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.
Source: PubMed


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.

8 Reads
  • Source
    • "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 [4] [5] [6], as well as a cooperation between the C-terminus and MSD0 [7], are crucial for proper trafficking of ABCC1. However, the specific targeting signals that direct ABCC1 to the basolateral plasma membrane are currently unknown. "
    [Show abstract] [Hide abstract]
    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.
    Biochemical and Biophysical Research Communications 10/2013; 441(1). DOI:10.1016/j.bbrc.2013.10.013 · 2.30 Impact Factor
  • Source
    • "ABCC1 expression can also affect the response of nonsmall cell lung cancer [7], breast cancer [8–10], and ovarian cancer [11] 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, 16]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Unlabelled: 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. Abbreviations: ABC - ATP-binding cassette, DHS - dehydrosilybin, MDR - multidrug resistance, NBD - nucleotide-binding domain, PDB - protein data bank.
    Bioinformation 10/2012; 8(19):907-11. DOI:10.6026/97320630008907 · 0.50 Impact Factor
  • Source
    • "The mechanistic interpretation is consistent with homologous structures [44] [45] [46] [47] [48] and single particle electron microscopy analysis of (SUR1-K IR 6.2) 4 [49] verifying that TMD0-L0 gatekeepers can fit between the K IR tetramer and four SUR1 cores and thus might transduce signals from the receptor core to the pore [12] [13]. A similar L0 helix was shown to interact with the conserved core in ABCC1 [23], and we provided biochemical (labeling) evidence for the "
    [Show abstract] [Hide abstract]
    ABSTRACT: Activating mutations in different domains of the ABCC8 gene-coded sulfonylurea receptor 1 (SUR1) cause neonatal diabetes. Here we show that a diabetogenic mutation in an unexplored helix preceding the ABC core of SUR1 dramatically increases open probability of (SUR1/Kir6.2)(4) channel (KATP) by reciprocally changing rates of its transitions to and from the long-lived, inhibitory ligand-stabilized closed state. This kinetic mechanism attenuates ATP and sulfonylurea inhibition, but not Mg-nucleotide stimulation, of SUR1/Kir6.2. The results suggest a key role for L0 helix in KATP gating and together with previous findings from mutant KATP clarify why many patients with neonatal diabetes require high doses of sulfonylureas.
    FEBS letters 11/2011; 585(22):3555-9. DOI:10.1016/j.febslet.2011.10.020 · 3.17 Impact Factor
Show more

Similar Publications