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.     

Functional importance of polar and charged amino acid residues in transmembrane helix 14 of multidrug resistance protein 1 (MRP1/ABCC1): identification of an aspartate residue critical for conversion from a high to low affinity substrate binding state

Human multidrug resistance protein 1 (MRP1) confers resistance to many chemotherapeutic agents and transports diverse conjugated organic anions. We previously demonstrated that Glu1089 in transmembrane (TM) 14 is critical for the protein to confer anthracycline resistance. We have now assessed the functional importance of all polar and charged amino acids in this TM helix. Asn1100, Ser1097, and Lys1092, which are all predicted to be on the same face of the helix as to Glu1089, are involved in determining the substrate specificity of the protein. Notably, elimination of the positively charged side chain of Lys1092, increased resistance to the cationic drugs vincristine and doxorubicin, but not the electroneutral drug etoposide (VP-16). In addition, mutations S1097A and N1100A selectively decreased transport of 17beta-estradiol 17-(beta-d-glucuronide) (E217betaG) but not cysteinyl leukotriene 4 (LTC4), demonstrating the importance of multiple residues in this helix in determining substrate specificity. In contrast, mutations of Asp1084 that eliminate the carboxylate side chain markedly decreased resistance to all drugs tested, as well as transport of both E217betaG and LTC4, despite the fact that LTC4 binding was unaffected. We show that these mutations prevent the ATP-dependent transition of the protein from a high to low affinity substrate binding state and drastically diminish ADP trapping at nucleotide binding domain 2. Based on results presented here and crystal structures of prokaryotic ATP binding cassette transporters, Asp1084 may be critical for interaction between the cytoplasmic loop connecting TM13 and TM14 and a region of nucleotide binding domain 2 between the conserved Walker A and ABC signature motifs.     

Identification of proline residues in the core cytoplasmic and transmembrane regions of multidrug resistance protein 1 (MRP1/ABCC1) important for transport function, substrate specificity, and nucleotide interactions

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-binding cassette transporter that confers resistance to drugs and mediates the transport of organic anions. MRP1 has a core structure of two membrane spanning domains (MSDs) each followed by a nucleotide binding domain. This core structure is preceded by a third MSD with five transmembrane (TM) helices, whereas MSD2 and MSD3 each contain six TM helices. We investigated the consequences of Ala substitution of 18 Pro residues in both the non-membrane and TM regions of MSD2 and MSD3 on MRP1 expression and organic anion transport function. All MRP1-Pro mutants except P1113A were expressed in human embryonic kidney cells at levels comparable with wild-type MRP1. In addition, five mutants containing substitutions of Pro residues in or proximal to the TM helices of MSD2 (TM6-Pro(343), TM8-Pro(448), TM10-Pro(557), and TM11-Pro(595)) and MSD3 (TM14-Pro(1088)) exhibited significantly reduced transport of five organic anion substrates. In contrast, mutation of Pro(1150) in the cytoplasmic loop (CL7) linking TM15 to TM16 caused a substantial increase in 17beta-estradiol-17-beta-(D-glucuronide) and methotrexate transport, whereas transport of other organic anions was reduced or unchanged. Significant substrate-specific changes in the ATP dependence of transport and binding by the P1150A mutant were also observed. Our findings demonstrate the importance of TM6, TM8, TM10, TM11, and TM14 in MRP1 transport function and suggest that CL7 may play a differential role in coupling the activity of the nucleotide binding domains to the translocation of different substrates across the membrane.     

Molecular modeling of the human multidrug resistance protein 1 (MRP1/ABCC1)

Multidrug resistance protein 1 (MRP1/ABCC1) is a 190 kDa member of the ATP-binding cassette (ABC) superfamily of transmembrane transporters that is clinically relevant for its ability to confer multidrug resistance by actively effluxing anticancer drugs. Knowledge of the atomic structure of MRP1 is needed to elucidate its transport mechanism, but only low resolution structural data are currently available. Consequently, comparative modeling has been used to generate models of human MRP1 based on the crystal structure of the ABC transporter Sav1866 from Staphylococcus aureus. In these Sav1866-based models, the arrangement of transmembrane helices differs strikingly from earlier models of MRP1 based on the structure of the bacterial lipid transporter MsbA, both with respect to packing of the twelve helices and their interactions with the nucleotide binding domains. The functional importance of Tyr³²⁴ in transmembrane helix 6 predicted to project into the substrate translocation pathway was investigated.     

Functional reconstitution of substrate transport by purified multidrug resistance protein MRP1 (ABCC1) in phospholipid vesicles

The 190-kDa multidrug resistance protein MRP1 (ABCC1) is a polytopic transmembrane protein belonging to the ATP-binding cassette transporter superfamily. In addition to conferring resistance to various antineoplastic agents, MRP1 is a transporter of conjugated organic anions, including the cysteinyl leukotriene C(4) (LTC(4)). We previously characterized the ATPase activity of reconstituted immunoaffinity-purified native MRP1 and showed it could be stimulated by its organic anion substrates (Mao, Q., Leslie, E. M., Deeley, R. G., and Cole, S. P. C. (1999) Biochim. Biophys. Acta 1461, 69-82). Here we show that purified reconstituted MRP1 is also capable of active transport of its substrates. Thus LTC(4) uptake by MRP1 proteoliposomes was osmotically sensitive and could be inhibited by two MRP1-specific monoclonal antibodies. LTC(4) uptake was also markedly reduced by the competitive inhibitor, S-decyl-glutathione, as well as by the MRP1 substrates 17 beta-estradiol 17-beta-(d-glucuronide), oxidized glutathione, and vincristine in the presence of reduced glutathione. The K(m) for ATP and LTC(4) were 357 +/- 184 microm and 366 +/- 38 nm, respectively, and 2.14 +/- 0.75 microm for 17 beta-estradiol 17-beta-(d-glucuronide). Transport of vincristine required the presence of both ATP and GSH. Conversely, GSH transport was stimulated by vincristine and verapamil. Our data represent the first reconstitution of transport competent purified native MRP1 and confirm that MRP1 is an efflux pump, which can transport conjugated organic anions and co-transport vincristine together with GSH.     

Role of GSH in estrone sulfate binding and translocation by the multidrug resistance protein 1 (MRP1/ABCC1)

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent efflux pump that can confer resistance to multiple anticancer drugs and transport conjugated organic anions. Unusually, transport of several MRP1 substrates requires glutathione (GSH). For example, estrone sulfate transport by MRP1 is stimulated by GSH, vincristine is co-transported with GSH, or GSH can be transported alone. In the present study, radioligand binding assays were developed to investigate the mechanistic details of GSH-stimulated transport of estrone sulfate by MRP1. We have established that estrone sulfate binding to MRP1 requires GSH, or its non-reducing analogue S-methyl GSH (S-mGSH), and further that the affinity (Kd) of MRP1 for estrone sulfate is 2.5-fold higher in the presence of S-mGSH than GSH itself. Association kinetics show that GSH binds to MRP1 first, and we propose that GSH binding induces a conformational change, which makes the estrone sulfate binding site accessible. Binding of non-hydrolyzable ATP analogues to MRP1 decreases the affinity for estrone sulfate. However, GSH (or S-mGSH) is still required for estrone sulfate binding, and the affinity for GSH is unchanged. Estrone sulfate affinity remains low following hydrolysis of ATP. The affinity for GSH also appears to decrease in the post-hydrolytic state. Our results indicate ATP binding is sufficient for reconfiguration of the estrone sulfate binding site to lower affinity and argue for the presence of a modulatory GSH binding site not associated with transport of this tripeptide. A model for the mechanism of GSH-stimulated estrone sulfate transport is proposed.     

Molecular modeling correctly predicts the functional importance of Phe594 in transmembrane helix 11 of the multidrug resistance protein, MRP1 (ABCC1)

The human ATP-binding cassette (ABC) transporter, multidrug resistance protein 1 (MRP1/ABCC1), confers resistance to a broad range of anti-cancer agents and transports a variety of organic anions. At present, essentially no structural data exists for MRP1 that might be used to elucidate its mechanism of transport. Consequently, we have applied a modeling strategy incorporating crystal and indirect structural data from other ABC transporters to construct a model of the transmembrane domains of the core region of MRP1 that includes the amino acid side chains. Three conserved Trp residues and one non-conserved Tyr residue, shown previously to be of functional importance (Koike, K., Oleschuk, C. J., Haimeur, A., Olsen, S. L., Deeley, R. G., and Cole, S. P. C. (2002) J. Biol. Chem. 277, 49495-49503), were found to line the "pore" in our model proximal to the membrane cytosol interface. A fifth aromatic residue (Phe594) was identified that, with the Trp and Tyr residues, completed a ring or "basket" of aromatic amino acids and, accordingly, we postulated that it would also be of functional importance. To test this idea, MRP1-Phe594 mutants were expressed in human embryonic kidney cells, and their properties were examined using membrane vesicles. Substitution of Phe594 with Ala substantially reduced or eliminated the transport of five organic anion substrates by MRP1 and abrogated the binding of leukotriene C4. On the other hand, the conservatively substituted F594W and F594Y mutants remained transport competent, although significant substrate- and substitution-specific changes were observed. These studies provide some structural insight into a possible substrate binding/transport site of MRP1 at the beginning of a putative substrate translocation pathway and demonstrate the usefulness of modeling for directing structure-function analyses of this transporter.     

ATP binding to the first nucleotide-binding domain of multidrug resistance protein MRP1 increases binding and hydrolysis of ATP and trapping of ADP at the second domain.

Multidrug resistance protein (MRP1) utilizes two non-equivalent nucleotide-binding domains (NBDs) to bind and hydrolyze ATP. ATP hydrolysis by either one or both NBDs is essential to drive transport of solute. Mutations of either NBD1 or NBD2 reduce solute transport, but do not abolish it completely. How events at these two domains are coordinated during the transport cycle have not been fully elucidated. Earlier reports (Gao, M., Cui, H. R., Loe, D. W., Grant, C. E., Almquist, K. C., Cole, S. P., and Deeley, R. G. (2000) J. Biol. Chem. 275, 13098-13108; Hou, Y., Cui, L., Riordan, J. R., and Chang, X. (2000) J. Biol. Chem. 275, 20280-20287) indicate that intact ATP is observed bound at NBD1, whereas trapping of the ATP hydrolysis product, ADP, occurs predominantly at NBD2 and that trapping of ADP at NBD2 enhances ATP binding at NBD1 severalfold. This suggested transmission of a positive allosteric interaction from NBD2 to NBD1. To assess whether ATP binding at NBD1 can enhance the trapping of ADP at NBD2, photoaffinity labeling experiments with [alpha-(32)P]8-N(3)ADP were performed and revealed that when presented with this compound labeling of MRP1 occurred at both NBDs. However, upon addition of ATP, this labeling was enhanced 4-fold mainly at NBD2. Furthermore, the nonhydrolyzable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP), bound preferentially to NBD1, but upon addition of a low concentration of 8-N(3)ATP, the binding at NBD2 increased severalfold. This suggested that the positive allosteric stimulation from NBD1 actually involves an increase in ATP binding at NBD2 and hydrolysis there leading to the trapping of ADP. Mutations of Walker A or B motifs in either NBD greatly reduced their ability to be labeled by [alpha-(32)P]8-N(3)ADP as well as by either [alpha-(32)P]- or [gamma-(32)P]8-N(3)ATP (Hou et al. (2000), see above). These mutations also strongly diminished the enhancement by ATP of [alpha-(32)P]8-N(3)ADP labeling and the transport activity of the protein. Taken together, these results demonstrate directly that events at NBD1 positively influence those at NBD2. The interactions between the two asymmetric NBDs of MRP1 protein may enhance the catalytic efficiency of the MRP1 protein and hence of its ATP-dependent transport of conjugated anions out of cells.     

Modulation of multidrug resistance protein (MRP1/ABCC1) expression: a novel physiological role for ouabain

Besides being a (Na⁺,K⁺)-ATPase inhibitor, high doses of the hormone ouabain have also been reported to modulate both the expression and activity of proteins belonging to the ATP binding cassette family of transporters, such as ABCC7 (CFTR), ABCB1 (P-glycoprotein), and ABCC1 (MRP1). Although these proteins are present in the kidney, only ABCB1 has a putative physiological role in this organ, secreting endobiotics and xenobiotics. In the present work, we studied the relationship between ouabain and ABCC1 expression and function, aiming to establish a physiological role for ouabain. It was observed that prolonged (24 h) but not short (30 min) incubation with 1 nmol/L or higher ouabain concentrations decreased the expression of ABCC1 protein and induced its mRNA expression. This decrease was rapidly reversible, reaching control levels after incubation of cells in ouabain-free medium for 3 h, denoting a hormonal action. Moreover, concentrations equal or higher than 100 nmol/L ouabain also induced impairment of ABCC1 activity, increasing the accumulation of carboxyfluorescein diacetate, an ABCC1 fluorescent substrate. Because ouabain is now accepted as an endogenous hormone, our results suggest that ABCC1 is regulated by hormones related to body volume control, which may have implications for the treatment of hypertensive cancer patients. Moreover, providing ABCC1 is expressed in several other tissues, such as brain, testis, and the immune system, and is related to the transport of glutathione, it is possible that ouabain release may control a number of functions within these organs and tissues by modulating both the expression and the activity of ABCC1.     

Expression and function of human MRP1 (ABCC1) is dependent on amino acids in cytoplasmic loop 5 and its interface with nucleotide binding domain 2

Multidrug resistance protein 1 (MRP1) is an ATP-binding cassette transporter that effluxes drugs and organic anions across the plasma membrane. The 17 transmembrane helices of MRP1 are linked by extracellular and cytoplasmic loops (CLs), but their role in coupling the ATPase activity of MRP1 to the translocation of its substrates is poorly understood. Here we have examined the importance of CL5 by mutating eight conserved charged residues and the helix-disrupting Gly(511) in this region. Ala substitution of Lys(513), Lys(516), Glu(521), and Glu(535) markedly reduced MRP1 levels. Because three of these residues are predicted to lie at the interface of CL5 and the second nucleotide binding domain (NBD2), a critical role is indicated for this region in the plasma membrane expression of MRP1. Further support for this idea was obtained by mutating NBD2 amino acids His(1364) and Arg(1367) at the CL5 interface, which also resulted in reduced MRP1 levels. In contrast, mutation of Arg(501), Lys(503), Glu(507), Arg(532), and Gly(511) had no effect on MRP1 levels. Except for K503A, however, transport by these mutants was reduced by 50 to 75%, an effect largely attributable to reduced substrate binding and affinity. Studies with (32)P-labeled azido-ATP also indicated that whereas ATP binding by the G511I mutant was unchanged, vanadate-induced trapping of azido-ADP was reduced, indicating changes in the catalytic activity of MRP1. Together, these data demonstrate the multiple roles for CL5 in the membrane expression and function of MRP1.     

The structure of the multidrug resistance protein 1 (MRP1/ABCC1). crystallization and single-particle analysis

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-binding cassette (ABC) polytopic membrane transporter of considerable clinical importance that confers multidrug resistance on tumor cells by reducing drug accumulation by active efflux. MRP1 is also an efficient transporter of conjugated organic anions. Like other ABC proteins, including the drug resistance conferring 170-kDa P-glycoprotein (ABCB1), the 190-kDa MRP1 has a core structure consisting of two membrane-spanning domains (MSDs), each followed by a nucleotide binding domain (NBD). However, unlike P-glycoprotein and most other ABC superfamily members, MRP1 contains a third MSD with five predicted transmembrane segments with an extracytosolic NH(2) terminus. Moreover, the two nucleotide-binding domains of MRP1 are considerably more divergent than those of P-glycoprotein. In the present study, the first structural details of MRP1 purified from drug-resistant lung cancer cells have been obtained by electron microscopy of negatively stained single particles and two-dimensional crystals formed after reconstitution of purified protein with lipids. The crystals display p2 symmetry with a single dimer of MRP1 in the unit cell. The overall dimensions of the MRP1 monomer are approximately 80 x 100 A. The MRP1 monomer shows some pseudo-2-fold symmetry in projection, and in some orientations of the detergent-solubilized particles, displays a stain filled depression (putative pore) appearing toward the center of the molecule, presumably to enable transport of substrates. These data represent the first structural information of this transporter to approximately 22-A resolution and provide direct structural evidence for a dimeric association of the transporter in a reconstituted lipid bilayer.     

Nonequivalent nucleotide trapping in the two nucleotide binding folds of the human multidrug resistance protein MRP1

Multidrug resistance protein 1 (MRP1) and P-glycoprotein, which are ATP-dependent multidrug efflux pumps and involved in multidrug resistance of tumor cells, are members of the ATP binding cassette proteins and contain two nucleotide-binding folds (NBFs). P-glycoprotein hydrolyzes ATP at both NBFs, and vanadate-induced nucleotide trapping occurs at both NBFs. We examined vanadate-induced nucleotide trapping in MRP1 stably expressed in KB cell membrane by using 8-azido-[alpha-(32)P]ATP. Vanadate-induced nucleotide trapping in MRP1 was found to be stimulated by reduced glutathione, glutathione disulfide, and etoposide and to be synergistically stimulated by the presence of etoposide and either glutathione. These results suggest that glutathione and etoposide interact with MRP1 at different sites and that those bindings cooperatively stimulate the nucleotide trapping. Mild trypsin digestion of MRP1 revealed that vanadate-induced nucleotide trapping mainly occurs at NBF2. Our results suggest that the two NBFs of MRP1 might be functionally nonequivalent.     

Allosteric interactions between the two non-equivalent nucleotide binding domains of multidrug resistance protein MRP1

Membrane transporters of the adenine nucleotide binding cassette (ABC) superfamily utilize two either identical or homologous nucleotide binding domains (NBDs). Although the hydrolysis of ATP by these domains is believed to drive transport of solute, it is unknown why two rather than a single NBD is required. In the well studied P-glycoprotein multidrug transporter, the two appear to be functionally equivalent, and a strongly supported model proposes that ATP hydrolysis occurs alternately at each NBD (Senior, A. E., al-Shawi, M. K., and Urbatsch, I. L. (1995) FEBS Lett 377, 285-289). To assess how applicable this model may be to other ABC transporters, we have examined adenine nucleotide interactions with the multidrug resistance protein, MRP1, a member of a different ABC family that transports conjugated organic anions and in which sequences of the two NBDs are much less similar than in P-glycoprotein. Photoaffinity labeling experiments with 8-azido-ATP, which strongly supports transport revealed ATP binding exclusively at NBD1 and ADP trapping predominantly at NBD2. Despite this apparent asymmetry in the two domains, they are entirely interdependent as substitution of key lysine residues in the Walker A motif of either impaired both ATP binding and ADP trapping. Furthermore, the interaction of ADP at NBD2 appears to allosterically enhance the binding of ATP at NBD1. Glutathione, which supports drug transport by the protein, does not enhance ATP binding but stimulates the trapping of ADP. Thus MRP1 may employ a more complex mechanism of coupling ATP utilization to the export of agents from cells than P-glycoprotein.     

Characterization of the role of polar amino acid residues within predicted transmembrane helix 17 in determining the substrate specificity of multidrug resistance protein 3

Human multidrug resistance protein (MRP) 3 is the most closely related homologue of MRP1. Like MRP1, MRP3 confers resistance to etoposide (VP-16) and actively transports 17 beta-estradiol 17-(beta-D-glucuronide) (E(2)17 beta G), cysteinyl leukotriene 4 (LTC(4)), and methotrexate, although with generally lower affinity. Unlike MRP1, MRP3 also transports monovalent bile salts. We have previously demonstrated that hydrogen-bonding residues predicted to be in the inner-leaflet spanning segment of transmembrane (TM) 17 of MRP1 are important for drug resistance and E(2)17 beta G transport. We have now examined the importance of the hydrogen-bonding potential of residues in TM17 of MRP3 on both substrate specificity and overall activity. Mutation S1229A reduced only methotrexate transport. Mutations S1231A and N1241A decreased resistance to VP-16 and transport of E(2)17 beta G and methotrexate but not taurocholate. Mutation Q1235A also reduced resistance to VP-16 and transport of E(2)17beta G but increased taurocholate transport without affecting transport of methotrexate. Mutations Y1232F and S1233A reduced resistance to VP-16 and the transport of all three substrates tested. In contrast, mutation T1237A markedly increased VP-16 resistance and transport of all substrates. On the basis of the substrates analyzed, residues Ser(1229), Ser(1231), Gln(1235), and Asn(1241) play an important role in determining the specificity of MRP3, while mutation of Tyr(1232), Ser(1233), and Thr(1237) affects overall activity. Unlike MRP1, the involvement of polar residues in determining substrate specificity extends throughout the TM helix. Furthermore, elimination of the hydrogen-bonding potential of a single amino acid, Thr(1237), markedly enhanced the ability of the protein to confer drug resistance and to transport all substrates examined.     

Role of the MRP1/ABCC1 multidrug transporter protein in cancer

Multidrug resistance is a major obstacle to cancer treatment and leads to poor prognosis for the patient. Multidrug resistance-associated protein 1 (MRP1) transports a wide range of therapeutic agents as well as diverse physiological substrates and may play a role in the development of drug resistance in several cancers including those of the lung, breast and prostate, as well as childhood neuroblastoma. The majority of patients with neuroblastoma present with widely disseminated disease at diagnosis and despite intensive treatment, the prognosis for such patients is dismal. There is increasing evidence that MRP1 is a MYCN target gene involved in the development of multidrug resistance in neuroblastoma. Given the importance of MRP1 overexpression in neuroblastoma, MRP1 inhibition may be a clinically relevant approach to improving patient outcome in this disease.     

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.     

Mutations of the Walker B motif in the first nucleotide binding domain of multidrug resistance protein MRP1 prevent conformational maturation

ATP-binding cassette (ABC) transporters couple the binding and hydrolysis of ATP to the translocation of solutes across biological membranes. The so-called "Walker motifs" in each of the nucleotide binding domains (NBDs) of these proteins contribute directly to the binding and the catalytic site for the MgATP substrate. Hence mutagenesis of residues in these motifs may interfere with function. This is the case with the MRP1 multidrug transporter. However, interpretation of the effect of mutation in the Walker B motif of NBD1 (D792L/D793L) was confused by the fact that it prevented biosynthetic maturation of the protein. We have determined now that this latter effect is entirely due to the D792L substitution. This variant is unable to mature conformationally as evidenced by its remaining more sensitive to trypsin digestion in vitro than the mature wild-type protein. In vivo, the core-glycosylated form of that mutant is retained in the endoplasmic reticulum and degraded by the proteasome. A different substitution of the same residue (D792A) had a less severe effect enabling accumulation of approximately equal amounts of mature and immature MRP1 proteins in the membrane vesicles but still resulted in defective nucleotide interaction and organic anion transport, indicating that nucleotide hydrolysis at NBD1 is essential to MRP1 function.     

Reconstitution of transport-active multidrug resistance protein 2 (MRP2; ABCC2) in proteoliposomes

The apical multidrug resistance protein MRP2 (symbol ABCC2) is an ATP-dependent export pump for anionic conjugates in polarized cells. MRP2 has only 48% amino acid identity with the paralog MRP1 (ABCC1). In this study we show that purified recombinant MRP2 reconstituted in proteoliposomes is functionally active in substrate transport. The Km values for ATP and LTC4 in the transport by MRP2 in proteoliposomes were 560 microM and 450 nM, respectively. This transport function of MRP2 in proteoliposomes was dependent on the amount of MRP2 protein present and was determined to 2.7 pmol x min(-1) x mg MRP2(-1) at 100 nM LTC4. Transport was competitively inhibited by the quinoline derivative MK571 with 50% inhibition at about 12 microM. Our data document the first reconstitution of transport-active purified recombinant MRP2. Binding and immunoprecipitation experiments indicated that MRP2 preferentially associates with the chaperone calnexin, but co-reconstitution studies using purified MRP2 and purified calnexin in proteoliposomes suggested that the LTC4 transport function of MRP2 is not dependent on calnexin. The purified, transport-active MRP2 may serve to identify additional interacting proteins in the apical membrane of polarized cells.     

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.     

The leucotriene C4 binding sites in multidrug resistance protein 1 (ABCC1) include the first membrane multiple spanning domain

The multiple drug resistance protein 1 (MRP1 or ABCC1) transports anticancer drugs and normal cell metabolites. Leucotriene C(4) (LTC(4)) is one of the highest affinity substrates of MRP1. In this study, we have synthesized and characterized a novel photoreactive azido analogue of LTC(4) (AALTC(4)). The specificity of AALTC(4) binding to MRP1 was confirmed using an LTC(4)-specific monoclonal antibody. Moreover, binding with radioiodinated [(125)I]AALTC(4) (or IAALTC(4)) to MRP1 was dramatically competed with unmodified LTC(4) and to a lesser degree by glutathione (GSH). Oxidized glutathione (GSSG) slightly increased IAALTC(4) binding to MRP1, while MK571, verapamil, and vincristine inhibited IAALTC(4) binding to MRP1. Using AALTC(4) together with a panel of epitope-specific and LTC(4)-specific monoclonal antibodies, we identified LTC(4) binding sites in MRP1. Western blotting of large tryptic fragments of MRP1 with three well-characterized epitope-specific mAbs (MRPr1, QCRL1, and MRPm6) showed LTC(4) binding in both the N- and C-terminal halves of MRP1. Furthermore, a peptide corresponding to the N-terminal membrane-spanning domain of MRP1 (MSD0) was photoaffinity labeled by AALTC(4), indicating that MSD0 contains an LTC(4) binding site. Higher resolution mapping of additional LTC(4) binding sites was obtained using eight MRP1 variants with each containing hemaglutanin A (HA) epitopes at different sites (at amino acid 4, 163, 271, 574, 653, 938, 1001, or 1222). MRP1 variants were photoaffinity labeled with IAALTC(4) and digested with trypsin to isolate specific regions of MRP1 that interact with LTC(4). These results confirmed that sequences in MSD0 interact with IAALTC(4). Other regions that were photoaffinity labeled by IAALTC(4) include TM 10-11, TM 16-17, and TM 12, shown previously to encode MRP1 drug binding site(s). Together, our results show a high-resolution map of LTC(4) binding domains in MRP1 and provide the first direct evidence for LTC(4) binding within MSD0.