Evidence for Two Interacting Ligand Binding Sites in Human Multidrug Resistance Protein 2 (ATP Binding Cassette C2)
2003; Elsevier BV; Volume: 278; Issue: 26 Linguagem: Inglês
10.1074/jbc.m303504200
ISSN1083-351X
AutoresNoam Zelcer, Maarten T. Huisman, Glen Reid, Peter R. Wielinga, Pauline Breedveld, Annemieke Kuil, Puck Knipscheer, Jan H.M. Schellens, Alfred H. Schinkel, Piet Borst,
Tópico(s)Pregnancy and Medication Impact
ResumoMultidrug 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-β-d-glucuronide (E217βG), 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 E217βG transport by MRP2 increases sigmoidally with substrate concentration indicative of homotropic cooperativity. Half-maximal transport was obtained at 120 μm E217βG, in contrast to values < 20 μm for MRP1 and 3. MRP2 stimulators, such as indomethacin and sulfanitran, strongly increased the affinity of MRP2 for E217βG (half-maximal transport rates at 65 and 16 μm E217βG, 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 E217βG transport, such as penicillin G or pantoprazole, are not detectably transported by MRP2, suggesting that they allosterically stimulate transport without being cotransported with E217βG. 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 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-β-d-glucuronide (E217βG), 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 E217βG transport by MRP2 increases sigmoidally with substrate concentration indicative of homotropic cooperativity. Half-maximal transport was obtained at 120 μm E217βG, in contrast to values < 20 μm for MRP1 and 3. MRP2 stimulators, such as indomethacin and sulfanitran, strongly increased the affinity of MRP2 for E217βG (half-maximal transport rates at 65 and 16 μm E217βG, 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 E217βG transport, such as penicillin G or pantoprazole, are not detectably transported by MRP2, suggesting that they allosterically stimulate transport without being cotransported with E217βG. 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. Members of the ABC 1The abbreviations used are: ABC, ATP binding cassette; E217βG, estradiol-17-β-d-glucuronide; GS-DNP, glutathione-S-dinitrophenol; MDCK, Madin-Darby canine kidney; MRP, multidrug resistance protein; MTX, methotrexate; NNAL-glucuronide, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol glucuronide; S1/2, apparent half-maximal rate; Sf9, Spodoptera frugiperda. family of membrane transporters mediate the transport of various substrates across biological membranes at the expense of ATP hydrolysis (1Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Google Scholar, 2Borst P. Oude Elferink R. Annu. Rev. Biochem. 2002; 71: 537-592Google Scholar). The ABCC subfamily (3Dean M. Rzhetsky A. Allikmets R. Genome Res. 2001; 11: 1156-1166Google Scholar) contains multidrug resistance proteins 1–9 (MRP1–9) along with SUR1, SUR2, and CFTR (1Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Google Scholar, 2Borst P. Oude Elferink R. Annu. Rev. Biochem. 2002; 71: 537-592Google Scholar, 4Borst P. Evers R. Kool M. Wijnholds J. J. Natl. Cancer Inst. 2000; 92: 1295-1302Google Scholar, 5Renes J. de Vries E.G. Jansen P.L. Muller M. Drug Resist. Update. 2000; 3: 289-302Google Scholar). Interest in the multidrug resistance proteins was sparked by their possible involvement in the clinical resistance of tumors to chemotherapeutic agents. The first member of this family to be cloned, MRP1, confers resistance to a broad spectrum of anticancer drugs when overproduced in cells (6Cole S.P. Bhardwaj G. Gerlach J.H. Mackie J.E. Grant C.E. Almquist K.C. Stewart A.J. Kurz E.U. Duncan A.M. Deeley R.G. Science. 1992; 258: 1650-1654Google Scholar). A common feature of MRPs is that they transport a wide variety of organic anions and compounds that are conjugated with sulfate, glucuronate, or glutathione (GSH) (7Jedlitschky G. Leier I. Buchholz U. Barnouin K. Kurz G. Keppler D. Cancer Res. 1996; 56: 988-994Google Scholar; for review, see Refs. 2Borst P. Oude Elferink R. Annu. Rev. Biochem. 2002; 71: 537-592Google Scholar and 8Konig J. Nies A.T. Cui Y. Leier I. Keppler D. Biochim. Biophys. Acta. 1999; 1461: 377-394Google Scholar, 9Jedlitschky G. Keppler D. Vitam. Horm. 2002; 64: 153-184Google Scholar, 10Leslie E.M. Deeley R.G. Cole S.P. Toxicology. 2001; 167: 3-23Google Scholar). How MRPs transport their substrates is not known in detail. MRPs are large membrane-associated proteins, and their structural analysis has proven difficult (11Varadi A. Tusnady G.E. Sarkadi B. Holland I.B. Cole S.P. Kuchler K. Higgins C.F. ABC Proteins from Bacteria to Man. Academic Press, London2002: 37-46Google Scholar). Although several high resolution structures of bacterial ABC transporters have been determined (12Hung L.W. Wang I.X. Nikaido K. Liu P.Q. Ames G.F. Kim S.H. Nature. 1998; 396: 703-707Google Scholar, 13Locher K.P. Lee A.T. Rees D.C. Science. 2002; 296: 1091-1098Google Scholar), only low resolution structures are available for the drug transporters MRP1 and MDR1 P-glycoprotein (14Rosenberg M.F. Callaghan R. Ford R.C. Higgins C.F. J. Biol. Chem. 1997; 272: 10685-10694Google Scholar, 15Rosenberg M.F. Kamis A.B. Callaghan R. Higgins C.F. Ford R.C. J. Biol. Chem. 2002; 278: 8294-8299Google Scholar, 16Rosenberg M.F. Mao Q. Holzenburg A. Ford R.C. Deeley R.G. Cole S.P. J. Biol. Chem. 2001; 276: 16076-16082Google Scholar). In the absence of a detailed structure, the mechanism of transport has been inferred from a combination of transport, binding and mutational studies. Models proposed for MDR1 P-glycoprotein predict three or four drug binding sites or a single complex substrate binding site in which the binding of one compound can affect the binding of another one, the induced-fit model (17Martin C. Berridge G. Higgins C.F. Mistry P. Charlton P. Callaghan R. Mol. Pharmacol. 2000; 58: 624-632Google Scholar, 18Shapiro A.B. Fox K. Lam P. Ling V. Eur. J. Biochem. 1999; 259: 841-850Google Scholar, 19Loo T.W. Clarke D.M. J. Biol. Chem. 2002; 277: 44332-44338Google Scholar, 20Kondratov R.V. Komarov P.G. Becker Y. Ewenson A. Gudkov A.V. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14078-14083Google Scholar). Also for MRP1 evidence for more than one ligand binding site was obtained (for review, see Refs. 2Borst P. Oude Elferink R. Annu. Rev. Biochem. 2002; 71: 537-592Google Scholar and 21Deeley R.G. Cole S.P. Holland I.B. Cole S.P. Kuchler K. Higgins C.F. ABC Proteins from Bacteria to Man. Academic Press, London2002: 393-422Google Scholar). The major canalicular organic anion transporter, MRP2 (ABCC2), is closely related to MRP1 (2Borst P. Oude Elferink R. Annu. Rev. Biochem. 2002; 71: 537-592Google Scholar, 8Konig J. Nies A.T. Cui Y. Leier I. Keppler D. Biochim. Biophys. Acta. 1999; 1461: 377-394Google Scholar). The substrate specificities of MRP1 and 2 overlap to a large extent (9Jedlitschky G. Keppler D. Vitam. Horm. 2002; 64: 153-184Google Scholar, 22Chen Z.S. Kawabe T. Ono M. Aoki S. Sumizawa T. Furukawa T. Uchiumi T. Wada M. Kuwano M. Akiyama S.I. Mol. Pharmacol. 1999; 56: 1219-1228Google Scholar, 23Cui Y. Konig J. Buchholz J.K. Spring H. Leier I. Keppler D. Mol. Pharmacol. 1999; 55: 929-937Google Scholar, 24Evers R. Kool M. van Deemter L. Janssen H. Calafat J. Oomen L.C. Paulusma C.C. Oude Elferink R.P. Baas F. Schinkel A.H. Borst P. J. Clin. Invest. 1998; 101: 1310-1319Google Scholar), but their tissue localizations differ. MRP1 is localized in the basolateral membranes of polarized cells and is present in all tissues, whereas MRP2 is found in the apical membranes of polarized cells and is expressed mainly in the liver, kidney, and intestine. Bakos et al. (25Bakos E. Evers R. Sinko E. Varadi A. Borst P. Sarkadi B. Mol. Pharmacol. 2000; 57: 760-768Google Scholar) demonstrated in vesicular transport assays that transport of the GSH conjugate of N-ethylmaleimide by MRP2 is stimulated by several organic anions. Experiments with polarized cells led to a model in which MRP2 cotransports drugs from two distinct drug binding sites (26Evers R. de Haas M. Sparidans R. Beijnen J. Wielinga P.R. Lankelma J. Borst P. Br. J. Cancer. 2000; 83: 375-383Google Scholar). Cotransport cannot account for recent observations on MRP1, however (27Leslie E.M. Ito K. Upadhyaya P. Hecht S.S. Deeley R.G. Cole S.P. J. Biol. Chem. 2001; 276: 27846-27854Google Scholar). In vectorial transport assays with MDCKII/MRP2 cells we recently observed that the transport of saquinavir is stimulated by probenecid (28Huisman M.T. Smit J.W. Crommentuyn K.M. Zelcer N. Wiltshire H.R. Beijnen J.H. Schinkel A.H. Aids. 2002; 16: 2295-2301Google Scholar). Such drug interactions could potentially affect the oral bioavailability and pharmacokinetics of drugs transported by MRP2. We have therefore studied drug interactions with MRP2 more in detail using transport assays with membrane vesicles from Spodoptera frugiperda (Sf9) insect cells that were infected with a baculovirus construct containing MRP2 (25Bakos E. Evers R. Sinko E. Varadi A. Borst P. Sarkadi B. Mol. Pharmacol. 2000; 57: 760-768Google Scholar). Using estradiol-17-β-d-glucuronide (E217βG), methotrexate (MTX), and glutathione-S-dinitrophenol (GS-DNP) as model substrates, we found stimulation of substrate transport by a range of compounds. We propose that MRP2 contains two distinguishable binding sites: one site from which drug is transported and a second site that allosterically regulates the former. Analogous results have been independently obtained by Bodo and colleagues and are presented in the accompanying manuscript. Materials—40.5 Ci/mmol [3H]E217βG, 21 Ci/mmol [3H]penicillin G, and 20 mCi/mmol [14C]indomethacin were obtained from PerkinElmer Life Sciences. 9 Ci/mmol [3H]MTX was obtained from Amersham Biosciences. 13.6 μCi/mg [14C]saquinavir was from Roche Applied Science. Vials of omeprazole (Losec®, AstraZeneca) and pantoprazole (Pantozol, Altana Pharma BV) were obtained from the pharmacy of The Netherlands Cancer Institute and dissolved in saline according to the manufacturers' instructions. Creatine phosphate and creatine kinase were obtained from Roche, and RC-L55 and OE-67 filters were from Schleicher & Schuell. All other chemicals and reagents were purchased from Sigma. Cell Lines and Culture Conditions—Sf9 insect cells in suspension were grown in Sf-900 II SFM medium in the absence of serum (Invitrogen). The MDCKII control and MRP2-overproducing lines were described previously (24Evers R. Kool M. van Deemter L. Janssen H. Calafat J. Oomen L.C. Paulusma C.C. Oude Elferink R.P. Baas F. Schinkel A.H. Borst P. J. Clin. Invest. 1998; 101: 1310-1319Google Scholar) and grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 100 units of penicillin/streptomycin/ml. Cells were grown at 37 °C with 5% CO2 under humidifying conditions. Protein Analysis and Immunoblotting—Membrane vesicle preparations were diluted in buffer (10 mm KCl, 1.5 mm MgCl2, 10 mm Tris-HCl, pH 7.4), supplemented by a mixture of protease inhibitors used at the concentration recommended by the manufacturer (Roche). The indicated amount of protein was size fractionized on a 7.5% SDS-polyacrylamide gel and subsequently blotted overnight in a tank blotting system. MRP1, 2, and 3 were detected with the monoclonal antibodies MRP-r1 (1:1000), M2II5 (1:250) and M3II9 (1:250), respectively, as described previously (24Evers R. Kool M. van Deemter L. Janssen H. Calafat J. Oomen L.C. Paulusma C.C. Oude Elferink R.P. Baas F. Schinkel A.H. Borst P. J. Clin. Invest. 1998; 101: 1310-1319Google Scholar, 29Zelcer N. Saeki T. Reid G. Beijnen J.H. Borst P. J. Biol. Chem. 2001; 276: 46400-46407Google Scholar). Signals were visualized with chemiluminescence (ECL, Amersham Biosciences). Transepithelial Transport Assays—Transepithelial transport assays were done as described previously (28Huisman M.T. Smit J.W. Crommentuyn K.M. Zelcer N. Wiltshire H.R. Beijnen J.H. Schinkel A.H. Aids. 2002; 16: 2295-2301Google Scholar). Briefly, Cells were seeded on microporous polycarbonate membrane filters (Transwell 3414, Costar, Corning, NY) at a density of 1.0 × 106 cells/well in 2 ml of complete medium. Cells were grown for 3 days with medium replacements every day. 2 h before the start of the experiment, complete medium was replaced from both compartments with Opti-MEM, without serum containing 5 μm GF120918 and the appropriate concentration of transport modulators. At t = 0 h the experiment was started by replacing the medium from both compartments containing 5 μm appropriate radiolabeled drug (∼ 3 kBq/well) and either 3H- or 14C-labeled inulin (∼ 3 kBq/well) in the appropriate compartment. The latter compound was added to check for leakage through the cell layers. Cells were incubated at 37 °C in 5% CO2, and 50-μl aliquots were taken each hour. The radioactivity in these aliquots was measured by the addition of 4 ml of scintillation fluid (Ultima-gold; Packard, Meriden, CT) and subsequent liquid scintillation counting. Inulin leakage did not exceed 3% over 4 h. The percentage of radioactivity appearing in the opposite compartment, of the total amount initially applied, was measured and plotted. The amount of radiolabeled drug in the cell layer at the end of the experiment was determined by liquid scintillation counting of the excised filter, after washing with ice-cold phosphate-buffered saline. Preparation of Membrane Vesicles—Membrane vesicles from Sf9 cells were obtained after infection with an MRP1 (25Bakos E. Evers R. Sinko E. Varadi A. Borst P. Sarkadi B. Mol. Pharmacol. 2000; 57: 760-768Google Scholar), MRP2 (25Bakos E. Evers R. Sinko E. Varadi A. Borst P. Sarkadi B. Mol. Pharmacol. 2000; 57: 760-768Google Scholar), MRP3 (29Zelcer N. Saeki T. Reid G. Beijnen J.H. Borst P. J. Biol. Chem. 2001; 276: 46400-46407Google Scholar), or MRP4 (30van Aubel R.A. Smeets P.H. Peters J.G. Bindels R.J. Russel F.G. J. Am. Soc. Nephrol. 2002; 13: 595-603Google Scholar, 31Zelcer N. Reid G. Wielinga P. Kuil A. Van Der Heijden I. Schuetz J.D. Borst P. Biochem. J. 2003; 371: 361-367Google Scholar) cDNA-containing baculovirus at a multiplicity of infection of 1. After incubation at 27 °C for 3 days, cells were harvested by centrifugation at 3,000 rpm for 5 min. The pellet was resuspended in ice-cold hypotonic buffer (0.5 mm sodium phosphate, 0.1 mm EDTA, pH 7.4) supplemented with protease inhibitors (2 mm phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 5 μg/ml leupeptin, 10 μm pepstatin) and incubated at 4 °C for 90 min. The suspension was centrifuged at 4 °C at 100,000 × g for 40 min, and the pellet was homogenized in ice-cold TS buffer (50 mm Tris-HCl, 250 mm sucrose, pH 7.4) using a tight fitting Dounce homogenizer. After centrifugation at 500 × g at 4 °C for 10 min, the supernatant was centrifuged at 4 °C at 100,000 × g for 40 min. The pellet was resuspended in TS buffer and passed through a 27-gauge needle 25 times. The vesicles were dispensed in aliquots, frozen in liquid nitrogen, and stored at –80 °C until use. Vesicular Transport Assays—Vesicular transport assays were performed in buffer consisting of 100 mm KCl, 50 mm HEPES/KOH, pH 7.4, in the presence or absence of 4 mm ATP (32Heijn M. Hooijberg J.H. Scheffer G.L. Szabo G. Westerhoff H.V. Lankelma J. Biochim. Biophys. Acta. 1997; 1326: 12-22Google Scholar). Similar results were obtained with a Tris/sucrose buffer (not shown). The time- and concentration-dependent uptake of substrates into membrane vesicles was studied following the rapid filtration method as described previously (29Zelcer N. Saeki T. Reid G. Beijnen J.H. Borst P. J. Biol. Chem. 2001; 276: 46400-46407Google Scholar). For all results presented here, accumulation of substrate increased with time for at least 10 min and was dependent on the presence of ATP. The ATP-dependent transport was calculated by subtracting the transport in the absence of ATP from that in its presence. In experiments where the effect of GSH on transport was studied, 10 mm dithiothreitol was in the reaction mixture. Concentration-dependent uptake was analyzed using a nonlinear regression algorithm. Effects of Drugs and Organic Anions on E217βG Transport by MRP2—Membrane vesicles were prepared from Sf9 insect cells transfected with a recombinant baculovirus coding for human MRP2. These vesicles contain high levels of MRP2 (Fig. 1) and were used to study the effect of various organic anions and commonly used drugs on the transport of 1 μm E217βG by MRP2. The compounds tested could be divided into four classes based on their interaction with MRP2: 1) compounds that only showed a stimulatory effect at the concentrations tested (Fig. 2, A and B); 2) compounds that stimulated transport at low concentrations but showed a decrease in their stimulation capacity at higher concentrations (Fig. 2, C and D); 3) compounds that only inhibited transport (Fig. 2E); and 4) compounds that had no substantial effect on the transport of E217βG by MRP2 (Fig. 2F).Fig. 2Effects of organic anions and drugs on the transport of 1 μm E217βG by MRP2. Membrane vesicles containing MRP2 were incubated with 1 μm [3H]E217βG for 2 min at 37 °C in the presence or absence of the indicated compounds. The ATP-dependent transport is plotted as a percentage of the control value. Each point and error are the mean ± S.E. of experiments in triplicate.View Large Image Figure ViewerDownload (PPT) Sulfanitran, the strongest stimulator of MRP2-mediated E217βG transport, also stimulated the vectorial transport of saquinavir, a recently described MRP2 substrate (28Huisman M.T. Smit J.W. Crommentuyn K.M. Zelcer N. Wiltshire H.R. Beijnen J.H. Schinkel A.H. Aids. 2002; 16: 2295-2301Google Scholar), across polarized MDCKII monolayers demonstrating that it also stimulates MRP2 in intact cells (Fig. 3). Comparison of B and D of Fig. 3 shows that sulfanitran increases transport of saquinavir in the apical direction, decreases transport in the basolateral direction, and substantially decreases the intracellular concentration of saquinavir. Saquinavir is too hydrophobic to study in the vesicular transport assay, but in transepithelial transport assays, we have shown previously that in addition to sulfanitran, other stimulators of vesicular transport also stimulate MRP2 in intact cells: transport of saquinavir is stimulated by both sulfinpyrazone and probenecid (28Huisman M.T. Smit J.W. Crommentuyn K.M. Zelcer N. Wiltshire H.R. Beijnen J.H. Schinkel A.H. Aids. 2002; 16: 2295-2301Google Scholar), and sulfinpyrazone and indomethacin stimulate transport of GSH (26Evers R. de Haas M. Sparidans R. Beijnen J. Wielinga P.R. Lankelma J. Borst P. Br. J. Cancer. 2000; 83: 375-383Google Scholar). The stimulation by the compounds studied was specific for MRP2. Neither lansoprazole nor saquinavir at their maximal MRP2-stimulatory concentration stimulated transport of E217βG in wild type, MRP1, MRP3, or MRP4 vesicles, and sulfanitran had no effect on transport of E217βG in wild type vesicles either (data not shown). Furosemide and acetaminophen-glucuronide even inhibited MRP3-mediated transport of E217βG (Fig. 4), whereas these compounds stimulated transport of E217βG by MRP2 (Fig. 2). Sulfanitran, the compound that stimulated MRP2 transport most, had only a minimal effect on MRP3 (Fig. 4). Effects of Drugs on Transport of GS-DNP by MRP2—GSH conjugates are another class of molecules transported by MRP2. We therefore tested whether transport of GS-DNP, a model GSH conjugate and a known substrate of MRP2, could be stimulated like transport of E217βG. The results are summarized in Table I. Like E217βG, GS-DNP transport is stimulated by sulfanitran and indomethacin albeit to a lower extent. Sulfinpyrazone stimulates GS-DNP transport in vesicular transport assays, similar to what we found previously in MDCKII/MRP2 cells (24Evers R. Kool M. van Deemter L. Janssen H. Calafat J. Oomen L.C. Paulusma C.C. Oude Elferink R.P. Baas F. Schinkel A.H. Borst P. J. Clin. Invest. 1998; 101: 1310-1319Google Scholar). Furosemide at its maximal stimulatory concentration (500 μm) has only a marginal effect on GS-DNP transport, in contrast to its effect on E217βG transport. Moreover, whereas probenecid strongly stimulated the transport of E217βG, it inhibited GS-DNP transport as is the case for MTX as well.Table IEffects of drugs and organic anions on GS-DNP transport by MRP2CompoundConcentrationATP-dependent transportμM%Sulfanitran1,000253 ± 10Indomethacin100274 ± 11Furosemide500111 ± 3MTX2,20083 ± 1Probenecid1,00062 ± 3E217βG5100 ± 3Sulfinpyrazone1,000152 ± 2 Open table in a new tab Effects of Stimulators of MRP2-mediated E217βG Transport on the Affinity of MRP2 for Substrate—Evers et al. (26Evers R. de Haas M. Sparidans R. Beijnen J. Wielinga P.R. Lankelma J. Borst P. Br. J. Cancer. 2000; 83: 375-383Google Scholar) demonstrated that sulfinpyrazone stimulates transport of GSH by MRP2 and that vinblastine transport is accompanied by GSH transport at an approximate ratio of 1:1. Sulfinpyrazone also stimulates transport of E217βG by MRP2 (Fig. 2C). Compounds that stimulate E217βG transport by MRP2 might therefore be cotransported with this substrate, as proposed previously (26Evers R. de Haas M. Sparidans R. Beijnen J. Wielinga P.R. Lankelma J. Borst P. Br. J. Cancer. 2000; 83: 375-383Google Scholar). We have tested this in vesicular and transepithelial transport assays. We did not detect vesicular transport of [3H]penicillin G at concentrations of up to 1 mm either in the absence or presence of varying concentrations of E217βG (data not shown). Similarly, In vesicular transport assays we did not detect transport of [14C]indomethacin (at concentrations up to 50 μm) by MRP2. Negative results in vesicular transport assays are not conclusive, however, as the substrate may leak out of the vesicles at high rate, preventing transport measurements. This is not a problem in transepithelial transport assays with MDCKII/MRP2 cells. Indeed, in these assays we detected marginal transport of indomethacin and probenecid by MRP2 (data not shown). In the same assays, we did not detect transport of penicillin G or pantoprazole, another stimulator of E217βG transport by MRP2 (Fig. 2, and data not shown). These results indicate that these compounds are either not transported by MRP2 or are poor substrates, even though they strongly stimulate transport of E217βG by MRP2, making cotransport unlikely. To investigate the mechanism of stimulation further, we determined the rate of transport of E217βG by MRP2 as a function of substrate concentration in the absence or presence of 100 μm indomethacin or 1 mm sulfanitran (Fig. 5). The transport of E217βG by MRP2 was not consistent with simple Michaelis-Menten kinetics, but the plot of reaction velocity versus substrate concentration was clearly sigmoidal with an estimated apparent half-maximal rate (S1/2) at 120 μm E217βG (Fig. 5A). In the presence of either of the two stimulators, the curve was shifted to a more hyperbolic shape with apparent S1/2 values of 65 and 16 μm in the presence of 100 μm indomethacin and 1 mm sulfanitran, respectively (Fig. 5, B and C). The maximal rate of transport remained relatively unchanged. The degree of stimulation of E217βG transport at low substrate concentration (1 μm) by these compounds correlates well with the increased affinity for this substrate (Figs. 2 and 5). At 200 μm E217βG transport was not stimulated by 100 μm indomethacin and was only stimulated by 10% by 1 mm sulfanitran (data not shown) suggesting that at this concentration of substrate MRP2 is close to saturation. We note, however, that Bodo et al.2 found higher rates of E217βG transport at 1 mm than at 200 μm, the maximal concentration that we were able to test because of solubility problems. As a control, we also determined the concentration-dependent transport of E217βG by MRP1, for which we found saturation kinetics with a Km of 3.1 ± 0.3 μm and a Vmax of 38 ± 1 pmol/mg/min. This further strengthens the notion that the requirements for optimal transport of the same substrate by MRP1 and MRP2 are different (27Leslie E.M. Ito K. Upadhyaya P. Hecht S.S. Deeley R.G. Cole S.P. J. Biol. Chem. 2001; 276: 27846-27854Google Scholar), even though the substrate specificity of these transporters largely overlaps. Characterization of MTX Transport by MRP2—MTX is transported by MRP2, but MRP2 has such a low affinity for this substrate that we were unable to determine reliable kinetic parameters for this transport process (not shown and Refs. 25Bakos E. Evers R. Sinko E. Varadi A. Borst P. Sarkadi B. Mol. Pharmacol. 2000; 57: 760-768Google Scholar and 33Chen Z.S. Lee K. Walther S. Raftogianis R.B. Kuwano M. Zeng H. Kruh G.D. Cancer Res. 2002; 62: 3144-3150Google Scholar). Sulfinpyrazone and indomethacin stimulate MTX transport, but to a much lower extent than the transport of E217βG (Fig. 6A). In contrast, E217βG, GSSG, and probenecid only inhibited MTX transport by MRP2 (Fig. 6B). E217βG at a concentration of 200 μm inhibits the transport of MTX by 80%, suggesting that these two substrates share a common step in transport. In this light, the absence of a substantial inhibitory or stimulatory effect of MTX (at a concentration up to 4.4 mm) on transport of 1 μm E217βG by MRP2 (Fig. 2F) is unexpected. A possible explanation is that MTX is a weak stimulator of E217βG transport and that at low E217βG concentrations (allosteric) stimulation and inhibition (by competition) of E217βG transport by MTX balance out. Indeed, MTX did inhibit the transport of high concentrations of E217βG (200 μm) and of 1 μm E217βG stimulated by 1 mm sulfanitran (Fig. 7). Moreover, trimetrexate, a structural analog of MTX, stimulates transport of 1 μm E217βG by 320 ± 10% at a concentration of 300 μm (data not shown). This is compatible with the hypothesis that MTX itself might have a weak stimulatory effect as well. Following the reasoning applied to MTX, GSH, another low affinity substrate of MRP2 (34Paulusma C.C. van Geer M.A. Evers R. Heijn M. Ottenhoff R. Borst P. Oude Elferink R.P. Biochem. J. 1999; 338: 393-401Google Scholar), should be able to inhibit E217βG and MTX transport under appropriate conditions. However, GSH at concentrations up to 10 mm had no effect on the transport of 200 μm E217βG or 100 μm MTX by MRP2 (not shown).Fig. 7Transport of E217βG by MRP2 at saturating substrate concentrations is inhibited by MTX. Membrane vesicles containing MRP2 were incubated at 37 °C for 30 s with 200 μm [3H]E217βG (black bars) or with 1 μm [3H]E217βG together with 1 mm sulfanitran (hatched bars) in the presence of increasing concentrations of MTX. The ATP-dependent transport of E217βG by MRP2 was determined, and each bar represents the mean ± S.E. of experiments in triplicate.View Large Image Figure ViewerDownload (PPT) Our work shows complex effects of various drugs and organic anions on MRP2. For transport of E217βG the plot of reaction velocity versus substrate concentration is sigmoidal (Fig. 5), indicative of at least two drug binding sites that interact in a positively cooperative manner. Many compounds stimulate E217βG transport at low substrate concentrations, and for two stimulators, sulfanitran and indomethacin, we have shown that they increase the affinity of MRP2 for substrate with no significant effect on the Vmax. Compounds that stimulate transport of substrates by MRP2 are not necessarily transported by MRP2. In vesicular transport assays we do not detect transport of [3H]penicillin G and [14C]indomethacin. Using vectorial transport assays with MDCKII/MRP2 cells we found only marginal transport of indomethacin and no transport of pantoprazole, another stimulator. Taurocholate is a good stimulator of E217βG transport by human (Fig. 2D) and rat Mrp2, but it is not transported by rat Mrp2 (35Akita H. Suzuki H. Ito K. Kinoshita S. Sato N. Takikawa H. Sugiyama Y. Biochim. Biophys. Acta. 2001; 1511: 7-16Google Scholar), as is the case with furosemide (36Chen C. Scott D. Hanson E. Franco J. Berryman E. Volberg M. Liu X. Pharm. Res. (N. Y.). 2003; 20: 31-37Google Scholar). Taken together, these observations indicate that transport of a compound by MRP2 is not a prerequisite for its ability to stimulate the t
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