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GSH Inhibits Trypsinization of the C-terminal Half of Human MRP1

2004; Elsevier BV; Volume: 280; Issue: 7 Linguagem: Inglês

10.1074/jbc.m409498200

1083-351X

Xiao‐Qin Ren, Tatsuhiko Furukawa, Yuichi Nakajima, Homare Takahashi, Shunji Aoki, Tomoyuki Sumizawa, Misako Haraguchi, Motomasa Kobayashi, Kazuo Chijiiwa, Shin‐ichi Akiyama,

Trace Elements in Health

MRP1 is a 190-kDa membrane glycoprotein that confers multidrug resistance to tumor cells. The accumulated evidence has proved that GSH interacts with MRP1 and stimulates drug transport. However, the mechanism of GSH-dependent drug transport by MRP1 remains unclear. In this study, we used limited tryptic digestion of MRP1 in isolated membrane vesicles, in the presence and absence of GSH, to investigate the influence of GSH on MRP1 conformation. We found that GSH inhibited the generation of an ∼35-kDa C-terminal tryptic fragment (including a C-terminal His tag) termed C2 from MRP1. This effect of GSH was not because of direct inhibition of trypsin activity, and agosterol A enhanced the inhibitory effect of GSH. The main cleavage site in MRP1 for the generation of the C2 fragment by trypsin resided between TMD2 and NBD2 of MRP1. Limited tryptic digestion of membrane vesicles expressing various truncated and co-expressed MRP1 fragments in the presence and absence of GSH revealed that GSH inhibited the production of the C2 fragment only in the presence of the L0 region of MRP1. Thus the L0 region is required for the inhibition of trypsinization of the C-terminal half of MRP1 by GSH. These findings, together with previous reports, suggest that GSH induces a conformational change at a site within the MRP1 that is indispensable for the interaction of MRP1 with its substrates. MRP1 is a 190-kDa membrane glycoprotein that confers multidrug resistance to tumor cells. The accumulated evidence has proved that GSH interacts with MRP1 and stimulates drug transport. However, the mechanism of GSH-dependent drug transport by MRP1 remains unclear. In this study, we used limited tryptic digestion of MRP1 in isolated membrane vesicles, in the presence and absence of GSH, to investigate the influence of GSH on MRP1 conformation. We found that GSH inhibited the generation of an ∼35-kDa C-terminal tryptic fragment (including a C-terminal His tag) termed C2 from MRP1. This effect of GSH was not because of direct inhibition of trypsin activity, and agosterol A enhanced the inhibitory effect of GSH. The main cleavage site in MRP1 for the generation of the C2 fragment by trypsin resided between TMD2 and NBD2 of MRP1. Limited tryptic digestion of membrane vesicles expressing various truncated and co-expressed MRP1 fragments in the presence and absence of GSH revealed that GSH inhibited the production of the C2 fragment only in the presence of the L0 region of MRP1. Thus the L0 region is required for the inhibition of trypsinization of the C-terminal half of MRP1 by GSH. These findings, together with previous reports, suggest that GSH induces a conformational change at a site within the MRP1 that is indispensable for the interaction of MRP1 with its substrates. MDR 1The abbreviations used are: MDR, multidrug resistance; P-gp, P-glycoprotein; MRP1, the human multidrug resistance protein; ABC transporter, ATP-binding cassette transporter; TMD, transmembrane domain; NBD, nucleotide-binding domain; GSH, glutathione; VCR, vincristine; ADM, adriamycin; LTC4, leukotriene C4; AG-A, Agosterol A; BAEE, N-(α-benzoyl)-l-arginine ethyl ester; mAb, monoclonal antibody; DTT, dithiothreitol.1The abbreviations used are: MDR, multidrug resistance; P-gp, P-glycoprotein; MRP1, the human multidrug resistance protein; ABC transporter, ATP-binding cassette transporter; TMD, transmembrane domain; NBD, nucleotide-binding domain; GSH, glutathione; VCR, vincristine; ADM, adriamycin; LTC4, leukotriene C4; AG-A, Agosterol A; BAEE, N-(α-benzoyl)-l-arginine ethyl ester; mAb, monoclonal antibody; DTT, dithiothreitol. is the major obstacle to successful cancer chemotherapy and is mediated by membrane proteins whose mechanism of action is not yet completely understood (1Gottesman M.M. Pastan I. Annu. Rev. Biochem. 1993; 62: 385-427Crossref PubMed Scopus (3546) Google Scholar). MRP1 (multidrug resistance protein 1) is a 190-kDa glycoprotein that belongs to a family of membrane proteins referred to as ATP-binding cassette (ABC) transporters that typically contain two transmembrane domains (TMD) and two nucleotide binding domains (NBD) (4Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3322) Google Scholar). In most ABC transporters, hydrolysis of ATP by the NBDs is believed to provide energy for substrate transport (4Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3322) Google Scholar). MRP1 is distinguished from the other ABC transporter, P-glycoprotein (P-gp), by an extra N-terminal TMD that is connected to the core region (ΔMRP) by a cytoplasmic linker region (L0) (5Cole 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: 1650-1654Crossref PubMed Scopus (2972) Google Scholar). The human MRP1 is frequently overexpressed in cells whose MDR is not mediated by P-gp (2Deeley R.G. Cole S.P. Semin. Cancer Biol. 1997; 8: 193-204Crossref PubMed Scopus (164) Google Scholar, 3Hipfner D.R. Deeley R.G. Cole S.P. Biochim. Biophys. Acta. 1999; 1461: 359-376Crossref PubMed Scopus (375) Google Scholar). As an organic anion transporter, MRP1 actively transports a wide variety of diverse anionic compounds (6Zaman G.J. Flens M.J. van Leusden M.R. de Haas M. Mulder H.S. Lankelma J. Pinedo H.M. Scheper R.J. Baas F. Broxterman H.J. Borst P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8822-8826Crossref PubMed Scopus (696) Google Scholar, 7Loe D.W. Almquist K.C. Deeley R.G. Cole S.P. J. Biol. Chem. 1996; 271: 9675-9682Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar, 8Loe D.W. Almquist K.C. Cole S.P. Deeley R.G. J. Biol. Chem. 1996; 271: 9683-9689Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). It was also found that although GSH is a poor substrate for MRP1, it can stimulate the ATP-dependent transport of certain non-anionic organic drugs such as vincristine (VCR) (9Loe D.W. Deeley R.G. Cole S.P. Cancer Res. 1998; 58: 5130-5136PubMed Google Scholar), adriamycin (ADM) (10Renes J. de Vries E.G. Nienhuis E.F. Jansen P.L. Muller M. Br. J. Pharmacol. 1999; 126: 681-688Crossref PubMed Scopus (243) Google Scholar, 11Ding G.Y. Shen T. Center M.S. Anticancer Res. 1999; 19: 3243-3248PubMed Google Scholar), and aflatoxin B1 (12Loe D.W. Stewart R.K. Massey T.E. Deeley R.G. Cole S.P. Mol. Pharmacol. 1997; 51: 1034-1041Crossref PubMed Scopus (187) Google Scholar) as well as certain endogenous hydrophilic anionic conjugates such as estrone 3-sulfate (13Qian Y.M. Song W.C. Cui H. Cole S.P. Deeley R.G. J. Biol. Chem. 2001; 276: 6404-6411Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar) and a tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (14Leslie E.M. Ito K. Upadhyaya P. Hecht S.S. Deeley R.G. Cole S.P. J. Biol. Chem. 2001; 276: 27846-27854Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Loe et al. (9Loe D.W. Deeley R.G. Cole S.P. Cancer Res. 1998; 58: 5130-5136PubMed Google Scholar) have shown that GSH is required for vesicular transport of VCR by MRP1 and for inhibition by VCR of the transport of organic anions by MRP1. ATP-dependent uptake of [3H]GSH into membrane vesicles from MRP1-transfected cells was stimulated by VCR in a dose-dependent manner (9Loe D.W. Deeley R.G. Cole S.P. Cancer Res. 1998; 58: 5130-5136PubMed Google Scholar). This experiment, however, did not answer the question of whether GSH is an activator or a co-transported substrate of MRP1. To clarify the role of GSH in MRP1 drug transport, we synthesized a photoaffinity analog of AG-A (azido-AG-A) that could reverse MRP1-mediated MDR (15Chen Z.S. Aoki S. Komatsu M. Ueda K. Sumizawa T. Furukawa T. Okumura H. Ren X.Q. Belinsky M.G. Lee K. Kruh G.D. Kobayashi M. Akiyama S. Int. J. Cancer. 2001; 93: 107-113Crossref PubMed Scopus (61) Google Scholar). We reported recently (16Ren X.Q. Furukawa T. Aoki S. Nakajima T. Sumizawa T. Haraguchi M. Chen Z.S. Kobayashi M. Akiyama S. J. Biol. Chem. 2001; 276: 23197-23206Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) that GSH was required for the binding of azido-AG-A to the C-terminal half of MRP1. A similar conclusion was reached in another study by using a drug known as LY475776 (17Mao Q. Qiu W. Weigl K.E. Lander P.A. Tabas L.B. Shepard R.L. Dantzig A.H. Deeley R.G. Cole S.P. J. Biol. Chem. 2002; 277: 28690-28699Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). However, the mechanism of the stimulation of azido-AG-A binding to MRP1 by GSH remains unclear. Recently, it has been reported that a photoactive analog of GSH (azidophenacyl-[35S]GSH) photolabels MRP1 at two sites that reside in the N- and C-terminal halves of the protein. No stimulation of photolabeling of MRP1 with azidophenacyl-[35S]GSH could be detected following treatment with varying concentrations of LY475776 (18Qian Y.M. Grant C.E. Westlake C.J. Zhang D.W. Lander P.A. Shepard R.L. Dantzig A.H. Cole S.P. Deeley R.G. J. Biol. Chem. 2002; 277: 35225-35231Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). These observations suggested that the first event in GSH-dependent drug transport is the interaction of GSH with MRP1. GSH may then induce a conformational change in MRP1 that is required for drug binding. Ligand-induced conformational change is a common mechanism for the modulation of protein function (19Jencks W.P. Catalysis in Chemistry and Enzymology. Dover Publications, Mineola, NY1987: 282-319Google Scholar). For example a cyclic pentapeptide ligand can induce a conformational change in the extracellular segment of the integrin αvβ3 as shown by crystal structure analysis (20Xiong J.P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S.L. Arnaout M.A. Science. 2002; 296: 151-155Crossref PubMed Scopus (1367) Google Scholar). Analyses of proteins by protease digestion in the presence and absence of ligands have proved to be extremely useful techniques for the detection of ligand-induced protein conformational changes (21Tan S. Richmond T.J. Cell. 1990; 62: 367-377Abstract Full Text PDF PubMed Scopus (80) Google Scholar, 22Ishii S. Hayashi H. Okamoto A. Kagamiyama H. Protein Sci. 1998; 7: 1802-1810Crossref PubMed Scopus (27) Google Scholar, 23Wang G. Pincheira R. Zhang M. Zhang J.T. Biochem. J. 1997; 328: 897-904Crossref PubMed Scopus (57) Google Scholar, 24Julien M. Gros P. Biochemistry. 2000; 39: 4559-4568Crossref PubMed Scopus (50) Google Scholar). In the present study, we have performed limited tryptic digestion of MRP1 in the presence or absence of GSH and found that GSH inhibited tryptic digestion of the C-terminal half of the protein. Deletion of the site in MRP1 that mediates GSH inhibition of tryptic cleavage had important functional consequences for drug transport and GSH-dependent drug binding. Materials—Cellfectin® and competent DH10Bac Escherichia coli cells were purchased from Invitrogen. Diphenylcarbamyl chloride-treated trypsin was obtained from ICN Biomedicals (St. Laurent, Quebec, Canada). GSH was obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan). TALON™ metal affinity resin was purchased from Clontech. Other drugs and chemicals were obtained from Sigma. Cell Culture and Membrane Vesicle Preparation—KB/MRP cells, human KB cells transfected with MRP1 cDNA, were cultured in minimum essential medium (Nissui Seiyaku co., Tokyo, Japan) containing 10% newborn calf serum as described previously (15Chen Z.S. Aoki S. Komatsu M. Ueda K. Sumizawa T. Furukawa T. Okumura H. Ren X.Q. Belinsky M.G. Lee K. Kruh G.D. Kobayashi M. Akiyama S. Int. J. Cancer. 2001; 93: 107-113Crossref PubMed Scopus (61) Google Scholar). Sf21 insect cells were cultured in serum-free Sf-900 II SFM medium (Invitrogen). Membrane vesicles were prepared from KB/MRP, KB/CV, and Sf21 insect cells infected with various recombinant baculoviruses as described previously (26Ren X.Q. Furukawa T. Chen Z.S. Okumura H. Aoki S. Sumizawa T. Tani A. Komatsu M. Mei X.D. Akiyama S. Biochem. Biophys. Res. Commun. 2000; 270: 608-615Crossref PubMed Scopus (18) Google Scholar). Membrane vesicles were suspended in dilution buffer containing 10 mm Tris-HCl (pH 7.5) and 250 mm sucrose. Protein concentrations were determined by the method of Bradford (27Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211805) Google Scholar). Generation of Constructs and Viral Infection—pFastBac MRP1-His containing the MRP1 coding region was constructed as described previously (26Ren X.Q. Furukawa T. Chen Z.S. Okumura H. Aoki S. Sumizawa T. Tani A. Komatsu M. Mei X.D. Akiyama S. Biochem. Biophys. Res. Commun. 2000; 270: 608-615Crossref PubMed Scopus (18) Google Scholar). Constructs expressing various truncated and co-expressed MRP1s have been described (16Ren X.Q. Furukawa T. Aoki S. Nakajima T. Sumizawa T. Haraguchi M. Chen Z.S. Kobayashi M. Akiyama S. J. Biol. Chem. 2001; 276: 23197-23206Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). MRP1 constructs encoding the MRP1-(1264–1531) fragment with a 10xHis tag were generated by PCR using 5′-ATGCTCAAGGAGTATTCAGAGA-3′ (forward) and 5′-CCCTAGTGATGGTGATGGTGATGGTGATGGTGATGCACCAAGCCGGCGTCTTTGGC-3′ (reverse) primers (boldface ATG encodes the start code for MRP1-(1264–1531); the underlined sequence encodes the 10xHis tag). Baculoviruses expressing the wild type and mutant MRP1s described above were generated using the Bac-to-Bac expression system (Invitrogen) as described previously (26Ren X.Q. Furukawa T. Chen Z.S. Okumura H. Aoki S. Sumizawa T. Tani A. Komatsu M. Mei X.D. Akiyama S. Biochem. Biophys. Res. Commun. 2000; 270: 608-615Crossref PubMed Scopus (18) Google Scholar). Trypsin Activity Assay—Trypsin activity was measured with the N-(α-benzoyl)-l-arginine ethyl ester (BAEE) as the substrate. Briefly, about 10–40 BAEE units of trypsin were incubated in the presence or absence of various agents for 5 min at room temperature in dilution buffer before mixing with substrate buffer (40 mm sodium phosphate (pH 7.6) and 0.06 mm HCl) containing BAEE (final concentration 0.15 mm). Following mixing, the rate of increase in absorbance at 253 nm (A253) was examined using a U-2001 spectrophotometer (Hitachi, Tokyo, Japan). ΔA253/min is the rate of A253 change representing trypsin activity. The residual trypsin activity in the presence of various reagents was calculated as a percentage of the activity of trypsin measured in the absence of reagents. Limited Trypsin Treatment of MRP1 in Inside-out Membrane Vesicles and Western Blotting—Trypsinization of membrane vesicles was carried out as described previously (16Ren X.Q. Furukawa T. Aoki S. Nakajima T. Sumizawa T. Haraguchi M. Chen Z.S. Kobayashi M. Akiyama S. J. Biol. Chem. 2001; 276: 23197-23206Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Membrane vesicles (50 μg) prepared from KB/MRP or Sf21 insect cells infected with viruses encoding various MRP1 mutants were digested with trypsin at the indicated concentrations in the presence or absence of GSH alone or in combination with other agents at 37 °C. Soybean trypsin inhibitor at a final concentration of 100 μg/ml was employed to terminate the trypsin activity. The samples were then immediately subjected to 8.5 or 11% SDS-PAGE. Immunoblotting was performed as described previously (26Ren X.Q. Furukawa T. Chen Z.S. Okumura H. Aoki S. Sumizawa T. Tani A. Komatsu M. Mei X.D. Akiyama S. Biochem. Biophys. Res. Commun. 2000; 270: 608-615Crossref PubMed Scopus (18) Google Scholar). Anti-MRP1 monoclonal antibodies MRPr1 (epitope amino acids 229–281) and MRPm6 (epitope amino acids 1389–1351) (28Flens M.J. Izquierdo M.A. Scheffer G.L. Fritz J.M. Meijer C.J. Scheper R.J. Zaman G.J. Cancer Res. 1994; 54: 4557-4563PubMed Google Scholar, 29Hipfner D.R. Gauldie S.D. Deeley R.G. Cole S.P. Cancer Res. 1994; 54: 5788-5792PubMed Google Scholar) were obtained from Progen Biotechnick (Heidelberg, Germany). Anti-His mAb was obtained from Qiagen (Hilden, Germany). Purification of 10xHis-tagged C-terminal Fragments of MRP1 and Protein Sequence of the Purified C2 Fragment—Membrane vesicles (2 mg, final concentration of the protein 2 mg/ml) prepared from Sf21 insect cells co-expressing N1–932 and C932–1531 of MRP1 were digested with trypsin (50 μg/ml) for 1 h at 37 °C. Soybean trypsin inhibitor at a final concentration of 50 μg/ml was employed to terminate the trypsin activity. The samples were then solubilized in 10 ml of buffer A (50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1% Triton X-100) and incubated for 1 h on ice. The solubilized membranes were centrifuged at 12,000 × g at 4 °C for 30 min. Sf21 insect cells (1 × 1010) infected with recombinant baculovirus encoding MRP1-(1264–1531) were sonicated in 20 ml of buffer A and centrifuged at 12,000 × g at 4 °C for 30 min. The supernatants were then incubated at 4 °C for 2 h with 100 μl of TALON metal affinity resin washed with phosphate-buffered saline. The bound resin was washed three times with buffer A containing 1% Triton X-100, washed one time with phosphate-buffered saline, and then solubilized in SDS sample buffer and subjected to 11% SDS-PAGE. The samples were then transferred to polyvinylidene difluoride membrane as described above. C2 tryptic fragment was detected by staining with Ponceau S. Bands were subjected to sequence analysis by automated Edman degradation on a 492 procise CLC Protein Sequencer (Applied Biosystems, Tokyo, Japan). Photoaffinity Labeling of MRP1 with 125I-Azido-AG-A—125I-Azido-AG-A (7.2 μCi/nmol) was used for photolabeling studies that were carried out as described previously (16Ren X.Q. Furukawa T. Aoki S. Nakajima T. Sumizawa T. Haraguchi M. Chen Z.S. Kobayashi M. Akiyama S. J. Biol. Chem. 2001; 276: 23197-23206Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Autoradiograms were exposed for 10 h. [3H] LTC4 Uptake by Membrane Vesicles—The extent of [3H] LTC4 uptake was measured using a rapid filtration technique as described previously (26Ren X.Q. Furukawa T. Chen Z.S. Okumura H. Aoki S. Sumizawa T. Tani A. Komatsu M. Mei X.D. Akiyama S. Biochem. Biophys. Res. Commun. 2000; 270: 608-615Crossref PubMed Scopus (18) Google Scholar). Briefly, isolated membrane vesicles (25 μg of protein) were incubated in the presence or absence of 4 mm ATP in 50 μl of transport buffer (0.25 m sucrose, 10 mm Tris-HCl (pH 7.5), 10 mm MgCl2,10 mm phosphocreatine, and 100 μg/ml creatine phosphokinase) with 100 nm [3H]LTC4 for the indicated times at 37 °C. The reaction was stopped at the indicated times with 3 ml of ice-cold stop solution (0.25 m sucrose, 0.1 m NaCl, and 10 mm Tris-HCl (pH 7.5)). The samples were passed through Millipore filters (GVWP, 0.22 μm pore size) under a light vacuum. Following three rinses with 3 ml of cold stop solution, the filters were immersed in liquid scintillation fluid, and their radioactivity was measured. GSH Inhibits the Generation of a C-terminal Tryptic Fragment of MRP1—To use protease digestion as a tool to monitor conformational changes induced by GSH in MRP1, we first established the cleavage fragments generated by limited tryptic digestion of MRP1. It has been shown, using N- and C-terminal MRP1-specific monoclonal antibodies, that the most accessible trypsin site on MRP1 lies within the cytoplasmic linker region between NBD1 and TMD2 (16Ren X.Q. Furukawa T. Aoki S. Nakajima T. Sumizawa T. Haraguchi M. Chen Z.S. Kobayashi M. Akiyama S. J. Biol. Chem. 2001; 276: 23197-23206Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Tryptic cleavage at this site generates a 67-kDa fragment termed C1. When the concentration of trypsin was increased, or the incubation time with trypsin was extended, the C-terminal C1 band was further degraded to an ∼35-kDa C2 fragment that could be detected with the C-terminal m6 antibody (Fig. 1, A and B, left). We then determined whether GSH-MRP1 interaction could induce a conformational change in MRP1 detectable by the generation of altered tryptic fragments. For this experiment KB/MRP membrane vesicles (50 μg of protein) were digested with trypsin at 20 ng/ml over a 90-min period in the presence or absence of 10 mm GSH and immunoblotted with the C-terminal antibody m6. As shown in Fig. 1B, the presence of GSH (right) suppressed the generation of the C2 tryptic fragment. The addition of GSH had no effect on the tryptic digestion pattern when assayed with an N-terminal MRP1 antibody r1 (data not shown). The inhibitory effect of GSH on the generation of the C2 fragment by trypsin was dose-dependent with decreasing amounts of the C2 fragment generated as the GSH concentration was increased from 0.5 to 10 mm (Fig. 1C). We next investigated whether the inhibitory effect of GSH on trypsin digestion of MRP1 might be due to a decreased enzymatic activity of trypsin in the presence of GSH. We therefore assayed the effect of GSH on trypsin activity by using BAEE as a substrate. As controls, we also tested the effect of other reducing agents such as cysteine and DTT, the nonreducing GSH analogs methyl-GSH and ethyl-GSH, and glycine and glutamic acids (the amino acids that constitute GSH) on trypsin activity. The mean of three independent experiments was analyzed. As shown in Table I, agents with reducing activities do inhibit trypsin activity. GSH, cysteine, and DTT at a concentration of 10 mm decreased trypsin activity by 15.5, 16.8, and 65.4%, respectively. In contrast, methyl-GSH at a concentration of 10 mm decreased trypsin activity by only 3.7%, and ethyl-GSH at a concentration of 10 mm had no effect on trypsin activity. We therefore determined whether 10 mm methyl-GSH or 10 mm ethyl-GSH could also suppress the generation of the C2 tryptic fragment of MRP1. As shown in Fig. 2A, both methyl- and ethyl-GSH inhibited the generation of the C2 tryptic fragment to a similar extent as GSH. In contrast, 10 mm DTT had a lesser effect on the generation of the C2 fragment, even though it inhibited trypsin activity to a greater extent than the GSH derivatives. Similarly the addition of cysteine, glycine, or glutamic acid, the amino acids that constitute GSH, had a much weaker effect on the generation of the C2 fragment by trypsin, even though cysteine could inhibit trypsin activity to a similar extent as GSH (Fig. 2B).Table IModulation of trypsin activity by GSH and other agents Trypsin activity was assayed using BAEE as substrate, as described under “Experimental Procedures,” in the absence (control) and in the presence of various agents. Residual trypsin activity (mean of three experiments ± S.E.) was calculated as described under “Experimental Procedures.”AgentsResidual activity% controlControl10050 mm GSH53.5 ± 6.810 mm GSH84.5 ± 3.52 mm GSH95.4 ± 1.310 mmS-methyl GSH96.3 ± 3.910 mmS-ethyl GSH100.7 ± 4.910 mm glycine98.1 ± 0.910 mm glutamic acid100.7 ± 0.510 mm cysteine83.2 ± 0.310 mm DTT34.6 ± 5.61 mm AG-A105.3 ± 2.91 mm VCR95.7 ± 6.0 Open table in a new tab These data are consistent with our previous observation that GSH stimulates azido-AG-A photolabeling of MRP1 independently of its reducing activity (16Ren X.Q. Furukawa T. Aoki S. Nakajima T. Sumizawa T. Haraguchi M. Chen Z.S. Kobayashi M. Akiyama S. J. Biol. Chem. 2001; 276: 23197-23206Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). They also indicate that GSH inhibition of the generation of the C2 tryptic fragment is not due to the direct inhibition of trypsin activity. The most likely explanation is that the binding of GSH to MRP1 may inhibit access of trypsin to MRP1 and the consequent cleavage reaction leading to generation of the C2 band. We have found previously (30Ren X.Q. Furukawa T. Aoki S. Sumizawa T. Haraguchi M. Nakajima T. Ikeda R. Kobayashi M. Akiyama S. Biochemistry. 2002; 41: 14132-14140Crossref PubMed Scopus (36) Google Scholar) that GSH was required for the binding of a photoactive analog of AG-A to the C-terminal half of MRP1, and that amino acids 1223–1295 of MRP1 were required for GSH-dependent binding of AG-A to MRP1. Based on the molecular size of the C2 fragment, the cleavage site that leads to the generation of C2 must be close to this region. Therefore, we investigated whether the VCR that is transported by MRP1 in the presence of GSH or AG-A might affect the generation of the C2 fragment. Limited trypsinization of MRP1 was conducted in the presence of the drugs alone or in combination with GSH, and the generation of the C2 fragment was monitored. As indicated in Fig. 3A, the addition of AG-A or VCR alone had no effect on the generation of the C2 fragment by trypsin. However, when AG-A was added in combination with GSH, AG-A considerably attenuated the generation of the C2 fragment (Fig. 3A). The effect of various concentrations of AG-A, ranging from 10 μm to 1 mm, on the generation of the C2 band in the presence of 2 mm GSH was investigated (Fig. 3B). Increasing AG-A concentrations enhanced the suppressive effect of 2 mm GSH on the tryptic generation of the C2 fragment. Because AG-A does not inhibit trypsin activity as shown in Table I, AG-A must enhance the suppressive effect of GSH by a different mechanism. The GSH-sensitive Tryptic Site Is Located in the Cytoplasmic Region of MRP1 between TMD2 and NBD2—We next investigated the location in MRP1 of the cleavage site that generates the C2 fragment. Because C2 has a molecular mass of about 35 kDa in SDS-PAGE, the cleavage site that generates the C2 fragment should be located about 350 amino acids upstream of the C-terminal end (1531) of MRP1. This would localize the C2 tryptic site within TMD2 of MRP1. If this is the case, then the C2 fragment should be associated with the membrane fraction. We therefore investigated the potential membrane association of the cleaved C2 fragment. KB/MRP membrane vesicles were digested with trypsin at 20 ng/ml for 30 min and then centrifuged to separate the membrane and soluble fractions. As indicated in Fig. 4A, most of the C2 tryptic fragments were detected in the soluble fraction, and the residual membrane-bound C2 fragments were released from the membrane by washing with the high concentration of NaCl, indicating that the tryptic site suppressed by GSH must be located in the cytoplasmic region of MRP1 between TMD2 and NBD2. To localize more exactly the cleavage site that generates the C2 fragment, we made use of a C-terminally His-tagged MRP1 expressed in Sf21 insect cells. This expression system allows dissection of the functional domains of MRP1. We have shown previously that a 10xHis tag inserted into the C terminus of MRP1 has no effect on the function of the transporter (26Ren X.Q. Furukawa T. Chen Z.S. Okumura H. Aoki S. Sumizawa T. Tani A. Komatsu M. Mei X.D. Akiyama S. Biochem. Biophys. Res. Commun. 2000; 270: 608-615Crossref PubMed Scopus (18) Google Scholar). We first determined the expression of the His-tagged MRP1 in insect cells. Because MRP1 in Sf21 insect cells is less glycosylated than in KB/MRP cells, it migrates as an ∼170-kDa band (26Ren X.Q. Furukawa T. Chen Z.S. Okumura H. Aoki S. Sumizawa T. Tani A. Komatsu M. Mei X.D. Akiyama S. Biochem. Biophys. Res. Commun. 2000; 270: 608-615Crossref PubMed Scopus (18) Google Scholar) (Fig. 4B). We then determined whether trypsin digestion of insect-expressed MRP1 could generate a C2 fragment similar to that generated in KB/MRP cells and, if so, whether this digestion was GSH-sensitive. KB/MRP and Sf/MRP membrane vesicles (50 μg of protein) were therefore digested with trypsin at 20 ng/ml for 90 min in the presence or absence of 10 mm GSH. Tryptic fragments were immunoblotted with either the C-terminal antibody m6 or an anti-His tag mAb as appropriate. As shown in Fig. 4C, the C2 fragment was detected with both of these antibodies, although the size of the C2 band derived from Sf/MRP membrane vesicles was slightly larger than that from KB/MRP probably due to the presence of the 10xHis tag in the C terminus of MRP1. The generation of the C2 fragment in Sf/MRP membrane vesicles was also inhibited in the presence of 10 mm GSH (Fig. 4C). Thus the His-tagged MRP1 expressed in insect cells can be used as a model of GSH suppression of MRP tryptic digestion. Because amino acid sequencing of the C2 fragment was difficult (data not shown) and the C2 fragment was not associated with membrane, the cleavage site probably resides within the cytoplasmic region close to the C terminus of TM17. We expressed a C-terminal fragment, MRP1-(1264–1531), containing a His tag in Sf21 insect cells. MRP1-(1264–1531) had an electrophoretic migration similar to that of the C2 tryptic fragment generated from Sf/MRP membrane vesicles (Fig. 4D). These data suggested that the main cleavage site in MRP1 for the generation of the C2 fragment resides between TMD2 and NBD2. L0 Region of MRP1 Is Required for Attenuation of the C2 Generation by GSH—It has been reported that the cytoplasmic region L0 between TMD0 and TMD1 of MRP1 is important for MRP1 function (16Ren X.Q. Furukawa T. Aoki S. Nakajima T. Sumizawa T. Haraguchi M. Chen Z.S. Kobayashi M. Akiyama S. J. Biol. Chem. 2001; 276: 23197-23206Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 31Gao M. Yamazaki M. Loe D.W. Westlake C.J. Grant C.E. Cole S.P. Deeley R.G. J. Biol. Chem. 1998; 273: 10733-10740Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 32Bakos E. Evers R. Szakacs G. Tusnady G.E. Welker E. Szabo K. de Haas M. van Deemter L. Borst P. Varadi A. Sarkadi B. J. Biol. Chem. 1998; 273: 32167-32175Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). We have shown previously that L0 is required for GSH-dependent binding of a photoanalog of AG-A to the C-terminal half of the protein, and we have suggested that L0 is the interaction site of GSH on MRP1 (16Ren X.Q. Furukawa T. Aoki S. Nakajima T. Sumizawa T. Haraguch