Mutation of Trp1254 in the multispecific organic anion transporter, multidrug resistance protein 2 (MRP2) (ABCC2), alters substrate specificity and results in loss of methotrexate transport activity

The ATP-binding cassette (ABC) proteins comprise a large superfamily of transmembrane transporters that utilize the energy of ATP hydrolysis to translocate their substrates across biological membranes. Multidrug resistance protein (MRP) 2 (ABCC2) belongs to subfamily C of the ABC superfamily and, when overexpressed in tumor cells, confers resistance to a wide variety of anticancer chemotherapeutic agents. MRP2 is also an active transporter of organic anions such as methotrexate (MTX), estradiol glucuronide (E217betaG), and leukotriene C4 and is located on the apical membrane of polarized cells including hepatocytes where it acts as a biliary transporter. We recently identified a highly conserved tryptophan residue in the related MRP1 that is critical for the substrate specificity of this protein. In the present study, we have examined the effect of replacing the analogous tryptophan residue at position 1254 of MRP2. We found that only nonconservative substitutions (Ala and Cys) of Trp1254 eliminated [3H]E217betaG transport by MRP2, whereas more conservative substitutions (Phe and Tyr) had no effect. In addition, only the most conservatively substituted mutant (W1254Y) transported [3H]leukotriene C4, whereas all other substitutions eliminated transport of this substrate. On the other hand, all substitutions of Trp1254 eliminated transport of [3H]MTX. Finally, we found that sulfinpyrazone stimulated [3H]E217betaG transport by wild-type MRP2 4-fold, whereas transport by the Trp1254 substituted mutants was enhanced 6-10-fold. In contrast, sulfinpyrazone failed to stimulate [3H]MTX transport by either wild-type MRP2 or the MRP2-Trp1254 mutants. Taken together, our results demonstrate that Trp1254 plays an important role in the ability of MRP2 to transport conjugated organic anions and identify this amino acid in the putative last transmembrane segment (TM17) of this ABC protein as being critical for transport of MTX.     

Evidence for two interacting ligand binding sites in human multidrug resistance protein 2 (ATP binding cassette C2)

Multidrug resistance protein 2 (MRP2) belongs to the ATP binding cassette family of transporters. Its substrates include organic anions and anticancer drugs. We have used transport assays with vesicles derived from Sf9 insect cells overproducing MRP2 to study the interactions of drugs, organic anions, and bile acids with three MRP2 substrates: estradiol-17-beta-d-glucuronide (E217betaG), methotrexate, and glutathione-S-dinitrophenol. Complex inhibition and stimulation patterns were obtained, different from those observed with the related transporters MRP1 and MRP3. In contrast to a previous report, we found that the rate of E217betaG transport by MRP2 increases sigmoidally with substrate concentration indicative of homotropic cooperativity. Half-maximal transport was obtained at 120 microm E217betaG, in contrast to values < 20 microm for MRP1 and 3. MRP2 stimulators, such as indomethacin and sulfanitran, strongly increased the affinity of MRP2 for E217betaG (half-maximal transport rates at 65 and 16 microm E217betaG, respectively) and shifted the sigmoidal dependence of transport rate on substrate concentration to a more hyperbolic one, without substantially affecting the maximal transport rate. Sulfanitran also stimulated MRP2 activity in cells, i.e. the transport of saquinavir through monolayers of Madin-Darby canine kidney II cells. Some compounds that stimulate E217betaG transport, such as penicillin G or pantoprazole, are not detectably transported by MRP2, suggesting that they allosterically stimulate transport without being cotransported with E217betaG. We propose that MRP2 contains two similar but nonidentical ligand binding sites: one site from which substrate is transported and a second site that regulates the affinity of the transport site for the substrate.     

Multidrug resistance protein 4 (MRP4/ABCC4) mediates efflux of bimane-glutathione

Multidrug resistance proteins (MRPs) are ATP-dependent export pumps that mediate the export of organic anions. ABCC1 (MRP1), ABCC2 (MRP2) and ABCC3 (MRP3) are all able to facilitate the efflux of anionic conjugates including glutathione (GSH), glucuronide and sulfate conjugates of xenobiotics and endogenous molecules. Earlier studies showed that ABCC4 functions as an ATP-driven export pump for cyclic AMP and cyclic GMP, as well as estradiol-17-beta-D-glucuronide. However, it was unclear if other conjugated metabolites can be transported by ABCC4. Hence in this study, a fluorescent substrate, bimane-glutathione (bimane-GS) was used to further examine the transport activity of ABCC4. Using cells stably overexpressing ABCC4, this study shows that ABCC4 can facilitate the efflux of the glutathione conjugate, bimane-glutathione. Bimane-glutathione efflux increased with time and >85% of the conjugate was exported after 15min. This transport was abolished in the presence of 2.5microM carbonylcyanide m-chlorophenylhydrasone (CCCP), an uncoupler of oxidative phosphorylation. Inhibition was also observed with known inhibitors of MRP transporters including benzbromarone, verapamil and indomethacin. In addition, 100microM methotrexate, an ABCC4 substrate or 100microM 6-thioguanine (6-TG), a compound whose monophosphate metabolite is an ABCC4 substrate, reduced efflux by >40%. A concentration-dependent inhibition of bimane-glutathione efflux was observed with 1-chloro-2,4-dinitrobenzene (CDNB) which is metabolized intracellularly to the glutathione conjugate, 2,4-dinitrophenyl-glutathione (DNP-GS). The determination that ABCC4 can mediate the transport of glucuronide and glutathione conjugates indicates that ABCC4 may play a role in the cellular extrusion of Phase II detoxification metabolites.     

Mammalian multidrug-resistance proteins (MRPs)

Subfamily C of the human ABC (ATP-binding cassette) superfamily contains nine proteins that are often referred to as the MRPs (multidrug-resistance proteins). The 'short' MRP/ABCC transporters (MRP4, MRP5, MRP8 and ABCC12) have a typical ABC structure with four domains comprising two membrane-spanning domains (MSD1 and MSD2) each followed by a nucleotide-binding domain (NBD1 and NBD2). The 'long' MRP/ABCCs (MRP1, MRP2, MRP3, ABCC6 and MRP7) have five domains with the extra domain, MSD0, at the N-terminus. The proteins encoded by the ABCC6 and ABCC12 genes are not known to transport drugs and are therefore referred to as ABCC6 and ABCC12 (rather than MRP6 and MRP9) respectively. A large number of molecules are transported across the plasma membrane by the MRPs. Many are organic anions derived from exogenous sources such as conjugated drug metabolites. Others are endogenous metabolites such as the cysteinyl leukotrienes and prostaglandins which have important signalling functions in the cell. Some MRPs share a degree of overlap in substrate specificity (at least in vitro), but differences in transport kinetics are often substantial. In some cases, the in vivo substrates for some MRPs have been discovered aided by studies in gene-knockout mice. However, the molecules that are transported in vivo by others, including MRP5, MRP7, ABCC6 and ABCC12, still remain unknown. Important differences in the tissue distribution of the MRPs and their membrane localization (apical in contrast with basolateral) in polarized cells also exist. Together, these differences are responsible for the unique pharmacological and physiological functions of each of the nine ABCC transporters known as the MRPs.     

Multidrug resistance proteins (MRPs/ABCCs) in cancer chemotherapy and genetic diseases

The ATP-binding cassette (ABC) transporters are a superfamily of membrane proteins that are best known for their ability to transport a wide variety of exogenous and endogenous substances across membranes against a concentration gradient via ATP hydrolysis. There are seven subfamilies of human ABC transporters, one of the largest being the 'C' subfamily (gene symbol ABCC). Nine ABCC subfamily members, the so-called multidrug resistance proteins (MRPs) 1-9, have been implicated in mediating multidrug resistance in tumor cells to varying degrees as the efflux extrude chemotherapeutic compounds (or their metabolites) from malignant cells. Some of the MRPs are also known to either influence drug disposition in normal tissues or modulate the elimination of drugs (or their metabolites) via hepatobiliary or renal excretory pathways. In addition, the cellular efflux of physiologically important organic anions such as leukotriene C(4) and cAMP is mediated by one or more of the MRPs. Finally, mutations in several MRPs are associated with human genetic disorders. In this minireview, the current biochemical and physiological knowledge of MRP1-MRP9 in cancer chemotherapy and human genetic disease is summarized. The mutations in MRP2/ABCC2 leading to conjugated hyperbilirubinemia (Dubin-Johnson syndrome) and in MRP6/ABCC6 leading to the connective tissue disorder Pseudoxanthoma elasticum are also discussed.     

Characterization of the transport properties of cloned rat multidrug resistance-associated protein 3 (MRP3).

We have previously cloned rat MRP3 as an inducible transporter in the liver (Hirohashi, T., Suzuki, H., Ito, K., Ogawa, K., Kume, K., Shimizu, T., and Sugiyama, Y. (1998) Mol. Pharmacol. 53, 1068-1075). In the present study, the function of rat MRP3 was investigated using membrane vesicles isolated from LLC-PK1 and HeLa cell population transfected with corresponding cDNA. The ATP-dependent uptake of both 17beta estradiol 17-beta-D-glucuronide ([3H]E217betaG) and glucuronide of [14C] 6-hydroxy-5, 7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole (E3040), but not that of [3H]leukotriene C4 and [3H]2, 4-dinitrophenyl-S-glutathione, was markedly stimulated by MRP3 transfection in both cell lines. The Km and Vmax values for the uptake of [3H]E217betaG were 67 +/- 14 microM and 415 +/- 73 pmol/min/mg of protein, respectively, for MRP3-expressing membrane vesicles and 3.0 +/- 0.7 microM and 3.4 +/- 0.4 pmol/min/mg of protein, respectively, for the endogenous transporter expressed on HeLa cells. [3H]E217betaG had also a similar Km value for MRP3 when LLC-PK1 cells were used as the host. All glucuronide conjugates examined (E3040 glucuronide, 4-methylumbelliferone glucuronide, and naphthyl glucuronide) and methotrexate inhibited MRP3-mediated [3H]E217betaG transport in LLC-PK1 cells. Moreover, [3H]methotrexate was transported via MRP3. The inhibitory effect of estrone sulfate, [3H]2,4-dinitrophenyl-S-glutathione, and [3H]leukotriene C4 was moderate or minimal, whereas N-acetyl-2,4-dinitrophenylcysteine had no effect on the uptake of [3H]E217betaG. The uptake of [3H]E217betaG was enhanced by E3040 sulfate and 4-methylumbelliferone sulfate. Thus we were able to demonstrate that several kinds of organic anions are transported via MRP3, although the substrate specificity of MRP3 differs from that of MRP1 and cMOAT/MRP2 in that glutathione conjugates are poor substrates for MRP3.     

Transport of the beta -O-glucuronide conjugate of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) by the multidrug resistance protein 1 (MRP1). Requirement for glutathione or a non-sulfur-containing analog

Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) play a crucial role in the induction of lung cancer, and NNAL-O-glucuronide formation and elimination are important steps in detoxification of these compounds. In the present study, we investigated the ATP-binding cassette (ABC) protein, MRP1 (ABCC1), as a candidate transporter responsible for NNAL-O-glucuronide export. MRP1 mediates the active transport of numerous GSH-, sulfate-, and glucuronide-conjugated organic anions and can transport certain xenobiotics by a mechanism that may involve co-transport with GSH. Using membrane vesicles prepared from transfected cells, we found that MRP1 transports [3H]NNAL-O-glucuronide but is dependent on the presence of GSH (Km 39 microm, Vmax 48 pmol x mg(-1) x min(-1)). We also found that the sulfur atom in GSH was dispensable because transport was supported by the GSH analog, gamma-glutamyl-alpha-aminobutyryl-glycine. Despite stimulation of NNAL-O-glucuronide transport by GSH, there was no detectable reciprocal stimulation of [3H]GSH transport. Moreover, whereas the MRP1 substrates leukotriene C4 (LTC4) and 17beta-estradiol 17beta-(d-glucuronide) (E(2)17betaG) inhibited GSH-dependent uptake of [3H]NNAL-O-glucuronide, only [3H]LTC4 transport was inhibited by NNAL-O-glucuronide (+GSH) and the kinetics of inhibition were complex. A mutant form of MRP1, which transports LTC4 but not E(2)17betaG, also did not transport NNAL-O-glucuronide suggesting a commonality in the binding elements for these two glucuronidated substrates, despite their lack of reciprocal transport inhibition. Finally, the related MRP2 transported NNAL-O-glucuronide with higher efficiency than MRP1 and unexpectedly, GSH inhibited rather than stimulated uptake. These studies provide further insight into the complex interactions of the MRP-related proteins with GSH and their conjugated organic anion substrates, and extend the range of xenotoxins transported by MRP1 and MRP2 to include metabolites of known carcinogens involved in the etiology of lung and other cancers.     

The multidrug resistance-associated protein (MRP/ABCC) subfamily of ATP-binding cassette transporters in plants

In many different plant species, genes belonging to the multidrug resistance-associated protein (MRP, ABCC) subfamily of ABC transporters have been identified. Following the discovery of vacuolar transport systems for xenobiotic or plant-produced conjugated organic anions, plant MRPs were originally proposed to be primarily involved in the vacuolar sequestration of potentially toxic metabolites. Indeed, heterologous expression of different Arabidopsis MRPs in yeast demonstrates their activity as ATP-driven pumps for structurally diverse substrates. Recent analysis of protein-protein interactions and the characterization of knockout mutants in Arabidopsis suggests that apart from transport functions plant MRPs play additional roles including the control of plant transpiration through the stomata. Here, we review and discuss the diverse functions of plant MRP-type ABC transporters and present an organ-related and developmental analysis of the expression of Arabidopsis MRPs using the publicly available full-genome chip data.     

Charged amino acids in the sixth transmembrane helix of multidrug resistance protein 1 (MRP1/ABCC1) are critical determinants of transport activity

The multidrug resistance protein, MRP1 (ABCC1), is an ATP-binding cassette transporter that confers resistance to chemotherapeutic agents. MRP1 also mediates transport of organic anions such as leukotriene C(4) (LTC(4)), 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG), estrone 3-sulfate, methotrexate (MTX), and GSH. We replaced three charged amino acids, Lys(332), His(335), and Asp(336), predicted to be in the sixth transmembrane (TM6) helix of MRP1 with neutral and oppositely charged amino acids and determined the effect on substrate specificity and transport activity. All mutants were expressed in transfected human embryonic kidney cells at levels comparable with wild-type MRP1, and confocal microscopy showed that they were correctly routed to the plasma membrane. Vesicular transport studies revealed that the MRP1-Lys(332) mutants had lost the ability to transport LTC(4), and GSH transport was reduced; whereas E(2)17betaG, estrone 3-sulfate, and MTX transport were unaffected. E(2)17betaG transport was not inhibited by LTC(4) and could not be photolabeled with [(3)H]LTC(4), indicating that the MRP1-Lys(332) mutants no longer bound this substrate. Substitutions of MRP1-His(335) also selectively diminished LTC(4) transport and photolabeling but to a lesser extent. Kinetic analyses showed that V(max) (LTC(4)) of these mutants was decreased but K(m) was unchanged. In contrast to the selective loss of LTC(4) transport in the Lys(332) and His(335) mutants, the MRP1-Asp(336) mutants no longer transported LTC(4), E(2)17betaG, estrone 3-sulfate, or GSH, and transport of MTX was reduced by >50%. Lys(332), His(335), and Asp(336) of TM6 are predicted to be in the outer leaflet of the membrane and are all capable of forming intrahelical and interhelical ion pairs and hydrogen bonds. The importance of Lys(332) and His(335) in determining substrate specificity and of Asp(336) in overall transport activity suggests that such interactions are critical for the binding and transport of LTC(4) and other substrates of MRP1.     

Mutation of a single conserved tryptophan in multidrug resistance protein 1 (MRP1/ABCC1) results in loss of drug resistance and selective loss of organic anion transport

Multidrug resistance protein 1 (MRP1/ABCC1) belongs to the ATP-binding cassette transporter superfamily and is capable of conferring resistance to a broad range of chemotherapeutic agents and transporting structurally diverse conjugated organic anions. In this study, we found that substitution of a highly conserved tryptophan at position 1246 with cysteine (W1246C-MRP1) in the putative last transmembrane segment (TM17) of MRP1 eliminated 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) transport by membrane vesicles prepared from transiently transfected human embryonic kidney cells while leaving the capacity for leukotriene C(4)- and verapamil-stimulated glutathione transport intact. In addition, in contrast to wild-type MRP1, leukotriene C(4) transport by the W1246C-MRP1 protein was no longer inhibitable by E(2)17betaG, indicating that the mutant protein had lost the ability to bind the glucuronide. A similar phenotype was observed when Trp(1246) was replaced with Ala, Phe, and Tyr. Confocal microscopy of cells expressing Trp(1246) mutant MRP1 molecules fused at the C terminus with green fluorescent protein showed that they were correctly routed to the plasma membrane. In addition to the loss of E(2)17betaG transport, HeLa cells stably transfected with W1246C-MRP1 cDNA were not resistant to the Vinca alkaloid vincristine and accumulated levels of [(3)H]vincristine comparable to those in vector control-transfected cells. Cells expressing W1246C-MRP1 were also not resistant to cationic anthracyclines (doxorubicin, daunorubicin) or the electroneutral epipodophyllotoxin VP-16. In contrast, resistance to sodium arsenite was only partially diminished, and resistance to potassium antimony tartrate remained comparable to that of cells expressing wild-type MRP1. This suggests that the structural determinants required for transport of heavy metal oxyanions differ from those for chemotherapeutic agents. Our results provide the first example of a tryptophan residue being so critically important for substrate specificity in a eukaryotic ATP-binding cassette transporter.     

Localization of a substrate specificity domain in the multidrug resistance protein.

Multidrug resistance protein (MRP) confers resistance to a number of natural product chemotherapeutic agents. It is also a high affinity transporter of some physiological conjugated organic anions such as cysteinyl leukotriene C(4) and the cholestatic estrogen, 17beta-estradiol 17(beta-D-glucuronide) (E(2)17betaG). We have shown that the murine orthologue of MRP (mrp), unlike the human protein, does not confer resistance to common anthracyclines and is a relatively poor transporter of E(2)17betaG. We have taken advantage of these functional differences to identify region(s) of MRP involved in mediating anthracycline resistance and E(2)17betaG transport by generating mrp/MRP hybrid proteins. All hybrid proteins conferred resistance to the Vinca alkaloid, vincristine, when transfected into human embryonic kidney cells. However, only those in which the COOH-terminal third of mrp had been replaced with the corresponding region of MRP-conferred resistance to the anthracyclines, doxorubicin, and epirubicin. Exchange of smaller segments of the COOH-terminal third of the mouse protein by replacement of either amino acids 959-1187 or 1188-1531 with those of MRP produced proteins capable of conferring some level of resistance to the anthracyclines tested. All hybrid proteins transported cysteinyl leukotriene C(4) with similar efficiencies. In contrast, only those containing the COOH-terminal third of MRP transported E(2)17betaG with an efficiency comparable with that of the intact human protein. The results demonstrate that differences in primary structure of the highly conserved COOH-terminal third of mrp and MRP are important determinants of the inability of the murine protein to confer anthracycline resistance and its relatively poor ability to transport E(2)17betaG.     

Identification of an amino acid residue in multidrug resistance protein 1 critical for conferring resistance to anthracyclines

Murine multidrug resistance protein 1 (mrp1), unlike human MRP1, does not confer resistance to anthracyclines. Previously, we have shown that a human/murine hybrid protein containing amino acids 959-1187 of MRP1 can confer resistance to these drugs. We have now examined the functional characteristics of mutant proteins in which we have converted individual amino acids in the comparable region of mrp1 to those present at the respective locations in MRP1. These mutations had no effect on the drug resistance profile conferred by mrp1 with the exception of converting glutamine 1086 to glutamate, as it is in the corresponding position (1089) in MRP1. This mutation created a protein that conferred resistance to doxorubicin without affecting vincristine resistance, or the ability of mrp1 to transport leukotriene C(4) (LTC(4)) and 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG). Furthermore, mutation Q1086D conferred the same phenotype as mutation Q1086E while the mutation Q1086N did not detectably alter the drug resistance profile of mrp1, suggesting that an anionic side chain was required for anthracycline resistance. To confirm the importance of MRP1 E1089 for conferring resistance to anthracyclines, we mutated this residue to Gln, Asp, Ala, Leu, and Lys in the human protein. The mutation E1089D showed the same phenotype as MRP1, while the E1089Q substitution markedly decreased resistance to anthracyclines without affecting LTC(4) and E(2)17betaG transport. Conversion of Glu-1089 to Asn, Ala, or Leu had a similar effect on resistance to anthracyclines, while conversion to a positive amino acid, Lys, completely eliminated resistance to anthracyclines and vincristine without affecting transport of LTC(4), E(2)17betaG, and the GSH-dependent substrate, estrone-3-sulfate. These results demonstrate that an acidic amino acid residue at position 1089 in predicted TM14 of MRP1 is critical for the ability of the protein to confer drug resistance particularly to the anthracyclines, but is not essential for its ability to transport conjugated organic anions such as LTC(4) and E(2)17betaG.     

Mutation of proline residues in the NH(2)-terminal region of the multidrug resistance protein,MRP1 (ABCC1): effects on protein expression,membrane localization,and transport function

The Multidrug Resistance Protein, MRP1 (ABCC1) confers drug resistance and transports organic anions such as leukotriene C(4) (LTC(4)) and 17beta-estradiol 17-(beta-D-glucuronide) (E(2)17betaG). Previous studies showed that portions of the first membrane spanning domain (MSD1) and the cytoplasmic loop (CL3) connecting it to MSD2 are important for MRP1 transport function. We have replaced 12 prolines in MSD1 and CL3 with alanine and determined the effects of these substitutions on MRP1 expression and transport activity. All singly substituted MRP1-Pro mutants could be expressed in HeLa cells, except MRP1-P104A. The expressed mutants also transported LTC(4) and E(2)17betaG, and their K(m) (LTC(4)) values were similar to wild-type MRP1. Expression of the double mutant MRP1-P42/51A was reduced by >80% although it localized to the plasma membrane and transported organic anions. MRP1 expression was also reduced when the first transmembrane helix (amino acids 37-54) was deleted. In contrast, the phenotypes of the multiply substituted CL3 mutants MRP1-P196/205/207/209A and MRP1-P235/255A were comparable to wild-type MRP1. However, Pro(255)-substituted MRP1 mutants showed reduced immunoreactivity with a monoclonal antibody (MAb) whose epitope is located in CL3. We conclude that certain prolines in MSD1 and CL3 play a role in the expression and structure of MRP1.     

MRP Subfamily Transporters and Resistance to Anticancer Agents

The MRP subfamily of ABC transporters from mammals consists of at least seven members, six of which have been implicated in the transport of amphipathic anions. MRP1, MRP2, and MRP3 bear a close structural resemblance, confer resistance to a variety of natural products as well as methotrexate, and have the facility for transporting glutathione and glucuronate conjugates. MRP1 is a ubiquitously expressed efflux pump for the products of phase II of xenobiotic detoxification, while MRP2, whose hereditary deficiency results in Dubin–Johnson syndrome, functions to extrude organic anions into the bile. MRP3 is distinguished by its capacity to transport the monoanionic bile constituent glycocholate, and may function as a basolateral back-up system for the detoxification of hepatocytes when the usual canalicular route is impaired by cholestatic conditions. MRP4 and MRP5 resemble each other more closely than they resemble MRPs 1–3 and confer resistance to purine and nucleotide analogs which are either inherently anionic, as in the case of the anti-AIDS drug PMEA, or are phosphorylated and converted to anionic amphiphiles in the cell, as in the case of 6-MP. Given their capacity for transporting cyclic nucleotides, MRP4 and MRP5 have also been implicated in a broad range of cellular signaling processes. The drug resistance activity and physiological substrates of MRP6 are unknown. However, its hereditary deficiency results in pseudoxanthoma elasticum, a multisystem disorder affecting skin, eyes, and blood vessels. It is hoped that elucidation of the resistance profiles and physiological functions of the different members of the MRP subfamily will provide new insights into the molecular basis of clinical drug resistance and spawn new strategies for combating this phenomenon.     

Conjugate export pumps of the multidrug resistance protein (MRP) family: localization, substrate specificity, and MRP2-mediated drug resistance

The membrane proteins mediating the ATP-dependent transport of lipophilic substances conjugated to glutathione, glucuronate, or sulfate have been identified as members of the multidrug resistance protein (MRP) family. Several isoforms of these conjugate export pumps with different kinetic properties and domain-specific localization in polarized human cells have been cloned and characterized. Orthologs of the human MRP isoforms have been detected in many different organisms. Studies in mutant rats lacking the apical isoform MRP2 (symbol ABCC2) indicate that anionic conjugates of endogenous and exogenous substances cannot exit from cells at a sufficient rate unless an export pump of the MRP family is present in the plasma membrane. Several mutations in the human MRP2 gene have been identified which lead to the absence of the MRP2 protein from the hepatocyte canalicular membrane and to the conjugated hyperbilirubinemia of Dubin-Johnson syndrome. Overexpression of recombinant MRP2 confers resistance to multiple chemotherapeutic agents. Because of its function in the terminal excretion of cytotoxic and carcinogenic substances, MRP2 as well as other members of the MRP family, play an important role in detoxification and chemoprevention.     

Identification of a nonconserved amino acid residue in multidrug resistance protein 1 important for determining substrate specificity: evidence for functional interaction between transmembrane helices 14 and 17.

Murine multidrug resistance protein 1 (mrp1), differs from its human ortholog (MRP1) in that it fails to confer anthracycline resistance and transports the MRP1 substrate, 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG), very poorly. By mutating variant residues in mrp1 to those present in MRP1, we identified Glu(1089) of MRP1 as being critical for anthracycline resistance. However, Glu(1089) mutations had no effect on E(2)17betaG transport. We have now identified a nonconserved amino acid within the highly conserved COOH-proximal transmembrane helix of MRP1/mrp1 that is important for transport of the conjugated estrogen. Converting Ala(1239) in mrp1 to Thr, as in the corresponding position (1242) in MRP1, increased E(2)17betaG transport 3-fold. Any mutation of mrp1 Ala(1239), including substitution with Thr, decreased resistance to vincristine and VP-16 without altering anthracycline resistance. However, introduction of a second murine to human mutation, Q1086E, which alone selectively increases anthracycline resistance, into mrp1A1239T restored resistance to both vincristine and VP-16. To confirm the importance of MRP1 Thr(1242) for E(2)17betaG transport and drug resistance, we mutated this residue to Ala, Cys, Ser, Leu, and Lys. These mutations decreased E(2)17betaG transport 2-fold. Conversion to Asp eliminated transport of the estrogen conjugate and also decreased leukotriene C(4) transport approximately 2-fold. The mutations also reduced the ability of MRP1 to confer resistance to all drugs tested. As with mrp1, introduction of a second mutation based on the murine sequence to create MRP1E1089Q/T1242A restored resistance to vincristine and VP-16, but not anthracyclines, without affecting transport of leukotriene C(4) and E(2)17betaG. These results demonstrate the important role of Thr(1242) for E(2)17betaG transport. They also reveal a highly specific functional relationship between nonconserved amino acids in TM helices 14 and 17 of both mrp1 and MRP1 that enables both proteins to confer similar levels of resistance to vincristine and VP-16.     

Substitution of Trp1242 of TM17 alters substrate specificity of human multidrug resistance protein 3

Multidrug resistance protein 3 (MRP3) is an ATP-dependent transporter of 17beta-estradiol 17beta(d-glucuronide) (E(2)17betaG), leukotriene C(4) (LTC(4)), methotrexate, and the bile salts taurocholate and glycocholate. In the present study, the role of a highly conserved Trp residue at position 1242 on MRP3 transport function was examined by expressing wild-type MRP3 and Ala-, Cys-, Phe-, Tyr-, and Pro-substituted mutants in human embryonic kidney 293T cells. Four MRP3-Trp(1242) mutants showed significantly increased E(2)17betaG uptake, whereas transport by the Pro mutant was undetectable. Similarly, the Pro mutant did not transport LTC(4). By comparison, LTC(4) transport by the Ala, Cys, Phe, and Tyr mutants was reduced by approximately 35%. The Ala, Cys, Phe, and Tyr mutants all showed greatly reduced methotrexate and leucovorin transport, except the Tyr mutant, which transported leucovorin at levels comparable with wild-type MRP3. In contrast, the MRP3-Trp(1242) substitutions did not significantly affect taurocholate transport or taurocholate and glycocholate inhibition of E(2)17betaG uptake. Thus Trp(1242) substitutions markedly alter the substrate specificity of MRP3 but leave bile salt binding and transport intact.     

Cholesterol but not association with detergent resistant membranes is necessary for the transport function of MRP2/ABCC2

MRP2(/ABCC2) excretes amphiphilic organic anions into bile, and associates with detergent-resistant bile canalicular membrane domains (DRM). Here, we have evaluated sensitivities of MRP2 transport function and DRM association by titrating the cellular cholesterol content. We demonstrate that the role of cholesterol in the partitioning of MRP2 to DRM can be separated from the role of cholesterol in the function of MRP2, such that (i) cholesterol is not necessary for the polarized distribution of MRP2 at the canalicular membrane, (ii) partitioning into DRM is not required for MRP2 function, yet (iii) the presence of cholesterol is necessary for transport activity.