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• W2068063192 abstract "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. 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. The development of resistance to chemotherapy remains one of the biggest obstacles to the successful treatment of cancer. One of the mechanisms by which tumors acquire resistance is the overexpression of membrane transport proteins such as multidrug resistance protein 1 (MRP1 2The abbreviations used are: MRP1, multidrug resistance protein 1; ABC, ATP binding cassette; GSH, reduced glutathione; S-mGSH, S-methyl glutathione; DTT, dithiothreitol; LTC4, leukotriene C4; E217βG, estradiol glucuronide; AMPPNP, adenylylimidodiphosphate; ATPγS, adenosine thiotriphosphate; Vi, orthovanadate ion; mAb, monoclonal antibody; BAEE, N-α-benzoyl-l-arginine ethyl ester; NBD, nucleotide binding domain. 2The abbreviations used are: MRP1, multidrug resistance protein 1; ABC, ATP binding cassette; GSH, reduced glutathione; S-mGSH, S-methyl glutathione; DTT, dithiothreitol; LTC4, leukotriene C4; E217βG, estradiol glucuronide; AMPPNP, adenylylimidodiphosphate; ATPγS, adenosine thiotriphosphate; Vi, orthovanadate ion; mAb, monoclonal antibody; BAEE, N-α-benzoyl-l-arginine ethyl ester; NBD, nucleotide binding domain. or ABCC1) (1Cole S.P.C. Bhardwaj G. Gerlach J.H. Mackie J.E. Grant C.E. Almquist K.C. Stewart A.J. Kurz E.U. Duncan A.M.V. Deeley R.G. Science. 1992; 258: 1650-1654Crossref PubMed Scopus (2986) Google Scholar, 2Gottesman M.M. Fojo T. Bates S.E. Nat. Rev. Cancer. 2002; 2: 48-58Crossref PubMed Scopus (4492) Google Scholar, 3Merino V. Jimenez-Torres N. Merino-Sanjuan M. Curr. Drug Deliv. 2004; 1: 203-212Crossref PubMed Scopus (25) Google Scholar). MRP1 is a member of the C subfamily of the ABC superfamily of transport proteins. It is expressed in most tissues in the body and utilizes energy from ATP hydrolysis to transport a wide variety of structurally and functionally distinct molecules across the plasma membrane. Many MRP1 substrates are conjugated organic anions such as LTC4 (4Leier I. Jedlitschky G. Buchholz U. Cole S.P.C. Deeley R.G. Keppler D. J. Biol. Chem. 1994; 269: 27807-27810Abstract Full Text PDF PubMed Google Scholar, 5Loe D.W. Almquist K.C. Deeley R.G. Cole S.P.C. J. Biol. Chem. 1996; 271: 9675-9682Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar), E217βG (6Loe D.W. Almquist K.C. Cole S.P.C. Deeley R.G. J. Biol. Chem. 1996; 271: 9683-9689Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 7Jedlitschky G. Leier I. Buchholz U. Barnouin K. Kurz G. Keppler D. Cancer Res. 1996; 56: 988-994PubMed Google Scholar), and estrone sulfate (8Qian Y.M. Song W.C. Cui H. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2001; 276: 6404-6411Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). In addition to transporting these endogenously formed metabolites, MRP1 has an important role in detoxification and protection of normal tissues from xenobiotics (9Lorico A. Rappa G. Finch R.A. Yang D. Flavell R.A. Sartorelli A.C. Cancer Res. 1997; 57: 5238-5242PubMed Google Scholar, 10Wijnholds J. Scheffer G.L. van der Valk M. van der Valk P. Beijnen J.H. Scheper R.J. Borst P. J. Exp. Med. 1998; 188: 797-808Crossref PubMed Scopus (186) Google Scholar, 11Wijnholds J. de Lange E.C.M. Scheffer G.L. van den Berg D.J. Mol C. A. A.M. van der Valk M. Schinkel A.H. Scheper R.J. Breimer D.D. Borst P. J. Clin. Invest. 2000; 105: 279-285Crossref PubMed Scopus (340) Google Scholar, 12Leslie E.M. Deeley R.G. Cole S.P.C. Toxicol. Appl. Pharmacol. 2005; 204: 216-237Crossref PubMed Scopus (1112) Google Scholar). However, this is problematic when MRP1 is expressed at elevated levels in cancer cells, because it causes resistance to anticancer agents, including anthracyclines, plant alkaloids, and anti-folates, by transporting them out of cells.An unusual feature of MRP1 (and some other members of the ABCC subfamily) is the requirement for the reducing tripeptide GSH to be present for transport of several of its substrates to occur, including certain anticancer drugs (5Loe D.W. Almquist K.C. Deeley R.G. Cole S.P.C. J. Biol. Chem. 1996; 271: 9675-9682Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar, 8Qian Y.M. Song W.C. Cui H. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2001; 276: 6404-6411Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 13Rappa G. Lorico A. Flavell R.A. Sartorelli A.C. Cancer Res. 1997; 57: 5232-5237PubMed Google Scholar, 14Loe D.W. Deeley R.G. Cole S.P.C. Cancer Res. 1998; 58: 5130-5136PubMed Google Scholar, 15Renes J. de Vries E.G.E. Nienhuis E.F. Jansen P.L.M. Muller M. Br. J. Pharm. 1999; 126: 681-688Crossref PubMed Scopus (244) Google Scholar, 16Leslie E.M. Ito K. Upadhyaya P. Hecht S.S. Deeley R.G. Cole S.P.C. J. Biol. Chem. 2001; 276: 27846-27854Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 17Peklak-Scott C. Townsend A.J. Morrow C.S. Biochemistry. 2005; 44: 4426-4433Crossref PubMed Scopus (42) Google Scholar). GSH is an essential cellular anti-oxidant, which reacts with electrophilic molecules to form conjugated hydrophilic metabolites that can be more readily eliminated from the body. Prior to elimination, such metabolites must be actively effluxed from the cells in which they are formed. MRP1 can transport many such GSH-conjugated anions (18Haimeur A. Conseil G. Deeley R.G. Cole S.P.C. Curr. Drug Metab. 2004; 5: 21-53Crossref PubMed Scopus (445) Google Scholar); however, the role of GSH in MRP1-mediated transport is not limited to forming conjugates. For example, transport of the Vinca alkaloid vincristine is markedly enhanced by GSH, and the interaction appears to be co-transport with the drug without formation of a conjugate (14Loe D.W. Deeley R.G. Cole S.P.C. Cancer Res. 1998; 58: 5130-5136PubMed Google Scholar). Estrone sulfate transport by MRP1 is also enhanced by GSH; however, in contrast to vincristine transport, GSH only stimulates the process and is not itself transported (8Qian Y.M. Song W.C. Cui H. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2001; 276: 6404-6411Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Some MRP1 inhibitors such as the tricyclic isoxazole derivative LY475776 also require GSH to photolabel to MRP1 (19Mao Q.C. Qiu W. Weigl K.E. Lander P.A. Tabas L.B. Shepard R.L. Dantzig A.H. Deeley R.G. Cole S.P.C. J. Biol. Chem. 2002; 277: 28690-28699Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), yet transport of some drugs, such as the antifolate methotrexate occur independently of GSH (20Bakos E. Evers R. Sinko E. Varadi A. Borst P. Sarkadi B. Mol. Pharm. 2000; 57: 760-768Crossref PubMed Scopus (288) Google Scholar). In contrast, GSH can be transported alone, and some xenobiotics, including verapamil, or bioflavonoids such as apigenin, stimulate GSH efflux without being transported themselves (21Loe D.W. Deeley R.G. Cole S.P.C. J. Pharmacol. Exp. Ther. 2000; 293: 530-538PubMed Google Scholar, 22Leslie E.M. Deeley R.G. Cole S.P.C. Drug Metab. Dispos. 2003; 31: 11-15Crossref PubMed Scopus (112) Google Scholar).Cellular GSH/oxidized glutathione levels are important for regulating the redox status of the cell and modulating responses to oxidative stress. Efflux of GSH in response to cell damage has been found to be part of an apoptotic signaling pathway (23Ghibelli L. Fanelli C. Rotilio G. Lafavia E. Coppola S. Colussi C. Civitareale P. Ciriolo M.R. FASEB J. 1998; 12: 479-486Crossref PubMed Scopus (297) Google Scholar, 24Coppola S. Ghibelli L. Biochem. Soc. Trans. 2000; 28: 56-61Crossref PubMed Scopus (140) Google Scholar, 25He Y.Y. Huang J.L. Ramirez D.C. Chignell C.F. J. Biol. Chem. 2003; 278: 8058-8064Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 26Mueller C.F. Widder J.D. McNally J.S. McCann L. Jones D.P. Harrison D.G. Circ. Res. 2005; 97: 637-644Crossref PubMed Scopus (108) Google Scholar), and it has recently been demonstrated that this efflux can be mediated by MRP1 (26Mueller C.F. Widder J.D. McNally J.S. McCann L. Jones D.P. Harrison D.G. Circ. Res. 2005; 97: 637-644Crossref PubMed Scopus (108) Google Scholar, 27Trompier D. Chang X.B. Barattin R. d'Hardemare A.D. Di Pietro A. Baubichon-Cortay H. Cancer Res. 2004; 64: 4950-4956Crossref PubMed Scopus (107) Google Scholar). However, it is not the reducing ability of GSH that is important for its interactions with MRP1, because non-reducing analogues such as ophthalmic acid or S-mGSH can functionally substitute for GSH (8Qian Y.M. Song W.C. Cui H. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2001; 276: 6404-6411Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 14Loe D.W. Deeley R.G. Cole S.P.C. Cancer Res. 1998; 58: 5130-5136PubMed Google Scholar, 16Leslie E.M. Ito K. Upadhyaya P. Hecht S.S. Deeley R.G. Cole S.P.C. J. Biol. Chem. 2001; 276: 27846-27854Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 17Peklak-Scott C. Townsend A.J. Morrow C.S. Biochemistry. 2005; 44: 4426-4433Crossref PubMed Scopus (42) Google Scholar, 19Mao Q.C. Qiu W. Weigl K.E. Lander P.A. Tabas L.B. Shepard R.L. Dantzig A.H. Deeley R.G. Cole S.P.C. J. Biol. Chem. 2002; 277: 28690-28699Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Thus it is clear that the role of GSH in MRP1-mediated transport is very complex, but as yet relatively little is known about the mechanism of interaction of GSH with this transporter.MRP1 function is often evaluated by measuring ATP-dependent uptake of a radiolabeled substrate into inside-out membrane vesicles prepared from cells expressing the transporter. Transmembrane transport of a substrate assayed in this way gives an overall measure of a multistep process. First, the substrate binds to a high affinity site on the cytosolic side of the membrane. This substrate binding site is subsequently reoriented to the extracellular face of the membrane where the binding affinity is decreased so that the substrate may be released. The final step of the transport cycle is the return of the binding site to its initial high affinity state (28Tanford C. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3701-3705Crossref PubMed Scopus (35) Google Scholar). For MRP1 (and other ABC transporters), these steps of the transport cycle are coupled to the binding and/or hydrolysis of ATP, but the precise details of this coupling are not known. Nor is it known when and how GSH participates in this process. Because conventional vesicular transport assays do not readily allow the individual constituent steps of the transport cycle to be examined, we have developed a radioligand binding assay to investigate directly the binding of estrone sulfate to MRP1, to measure the effect of GSH on the binding of this substrate, and to determine how the binding of estrone sulfate and GSH is affected by nucleotide binding and hydrolysis.EXPERIMENTAL PROCEDURESMaterials—[6,7-3H]Estrone sulfate (57.3 Ci/mmol) and [14,15,19,20-3H]LTC4 were purchased from PerkinElmer Life Sciences. LTC4 was from Calbiochem (La Jolla, CA). Methotrexate sodium solution was from Faulding (Montreal, Quebec, Canada). mAbs MRPm6 and MRPr1 were a kind gift from Drs. R. J. Scheper and G. L. Scheffer (Amsterdam, Netherlands). Diphenylcarbamyl chloride-treated trypsin was from ICN Biomedicals (Solon, OH). Complete, EDTA-free protease inhibitor mixture was from Roche Diagnostics (Laval, Quebec, Canada). GSH, S-mGSH, ATP, AMPPNP, ATPγS, sodium orthovanadate, E217βG, estrone 3-sulfate, DTT, and BAEE were from Sigma.Cell Culture and Membrane Preparation—The H69 small cell lung cancer cell line and its doxorubicin-selected, MRP1-overexpressing H69AR-derivative cell line were cultured as described previously (29Cole S.P. Pinkoski M.J. Bhardwaj G. Deeley R.G. Br. J. Cancer. 1992; 65: 498-502Crossref PubMed Scopus (96) Google Scholar). To prepare plasma membranes, cells (∼5 × 108) were harvested, resuspended in buffer 1 (50 mm Tris, pH 7.4, 250 mm sucrose) containing 250 μm CaCl2 and protease inhibitors, and subjected to argon cavitation (250 p.s.i., 5 min, 4 °C). Unexploded cells and debris were removed by low speed centrifugation (750 × g, 10 min, 4 °C), and the supernatant was loaded onto a 35% sucrose cushion. Following centrifugation (100,000 × g, 1 h, 4 °C), plasma membranes were harvested from the sucrose interface, diluted with buffer 2 (50 mm Tris, pH 7.4, 25 mm sucrose), and centrifuged again. The membrane pellet was resuspended in buffer 1 at ∼10 mg of protein per ml and stored at –70 °C for up to 3 months. Protein concentrations were measured using a Bio-Rad Bradford assay, with bovine serum albumin as a standard.[3H]Estrone Sulfate Binding Assays—Membrane protein (10 μg) was allowed to equilibrate with [3H]estrone sulfate in the presence or absence of organic anion substrates and/or nucleotides in a total volume of 50 μlof hypotonic buffer 3 (50 mm HEPES, pH 7.4) at 23 °C for 1 h. The use of hypotonic buffer ensures that the membrane vesicles are burst open and, thus, that the MRP1 protein is accessible from both sides of the membrane. Following equilibration, 1.5 ml of ice-cold buffer 4 (20 mm HEPES, pH 7.4, 20 mm MgCl2) was added, and samples were filtered immediately through a PerkinElmer Life Sciences unifilter GF/B plate using a Packard Filtermate Harvester, and washed twice with buffer 4. Tritium bound to the filter was determined using a PerkinElmer Life Sciences Top Count NXT Microplate Scintillation counter.Displacement/Stimulation Assays—Binding assays were used to measure the displacement of estrone sulfate binding by other substrates of MRP1 or the increase in estrone sulfate binding in the presence of GSH analogues. Membrane proteins (10 μg) were incubated with [3H]estrone sulfate (35–40 nm) in the presence of (a) 3 mm S-mGSH and various concentrations of LTC4 (3 nm to 10 μm), E217βG (300 nm to 3 mm), or methotrexate (1 μm to 3 mm) or (b) various concentrations of GSH or S-mGSH (1 μm to 10 mm), before filtering as described above. DTT (10 mm) was present in all experiments containing GSH to prevent oxidation to oxidized glutathione. Data were fitted with a sigmoidal dose-response curve by non-linear regression analysis (Equation 1), B=Bmin+(Bmax−Bmin)/(1+10(log EC50−log[S]))Eq. 1 where B is the amount of estrone sulfate bound, Bmax is maximal binding at this concentration of estrone sulfate, Bmin is nonspecific binding, [S] = concentration of substrate, and EC50 is the substrate concentration at half-maximal estrone sulfate binding.Saturation Binding Isotherms—To determine parameters of maximal binding capacity (Bmax) and binding dissociation constant (Kd), 10 μgof membrane protein was incubated with various concentrations of [3H]estrone sulfate (10 nm to 30 μm). This assay was done in the presence of 0.3, 1, 3, or 10 mm S-mGSH or GSH (plus 10 mm DTT) in the presence or absence of 1 mm E217βG. Nonspecific binding (in the presence of 1 mm E217βG) was subtracted from total binding to determine specific binding, and specific data were fitted with a one-site binding hyperbola (Equation 2), B=Bmax·[L]/(Kd+[L])Eq. 2 where B is the amount of estrone sulfate bound, Bmax is maximal binding capacity for estrone sulfate, [L] is the concentration of estrone sulfate, and Kd is the binding dissociation constant. The commercially available radioligand could not be used to generate [3H]estrone sulfate concentrations above 3 μm, and this was therefore achieved by supplementing with unlabeled estrone sulfate as previously described (30Martin C. Higgins C.F. Callaghan R. Biochemistry. 2001; 40: 15733-15742Crossref PubMed Scopus (94) Google Scholar).Association Kinetics—The rate of association of estrone sulfate with MRP1 in the presence of S-mGSH was determined by using the following three protocols. Comparison of the data from each protocol was used to ascertain the order of binding of GSH and estrone sulfate to MRP1. Firstly, membrane proteins (10 μg) were preincubated with 3 mm S-mGSH for 30 min in the presence or absence of 1 mm E217βG (for determination of nonspecific estrone sulfate binding). [3H]Estrone sulfate (40 nm) was added at selected time points (0–60 min) and then stopped by addition of buffer 4 and filtration as described above. Secondly, [3H]estrone sulfate (40 nm) and S-mGSH (3 mm) were mixed, with or without E217βG (1 mm), and membrane proteins (10 μg) were added at selected times (0–60 min) before filtering. Lastly, membrane proteins (10 μg) were preincubated with [3H]estrone sulfate (40 nm) for 30 min, with or without 1 mm E217βG, and S-mGSH (3 mm) was added at selected times (0–60 min) before filtering. For each of the above three protocols, specific estrone sulfate binding was calculated by subtracting nonspecific binding (in the presence of 1 mm E217βG) from total binding (in the absence of E217βG), for each time point. An exponential association curve (Equation 3) was fitted to the specific binding data by non-linear regression, B=Bmax(1−e−kobst)Eq. 3 where B is the estrone sulfate bound, Bmax is maximal binding at this concentration of estrone sulfate, t is time, and kobs is the observed rate constant.Addition of Nucleotides—AMPPNP or ATPγS was added to membrane proteins to a final concentration of 4 mm in the presence of 5 mm MgSO4. Vanadate trapping of nucleotide was achieved by incubating membrane proteins at 37 °C for 30 min with 4 mm ATP, 5 mm MgSO4, and 1 mm sodium orthovanadate (prepared as a 100 mm stock solution, pH 10, and boiled prior to use (31Goodno C.C. Method Enzymol. 1982; 85: 116-123Crossref PubMed Scopus (179) Google Scholar)). Excess ATP was removed by centrifugation (25,000 × g, 15 min, 4 °C), and the membranes were resuspended in buffer 3 containing 1 mm orthovanadate, ready for use in the estrone sulfate binding assays. Membrane proteins were subjected to the same conditions but without ATP as a control and were included in each assay to ensure there was no spurious effect of the nucleotide-trapping conditions.[3H]LTC4 Transport to Measure Vanadate-induced Nucleotide Trapping Efficiency—Vanadate-induced nucleotide trapping by MRP1 was carried out as described above, except buffer 1 (sucrose buffer) was used in place of buffer 3 (hypotonic buffer), and following centrifugation to remove unbound ATP, the vesicles were resuspended in buffer 1 without vanadate and immediately used in LTC4 transport assays. As before, a sample subjected to the same conditions but without ATP was prepared as a control. [3H]LTC4 transport was carried out as described previously (5Loe D.W. Almquist K.C. Deeley R.G. Cole S.P.C. J. Biol. Chem. 1996; 271: 9675-9682Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar, 32Tabas L.B. Dantzig A.H. Anal. Biochem. 2002; 310: 61-66Crossref PubMed Scopus (29) Google Scholar). Briefly, 50 nm [3H]LTC4 (20 nCi per point), 10 mm MgCl2, and 4 mm ATP (plus an ATP-regenerating system consisting of creatine kinase and creatine phosphate) or 4 mm AMP were incubated with 2 μg of membrane protein at 23 °C for 1 min. The reaction was stopped by dilution in ice-cold buffer 1 and rapidly filtered as described for the binding assays. Uptake in the presence of AMP was subtracted from uptake in the presence of ATP to determine ATP-dependent transport. The residual ATP-dependent [3H]LTC4 uptake in the vanadate-trapped sample was compared with the control sample as a measure of vanadate-induced nucleotide trapping efficiency.Limited Trypsin Digestion of MRP1—Membranes (0.25 mg of protein ml–1) in buffer 3 were incubated alone, with 10 mm DTT, 50 μm estrone sulfate, 10 mm GSH (plus 10 mm DTT), 10 mm S-mGSH or various combinations of these reagents, for 30 min on ice. Diphenylcarbamyl chloride-treated trypsin was then added at trypsin:protein ratios of 1:5000 to 2.5:1 (w/w) for 15 min at 37 °C. Reactions were stopped by addition of Laemmli sample buffer containing leupeptin (16.7 μgml–1) and phenylmethylsulfonyl fluoride (10 mm). Samples (2 μg of protein) were resolved on a 7% acrylamide gel and immunoblotted. Tryptic fragments of MRP1 were detected by chemiluminescence using the primary mAbs MRPm6 (1:1000) and MRPr1 (1:5000), whose epitopes lie at the COOH terminus (amino acids 1511–1520) and in cytoplasmic loop 3 connecting the first and second membrane spanning domains (amino acids 238–247), respectively (33Hipfner D.R. Gao M. Scheffer G. Scheper R.J. Deeley R.G. Cole S.P.C. Br. J. Cancer. 1998; 78: 1134-1140Crossref PubMed Scopus (71) Google Scholar).The direct effect of estrone sulfate, GSH, and/or DTT on trypsin activity per se was assayed using the model substrate BAEE. Diphenylcarbamyl chloride-treated trypsin (500 units ml–1 in 1 mm HCl) was incubated alone, or in the presence of 50 μm estrone sulfate, 10 mm DTT, 10 mm GSH, 10 mm S-mGSH or combinations of these reagents at 23 °C for 30 min. The trypsin (200 μl) was then added to 0.25 mm BAEE (3 ml), and trypsin activity was determined as the rate of increase of absorbance at 250 nm over a 10-min period. 3Available at www.sigmaaldrich.com/sigma/enzyme%20assay/t9253enz.pdf. Data Analysis—All non-linear regression analyses were carried out using GraphPad Prism 3.0 (San Diego, CA). Data sets contain a minimum of three independent experiments, and data are depicted as means ± S.E. Statistical comparisons were carried out using one-way analysis of variance with a Tukey post-hoc test. Differences were considered statistically significant when p was <0.05.RESULTSRequirement of GSH for Specific Estrone Sulfate Binding—Radioligand binding assays were developed to study the interaction of estrone sulfate and GSH with MRP1. Approximately 250,000 dpm of [3H]estrone sulfate was added to each reaction (10 μg of membrane), of this <1% was bound by the filters. As shown in Fig. 1A, the level of [3H]estrone sulfate binding to H69AR (MRP1+) membranes in the absence of GSH was ∼1,100 dpm, the same as that to membranes from parental H69 (MRP1–) cells, indicating low level nonspecific binding to non-MRP1 components of the membrane preparation. Consistent with this conclusion, the amount of estrone sulfate binding could not be displaced by the MRP1 substrate E217βG. The inability to measure specific estrone sulfate binding could also not be overcome by increasing concentrations of [3H]estrone sulfate, at least up to 10 μm (data not shown). In contrast, the addition of GSH produced a substantial increase in [3H]estrone sulfate binding to the MRP1+ membranes while having no effect on the MRP1– membranes (Fig. 1A), indicating that specific binding of estrone sulfate to MRP1 occurs in the presence of GSH. The specificity of this GSH-induced binding was confirmed by the ability of excess E217βG to reduce [3H]estrone sulfate binding to the nonspecific level seen in the absence of GSH while having no effect on the MRP1– membranes. The specific binding of estrone sulfate to MRP1 could also be readily measured in the presence of the non-reducing GSH analogue S-mGSH. Together these data indicate that membranes from H69AR cells display a specific binding site for estrone sulfate but only in the presence of GSH (or S-mGSH). The lack of binding to H69 membranes indicates that the binding to membranes from resistant cells was to the MRP1 transporter. Support for the specific binding component as MRP1 was provided through heterologous displacement assays using several other known substrates of MRP1. Thus, LTC4 (IC50 0.95 μm), E217βG (IC50 90 μm), and methotrexate (IC50 1.8 mm) all produced concentration-dependent reductions in estrone sulfate binding to levels observed in the H69 (MRP1–) membranes (Fig. 1B). The order of potency to displace estrone sulfate binding matched the relative uptake affinities for MRP1 displayed by these substrates (5Loe D.W. Almquist K.C. Deeley R.G. Cole S.P.C. J. Biol. Chem. 1996; 271: 9675-9682Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar, 6Loe D.W. Almquist K.C. Cole S.P.C. Deeley R.G. J. Biol. Chem. 1996; 271: 9683-9689Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 20Bakos E. Evers R. Sinko E. Varadi A. Borst P. Sarkadi B. Mol. Pharm. 2000; 57: 760-768Crossref PubMed Scopus (288) Google Scholar).Estrone Sulfate Binding to MRP1 Is Greater in the Presence of S-mGSH than GSH—The increase in estrone sulfate binding to MRP1 in the presence of increasing concentrations of GSH or S-mGSH is shown in Fig. 2. The level of estrone sulfate binding in the presence of S-mGSH was significantly higher than in the presence of GSH itself, a difference consistent with the relative abilities of these two tripeptides to stimulate estrone sulfate vesicular transport by MRP1 (8Qian Y.M. Song W.C. Cui H. Cole S.P.C. Deeley R.G. J. Biol. Chem. 2001; 276: 6404-6411Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 34Leslie E.M. Bowers R.J. Deeley R.G. Cole S.P.C. J. Pharmacol. Exp. Ther. 2003; 304: 643-653Crossref PubMed Scopus (48) Google Scholar). This difference is not due to the presence of DTT, which is added to all experiments using GSH to prevent oxidation to oxidized glutathione, because comparable results are obtained with S-mGSH whether supplemented with DTT or not (data not shown). Despite differences in the amount of estrone sulfate bound, the potencies of GSH and S-mGSH to increase estrone sulfate binding were comparable, with EC50 values of 0.75 ± 0.12 mm and 0.77 ± 0.06 mm, respectively. These observations indicate that MRP1 displays no difference in its affinity for GSH or S-mGSH.FIGURE 2Effect of GSH and S-mGSH on [3H]estrone sulfate binding to MRP1. Binding of [3H]estrone sulfate (40 nm) to H69AR (MRP1+) membrane (10 μg of protein) was measured in the presence of increasing concentrations (1 μm to 10 mm) of S-mGSH (closed circles) or GSH (open squares). Data points are means ± S.E. from at least six independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Affinity and Binding Capacity of MRP1 for Estrone Sulfate—To determine the affinity and maximal binding capacity of MRP1, saturation binding isotherms for estrone sulfate, in the presence of either GSH or S-mGSH (3 mm), were determined (Fig. 3). A statistically significant difference between the affinity (Kd) for estrone sulfate binding to MRP1 was observed in the presence of 3 mm GSH (1.48 ± 0.06 μm) compared with 3 mm S-mGSH (0.59 ± 0.05 μm, p < 0.001). However, there was no difference in the maximal binding capacity for estrone sulfate (Bmax 83 ± 8 pmol mg–1 with GSH versus 87 ± 7 pmol mg–1 with S-mGSH). The difference in affinity accounts for the higher level of estrone sulfate binding in the presence of S-mG" @default.
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• W2068063192 date "2006-05-01" @default.
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• W2068063192 title "Role of GSH in Estrone Sulfate Binding and Translocation by the Multidrug Resistance Protein 1 (MRP1/ABCC1)" @default.
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• W2068063192 doi "https://doi.org/10.1074/jbc.m600869200" @default.
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