing predicted binding affinities greater than ATP. Approximately 5% (13/265) of the hit compounds possessed lower docking scores than ATP in ABCC1 NBD1 (NSC93033, NSC662377, NSC319661, NSC333748, NSC683893, NSC226639, NSC94231, NSC55979, NSC169121, NSC166574, NSC73380, NSC127738, NSC115534), whereas approximately 7% (7/104) of docked NCI compounds were predicted to possess lower docking scores than ATP in ABCC1 NBD2 (NSC91789, NSC529483, NSC211168, NSC318214, NSC116519, NSC372332, NSC526974). Analyses of docking orientations revealed P-loop residues of each NBD and the aromatic amino acids Trp653 (NBD1) and Tyr1302 (NBD2) were key in interacting with high-affinity compounds. On the basis of docked orientation and docking score the compounds identified may be potential inhibitors of ABCC1 and require further pharmacological analysis.
ABC - ATP-binding cassette, DHS - dehydrosilybin, MDR - multidrug resistance, NBD - nucleotide-binding domain, PDB - protein data bank.
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