Characterization of the amino-terminal regions in the human multidrug resistance protein (MRP1).
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 (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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ABSTRACT: Cardiac ATP-sensitive potassium (K(ATP)) channels are metabolic sensors formed by the association of the inward rectifier potassium channel Kir6.2 and the sulphonylurea receptor SUR2A. SUR2A adjusts channel gating as a function of intracellular ATP and ADP and is the target of pharmaceutical openers and blockers which, respectively, up- and down-regulate Kir6.2. In an effort to understand how effector binding to SUR2A translates into Kir6.2 gating modulation, we examined the role of a 65-residue SUR2A fragment linking transmembrane domain TMD2 and nucleotide-binding domain NBD2 that has been shown to interact with Kir6.2. This fragment of SUR2A was replaced by the equivalent residues of its close homologue, the multidrug resistance protein MRP1. The chimeric construct was expressed in Xenopus oocytes and characterized using the patch-clamp technique. We found that activation by MgADP and synthetic openers was greatly attenuated although apparent affinities were unchanged. Further chimeragenetic and mutagenetic studies showed that mutation of three residues, E1305, I1310 and L1313 (rat numbering), was sufficient to confer this defective phenotype. The same mutations had no effects on channel block by the sulphonylurea glibenclamide or by ATP, suggesting a role for these residues in activatory--but not inhibitory--transduction processes. These results indicate that, within the K(ATP) channel complex, the proximal C-terminal of SUR2A is a critical link between ligand binding to SUR2A and Kir6.2 up-regulation.The Journal of Physiology 08/2008; 586(13):3075-85. DOI:10.1113/jphysiol.2008.152744 · 4.54 Impact Factor
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ABSTRACT: We present an overview of the architecture of ATP-binding cassette (ABC) transporters and dissect the systems in core and accessory domains. The ABC transporter core is formed by the transmembrane domains (TMDs) and the nucleotide binding domains (NBDs) that constitute the actual translocator. The accessory domains include the substrate-binding proteins, that function as high affinity receptors in ABC type uptake systems, and regulatory or catalytic domains that can be fused to either the TMDs or NBDs. The regulatory domains add unique functions to the transporters allowing the systems to act as channel conductance regulators, osmosensors/regulators, and assemble into macromolecular complexes with specific properties.FEBS Letters 03/2006; 580(4):1023-35. DOI:10.1016/j.febslet.2005.11.079 · 3.34 Impact Factor
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ABSTRACT: Multidrug resistance protein (MRP) 1 belongs to the 'C' branch of the ABC transporter superfamily. MRP1 is a high-affinity transporter of the cysteinyl leukotriene C(4) and is responsible for the systemic release of this cytokine in response to an inflammatory stimulus. However, the substrate specificity of MRP1 is extremely broad and includes many organic anion conjugates of structurally unrelated endo- and xenobiotics. In addition, MRP1 transports unmodified hydrophobic compounds, such as natural product type chemotherapeutic agents and mutagens, such as aflatoxin B(1). Transport of several of these compounds has been shown to be dependent on the presence of reduced glutathione (GSH). More recently, GSH has also been shown to stimulate the transport of some conjugated compounds, including sulfates and glucuronides. Here, we summarize current knowledge of the substrate specificity and modes of transport of MRP1 and discuss how the protein may recognize its structurally diverse substrates.FEBS Letters 03/2006; 580(4):1103-11. DOI:10.1016/j.febslet.2005.12.036 · 3.34 Impact Factor