Glutathione-dependent Binding of a Photoaffinity Analog of Agosterol A to the C-terminal Half of Human Multidrug Resistance Protein*

Xiao-Qin RenDagger §, Tatsuhiko FurukawaDagger , Shunji Aoki, Tatsuo Nakajima, Tomoyuki SumizawaDagger , Misako HaraguchiDagger , Zhe-Sheng ChenDagger , Motomasa Kobayashi, and Shin-ichi AkiyamaDagger ||

From the Dagger  Department of Cancer Chemotherapy, Institute for Cancer Research, Faculty of Medicine, Kagoshima University, Sakuragaoka 8-35-1, Kagoshima 890-8520 and the  Graduate School of Pharmaceutical Sciences, Osaka University, Yamada-oka 1-6, Suita, Osaka 565-0871, Japan

Received for publication, February 20, 2001, and in revised form, April 4, 2001

    ABSTRACT
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MRP1 is a 190-kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells. MRP1 is characterized by an N-terminal transmembrane domain (TMD0), which is connected to a P-glycoprotein-like core region (Delta MRP) by a cytoplasmic linker domain zero (L0). It has been demonstrated that GSH plays an important role in MRP1-mediated MDR. However, the mechanism by which GSH mediates MDR and the precise roles of TMD0 and L0 are not known. We synthesized [125I]11-azidophenyl agosterol A ([125I]azidoAG-A), a photoaffinity analog of the MDR-reversing agent, agosterol A (AG-A), to photolabel MRP1, and found that the analog photolabeled the C-proximal molecule of MRP1 (C932-1531) in a manner that was GSH-dependent. The photolabeling was inhibited by anticancer agents, reversing agents and leukotriene C4. Based on photolabeling studies in the presence and absence of GSH using membrane vesicles expressing various truncated, co-expressed, and mutated MRP1s, we found that L0 is the site on MRP1 that interacts with GSH. This study demonstrated that GSH is required for the binding of an unconjugated agent to MRP1 and suggested that GSH interacts with L0 of MRP1. The photoanalog of AG-A will be useful for identifying the drug binding site within MRP1, and the role of GSH in transporting substrates by MRP1.

    INTRODUCTION
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INTRODUCTION
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Following exposure to a natural product chemotherapeutic agent, tumor cells often acquire resistance to several structurally and functionally unrelated drugs, so-called multidrug resistance (MDR).1 MDR is the major obstacle to successful cancer chemotherapy. Two membrane proteins, P-glycoprotein (P-gp) and the human multidrug resistance protein (MRP1) are frequently overexpressed in MDR cells (for reviews see Refs. 1-4). Although both MRP1 (190 kDa) and P-gp (170 kDa) are members of the family of ATP-binding cassette transporters (5), they share only 15% amino acid sequence identity (6). The amino acid sequence suggests that P-gp consists of two homologous halves and a variable linker region. Each half of the protein has six transmembrane segments and one nucleotide binding domain (NBD). MRP1 differs from P-gp by the presence of an extra N-terminal extension with five transmembrane segments (TMD0), which is connected to the P-gp-like core (Delta MRP) by a cytoplasmic linker domain zero (L0) (7, 8). The precise roles of TMD0 and L0 are unknown. Although MRP1 and P-gp both confer multidrug resistance by actively effluxing drugs from the cells (9, 10), there is compelling evidence that they function very differently in drug transport (6, 11). P-gp confers multidrug resistance by directly binding and transporting unmodified drugs (12, 13). MRP1, however, is an active transporter of amphiphilic conjugated organic anions, including a number of compounds conjugated with GSH, glucuronide, and sulfate. To date, leukotriene C4 (LTC4) is the best substrate for MRP1 (14).

It has been reported that GSH plays a critical role in MRP1-mediated MDR (15). Reduction of GSH level in MRP1-expressing cells by buthionine sulfoximine, an inhibitor of GSH synthesis, resulted in a decrease in the active efflux of some anticancer agents, and an increase in cellular sensitivity to adriamycin (ADM), vincristine (VCR), and etoposide (VP-16) (15). In addition to conjugated anions, MRP1 has been shown to actively transport some unmodified compounds such as VCR, only in the presence of GSH (16). There is also evidence that VCR and GSH are co-transported by MRP1 (16). ATP-dependent VCR transport into membrane vesicles overexpressing MRP1 could be stimulated by GSH, and ATP-dependent uptake of [3H]GSH was stimulated by VCR. Although all of the above data strongly suggest that GSH is essential for MRP1-mediated MDR, the role of GSH in binding and/or transport of drugs by MRP1 is unclear. In contrast to the above studies, Vezmar et al. (17, 18) reported that a quinoline-based photoactive drug, N-{4-[1-hydroxy-2-(dibutylamino) ethyl] quinolin-8-yl}-4-azidosalicylamide (IAAQ), photolabeled MRP1 in both MRP1-overexpressing intact cells and membrane vesicles, and that GSH is not required for the binding of the drug. More recently, another quinoline-based photoaffinity drug, N-(hydrocinchonidin-8'-yl)-4-azido-2-hydroxybenzamide (IACI), which has a structure very similar to that of IAAQ, has also been shown to photolabel multiple sites of MRP1 in the absence of GSH (19). These findings raise the question of whether GSH is essential for MRP1-mediated MDR, and, if it is essential, which step in the drug transport is stimulated by GSH: the drug binding step or subsequent steps.

Photoaffinity analogs of some xenobiotics are powerful tools for studying drug binding sites on proteins (20). Photoaffinity analogs of some anti-cancer and other reversing agents, such as azidopine (21), N-(p-azido-(3-I)-salicyl)-N'-beta -aminoethylvindesine (12, 22), N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine (13), and iodoarylazidoprazosin (23), have been successfully used to photolabel P-gp and determine its drug binding site(s). Although the resistance phenotypes conferred by expression of MRP1 are similar to those of P-gp expression, a photoaffinity labeling study using known photoaffinity analogs for P-gp failed to photolabel MRP1 under the same conditions used for P-gp (24). Leier et al. (25, 26) reported that LTC4 photolabeled MRP1; however, the labeling site of LTC4 on MRP1 has not been determined.

Recently, we isolated a novel polyhydroxylated sterol acetate, named agosterol A (AG-A), from a marine sponge (Spongia sp.). We found that AG-A completely reversed MDR in human KB carcinoma cells overexpressing MRP1 (27), and that AG-A competitively inhibited the transport of LTC4 by MRP1 (28). This suggests that AG-A is a new MDR-reversing agent.

In the present study, we synthesized a photoaffinity analog of AG-A, [125I]11-azidophenyl agosterol A ([125I]azidoAG-A), and used it to photolabel MRP1. We found that GSH is required for [125I]azidoAG-A binding to the C-terminal half of MRP1. In order to clarify the site of GSH interaction on MRP1, a series of truncated and mutated MRP1 constructs and co-expressed half-molecules of MRP1 were used to see if they are photolabeled by the photoanalog of AG-A in the presence or absence of GSH. We found that the C-proximal fragment of MRP1 (C932-1531) is photolabeled by the photoanalog of AG-A in the presence of GSH and GSH interacts with the L0 region of MRP1.

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Materials-- [125I]NaI (3.7 GBq (100 mCi)/ml) was purchased from PerkinElmer Life Sciences. AG-A was isolated from a marine sponge (Spongia sp). collected in Mie Prefecture, Japan. Details of its isolation and determination of its structure were described in a previous paper (27). 2-Azido-5-(trimethylstannyl) benzoic acid was attached to the 11-hydroxy group of AG-A with a C-4 linker and then iodinated with [125I]NaI and chloramine T (Fig. 1) (29). The product was confirmed as [125I]azidoAG-A by comparison with a standard sample on thin layer chromatography. 7.2 µ Ci/nmol [125I]azidoAG-A was used for the experiment. PAK-104P, MK571, and ONO-1078 were obtained as described previously (30). Mouse IgG2a and Protein G-Sepharose 4B were obtained from Zymed Laboratories Inc. (San Francisco, CA). Cellfectin and competent DH10Bac Escherichia coli cells were purchased from Life Technologies, Inc. Other drugs and chemicals were obtained from Sigma.

Cell Culture and Membrane Vesicle Preparation-- KB/MRP, human KB cells transfected with MRP1 cDNA, and KB/CV, KB cells transfected with an empty vector, were cultured in minimum essential medium (Nissui Seiyaku Co., Tokyo, Japan) containing 10% newborn calf serum as described previously (31, 32). Sf21 insect cells were cultured in serum-free Sf-900 II SFM medium (Life Technologies, Inc.). Membrane vesicles were prepared from KB/MRP, KB/CV cells, and Sf21 insect cells infected with various recombinant baculoviruses as described previously (30). Membrane vesicles were suspended in buffer containing 10 mM Tris-HCl (pH 7.5), and 250 mM sucrose. Protein concentrations were determined by the method of Bradford (33).

Generation of Constructs and Viral Infection-- pFastBac MRP1-His containing the MRP1 coding region was constructed as described previously (30).

To generate a construct expressing Delta MRP, an adaptor was constructed using two oligonucleotides (5'-TCGACGCCGCCATGGATGCATCG-3', 5'-GATCCGATGCATCCATGGCGGCG-3') encoding a consensus Kozak sequence (bold and underlined), a NsiI site (underlined), and cohesive ends of SalI (italic) and BamHI (bold). The adaptor was then ligated to pFastBac MRP1-His in which the SalI/BamHI fragment encoding MRP1 amino acids 1-280 was removed. The correct insertion of the adaptor was identified by the presence of the NsiI site and sequence analysis.

To generate constructs for expression of the N1-932 and C932-1531 of MRP1, nucleotides 2505-2995 and 2993-4399 of MRP1 were amplified by PCR from an MRP1 cDNA template. The primers used for the 2505-2995 fragment were 5'-CTGGGGGCCAGAAGCAGCGCGTGAG-3' (forward) and 5'-TTATGCGGTGCTGTTGTGGTGCC-3' (reverse). A stop codon (underlined) was introduced into the reverse primer. The primers used for the 2993-4399 fragment were: 5'-ATGGCAGAACTGCAGAAAGCTGAG-3' (forward) and 5'-CCAGACTTCTTCATCCGAGT-3' (reverse). In the forward primer, a start codon was introduced (underlined). The two PCR fragments were first cloned into a pBluescript T-vector to make pBluescript MRPN and pBluescript MRPC. The orientation and fidelity of the PCR fragments were verified by sequence analysis and were then excised from the plasmid as XhoI/NotI and SalI/NcoI fragments, respectively. The XhoI/NotI fragment was then ligated to pFastBac MRP1-His predigested with XhoI and NotI to construct pFastBac1 MRPN encoding N1-932. The SalI/NcoI fragment, together with the MRP1 NcoI/KpnI fragment, was cloned between the XhoI and KpnI sites in the multiple cloning site II of pFastBac DUAL donor plasmid (Life Technologies, Inc.) to construct pFastBac DUAL MRPC for C932-1531. The construct pFastBac DUAL MRP full resulted from ligation of the two large BssHII/PvuI fragments of the N and C donor plasmids.

To express TMD0L0 and Delta MRP from a dual vector, pFastBac MRP1-His was digested with BamHI and SphI. After being blunted by T4 polymerase, the two sites were then ligated to each other. The fidelity of the blunting was confirmed by reconstitution of a BamHI site. The fragment encoding TMD0L0 of MRP1 was removed from the plasmid as a SalI/KpnI fragment and then cloned between the XhoI and KpnI sites in multiple cloning site II of the pFastBac DUAL donor plasmid. Translation of the inserted fragment terminated at a stop codon in the DUAL vector resulting in the addition of eight amino acids, PVPGDGGG. The BssHII/PvuI fragment was then ligated with the large BssHII/PvuI fragment of the Delta MRP expression construct.

To facilitate detection of the expressed CoreN of MRP1, a sequence encoding a myc tag was inserted by PCR using pBluescript MRPN as a template. For this purpose, two primers: 5'-TCTGAAGAGGATCTGTAAACTAGTTCTAGAGCGGCCGC-3' (forward) and 5'-GATGAGTTTCTGTTCTGCGGTGCTGTTGTGGTGCC-3' (reverse), encoding the myc epitope EQKLISEEDL (underlined) were used. The PCR products were digested with DpnI to remove template plasmid DNA and then self-ligated in the presence of T4 kinase (34). Following verification by DNA sequencing, the insertion was removed as an XhoI/NotI fragment and exchanged with the corresponding fragment in the Delta MRP expression construct to construct pFastBac MRP CoreN-myc. In order to co-express the CoreN of MRP1 with C932-1531, the BssHII/PvuI vector part of the above plasmid was exchanged with the large BssHII/PvuI fragment of the pFastBac DUAL MRPC.

Constructs encoding L0Delta MRP, ML0Delta MRP, and Delta L0full were prepared as described above. The PCR primer used for L0Delta MRP was 5'-GCCGCCATGGACCCTAATCCCTGCCCAGAG-3' encoding a consensus Kozak sequence (underlined). The primers used to introduce two mutations, K267M and W261A, into MRP1 were 5'-GAGTGCGCCATGACTAGGAAG-3' (forward) and 5'-CTTCTTCGCGTTCTTTACCAAAAC-3' (reverse) encoding mismatched bases (bold). The underlined mismatched base introduces a silent mutation, which destroys an original BsmI site in MRP1 and acts as a marker for the mutant cDNA. The primer used for Delta L0full was a reverse primer: 5'-GGATCCTGGATGGTTTCCGAGAAC-3'.

Baculoviruses encoding the above wild type and mutant MRP1s were generated using the Bac to Bac expression system (Life Technologies, Inc.) as described previously (30).

Photoaffinity Labeling of MRP1 with [125I]AzidoAG-A-- Membrane vesicles (100 µg of protein) were incubated for 20 min at room temperature with 5 µM photoanalog of AG-A labeled with 125I (1.2 × 106 cpm) in the absence or presence of various agents and different concentrations of GSH as indicated. Following continuous irradiation of the samples with a short wavelength (366 nm) UV lamp for 20 min at 25 °C, the samples were solubilized in SDS sample buffer as described by Debenham et al. (35) and subjected to SDS-PAGE. Autoradiograms were developed after 6-24 h of exposure. Gel slices corresponding to specific bands on the autoradiogram were cut, and the radioactivity (cpm) was measured with an Auto Well Gamma System (ARC-380, Aloka Co., Ltd., Tokyo, Japan). Background radioactivity in equal areas of the gel in each lane was measured and subtracted from the radioactivity of the band.

Immunoprecipitation of MRP1 and Inhibition of Photoaffinity Labeling-- Membrane proteins (300 µg) were photolabeled by [125I]azidoAG-A as described above. Membrane vesicles were then solubilized in 2 ml of Buffer A (50 mM Tris-HCl (pH 8.0), 150 mM NH4Cl, 2 mM MgCl2) containing 1% CHAPS and incubated for 30 min at 4 °C. The solubilized membranes were centrifuged at 12,000 × g, and the supernatant was incubated at 4 °C for 2 h with 4 µg of anti-MRP1 monoclonal antibody, QCRL-3, or 8 µg of control mouse IgG2a. The mixture was then incubated with 200 µg of 20% Protein G-Sepharose in buffer A for 30 min at 4 °C with continuous mixing, and the precipitates were washed four times with Buffer A containing 1% CHAPS. The precipitates were then analyzed by SDS-PAGE and autoradiography.

Treatment of Vesicles with Trypsin and Immunoblotting-- Membrane vesicles were treated with diphenylcarbamyl chloride-treated trypsin (ICN Biomedicals, St. Laurent, Quebec, Canada) according to Hipfner et al. (7) with some modifications. Briefly, membrane vesicles (50 µg) were diluted in buffer (10 mM Tris-HCl (pH 7.5), 250 mM sucrose) to a final concentration of 5 mg/ml and then treated with 2-50 µg/ml trypsin for 30 min at 37 °C. The reaction was stopped by the addition of 2 mM aminophenylmethylsulfonyl fluoride (Wako Chemical, Osaka, Japan) and 0.5 TIU (trypsin inhibitory unit)/ml aprotinin, and the samples were subjected to SDS-PAGE and immunoblotting as described previously (30). The N- and C-terminal fragments of trypsinized MRP1 were detected with specific monoclonal antibodies MRPr1 (epitope amino acids MRPr1 (epitope aa 238-247) (36, 41) and MRPm6 (epitope aa 1511-1520) (36, 41), respectively. Treatment of membrane vesicles with trypsin was carried out either before or after the photolabeling as indicated.

    RESULTS
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INTRODUCTION
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DISCUSSION
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GSH-dependent Binding of [125I]AzidoAG-A to a Drug Binding Site on MRP1-- We recently demonstrated that AG-A reverses MDR in human KB carcinoma cells overexpressing MRP1. AG-A competitively interacts with the binding site for LTC4 on MRP1 and inhibited the transport of LTC4 by MRP1 (28).

To examine whether AG-A directly binds to the drug binding site of MRP1, we synthesized the photoaffinity analog of AG-A, [125I]azidoAG-A (Fig. 1). [125I]AzidoAG-A photolabeled the 190-kDa cellular component only in the presence of GSH. The labeled band was not detected even in the presence of GSH in membrane vesicles from KB/CV cells that did not express detectable levels of MRP1 (Fig. 2A). The photolabeling increased with increasing concentrations of GSH up to 2 mM (Fig. 2B).


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Fig. 1.   The structural formulas of AG-A and [125I]azidoAG-A.


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Fig. 2.   The effect of GSH on [125I]azidoAG-A binding to MRP1-enriched membrane vesicles. Membrane vesicles were photolabeled with [125I]azidoAG-A as indicated, detergent-solubilized, and analyzed by SDS-PAGE and autoradiography. A, KB/MRP and KB/CV membrane vesicles were photolabeled with 5 µM [125I]azidoAG-A in the presence or absence of 2 mM GSH. An arrow indicates the 190-kDa labeled protein. B, KB/MRP membrane vesicles were photolabeled with [125I]azidoAG-A in the presence of increasing concentrations of GSH (0.1-5 mM) (inset). The data were quantified by removing gel slices corresponding to the 190-kDa bands on the autoradiogram and determining their radioactivity. The background activity was measured and subtracted from the radioactivity of the band. C, SDS-PAGE of immunoprecipitates of [125I]azidoAG-A-labeled, detergent-solubilized, KB/MRP membrane vesicles. Photoaffinity labeling of KB/MRP membrane vesicles with the photoanalog of AG-A was carried out as described under "Experimental Procedures." Photolabeled membrane vesicles (300 µg) were solubilized with a buffer containing CHAPS, and immunoprecipitated with QCRL-3 (left lane) or nonimmune mouse IgG2a (right lane). The immune complex precipitated with protein G-Sepharose was collected and subjected to 7.5% SDS-PAGE and autoradiography. Molecular size markers at the left are in kDa. Autoradiograms were developed after 3 days (left lane) and 6 days (right lane) of exposure, respectively.

To determine if the 190-kDa component was MRP1, photolabeled membrane vesicles from KB/MRP cells were solubilized with CHAPS and immunoprecipitated with a monoclonal anti-MRP1 antibody, QCRL-3 (37). The labeled protein was immunoprecipitated by the monoclonal antibody, but not by nonimmune mouse IgG2a confirming that the [125I]azidoAG-A-binding protein is MRP1 (Fig. 2C).

We then characterized the effect of GSH on [125I]azidoAG-A binding to MRP1. Loe et al. (16) reported that, in addition to GSH, some short S-alkyl-GSH derivatives stimulated [3H]VCR uptake by MRP1. We therefore investigated whether GSH derivatives could also stimulate [125I]azidoAG-A binding to MRP1. As shown in Fig. 3A, S-methyl-GSH stimulated photolabeling of MRP1 to an extent similar to that for GSH. Stimulation of labeling was less efficient for S-ethyl-GSH, and S-octyl-GSH was ineffective. The role of the reducing activity of GSH in stimulation of the AG-A-MRP1 binding was tested by determining the ability of the sulfhydryl reducing agents, dithiothreitol, 2-mercaptoethanol, and thioredoxin, and a glucuronic acid, to substitute for GSH in binding assay (Fig. 3B). It seems that the reducing activity of GSH is not essential for the photolabeling.


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Fig. 3.   The effect of various GSH derivatives, sulfhydryl reducing agents, and glucuronic acid on [125I]azidoAG-A binding to MRP-enriched membrane vesicles. Photolabeling was carried out as described in the legend to Fig. 2 in the presence of 2 mM GSH or its derivatives (A), or 2 mM glucuronic acid, dithiothreitol, 2-mercaptoethanol, or thioredoxin (25 mg/ml) (B). Autoradiograms were developed after 8 h of exposure.

In order to determine if [125I]azidoAG-A binds to the biologically relevant site(s) of MRP1, quantitative inhibition studies were performed with a molar excess of AG-A, LTC4, or several anti-cancer and reversing agents and conjugated organic anions. The effect of AG-A, ADM, VCR, and LTC4 on photolabeling of MRP1 in membrane vesicles from KB/MRP cells is shown in Fig. 4A. The data for all agents used were quantified by removing slices of the gel corresponding to the bands on the autoradiograms (including those not shown) and measuring their radioactivity (Fig. 4B). AG-A inhibited the photolabeling in a dose-dependent manner and completely inhibited it at 200 µM. Inhibition by LTC4 was similar to that of AG-A. Among the anticancer agents, VCR and ADM inhibited photolabeling most strongly, with half-maximal inhibition at 33.9 and 30.4 µM, respectively, and almost complete inhibition at 200 µM. VP-16 and cisplatin inhibited the labeling by only 30% and 7% at 200 µM, respectively. Among the reversing agents, 4-oxo-8-[p-(4-phenylbutyl-oxy)benzoylamino]-2-(tetrazol-5-yl)-4H-1-benzopyran hemihydrate (ONO-1078) (38) had the highest inhibitory activity, and completely inhibited the labeling at 200 µM. ONO-1078, 3-([{3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl}-{(3-dimethylamino-3-oxopropyl)-thio}-methyl]thio)propanoic acid (MK571) (39), and 2-[4-(diphenylmethyl)-1-piperazinyl]-5-(trans-4,6-dimethyl-1,3,2-dioxaphosphorinan-2-yl)-2,6-dimethyl-4-(3-nitrophenyl)-3-pyridinecarboxylate P-oxide (PAK-104P) (40) inhibited the labeling by 50% at 10.6, 18, and 24.9 µM, respectively. Among the conjugated organic anions, LTC4 inhibited the photolabeling most strongly, with half-maximal inhibition at 1.3 µM. 17-beta -Estradiol-17 (beta -D-glucuronate) (E217beta G) inhibited the photolabeling by 50% at 129 µM and by 40% at 200 µM. These results indicate that [125I]azidoAG-A bound to the biologically relevant site on MRP1.


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Fig. 4.   Inhibition of [125I]azidoAG-A photolabeling of MRP1 in KB/MRP membrane vesicles by various agents. A, KB/MRP membrane vesicles were photolabeled with 5 µM [125I]azidoAG-A and 2 mM GSH in the absence or presence of the indicated concentrations of AG-A, ADM, VCR, and LTC4. Autoradiograms were developed after 12 h of exposure. B, inhibition of [125I]azidoAG-A labeling of MRP1 by anti-cancer agents, MDR-reversing agents, E217beta G, and LTC4. Quantification was achieved as described in the legend to Fig. 2. Data are expressed as the percentage of photolabeling of MRP1 in the absence of agents.

GSH Stimulates AG-A Binding to the C-terminal Half of MRP1-- In order to determine the drug binding site of AG-A on MRP1, the MRP1 molecule was cleaved into defined fragments by trypsin digestion and the fragments tested for binding to [125I]azidoAG-A. Membrane vesicles from KB/MRP cells were digested with trypsin and immunoblotted with antibodies specific for the N- and C-terminal half of MRP1, i.e. MRPr1 (epitope aa 238-247) and MRPm6 (epitope aa 1511-1520) (36, 41). Fig. 5 shows that both antibodies detect the intact 190-kDa MRP1 prior to trypsin digestion. In addition, a 120-kDa band (N1) was detected with MRPr1 and a minor band at 78 kDa was detected with MRPm6. Following treatment with 10 µg/ml trypsin, the amounts of the 120-kDa (N1) and 78-kDa (C1) peptides increased. When the concentration of trypsin was increased to 20 µg/ml, other diffuse bands (N2 (50-60 kDa) and C2 (40 kDa)) appeared, which were detected with MRPr1 and MRPm6, respectively. Fig. 5B shows the time courses of the appearance of N1 and C1 during treatment with trypsin at 10 µg/ml. N1 and C1 increased simultaneously during 5-30 min of incubation, indicating that both of them came from MRP1. These results are in accordance with previous studies (7) and suggest that the trypsin-accessible sites of MRP1 are located on the cytoplasmic NBD1 and L0 of MRP1. Thus, N1 and C1 bands represent the N-terminal half (i.e. TMD0 +TMD1+NBD1), and the C-terminal half (i.e. TMD2+NBD2) of MRP1, respectively.


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Fig. 5.   Immunoblots of the proteolytic products of MRP1 generated by trypsin digestion. A, membrane vesicles (50 µg) from KB/MRP cells were treated at 37 °C for 30 min with the indicated concentrations of trypsin and then subjected to 7.5% SDS-PAGE followed by immunoblotting with MRPr1 and MRPm6. B, membrane vesicles were treated at 37 °C for the indicated periods with 10 µg/ml trypsin and then subjected to 7.5% SDS-PAGE and immunoblotting with MRPr1 and MRPm6. N1, N2, C1, and C2 are 120-, 50-60-, 78-, and 40-kDa tryptic fragments, respectively.

Since considerable amounts of N- and C-terminal halves of MRP1 were produced by digestion of KB/MRP membrane vesicles with 10 µg/ml trypsin, it was possible to use these fragments to characterize the drug binding sites on MRP1. Membrane vesicles were photolabeled with [125I]azidoAG-A and then trypsinized under the same conditions used for immunoblotting (Fig. 6A). We found that the photoaffinity analog of AG-A labeled MRP1 and a 78-kDa fragment (C1). Together with previous reports (7, 42), this indicates that AG-A photolabeled the C-terminal half of MRP1. To determine whether the N-terminal half is photolabeled with increasing concentrations of the photoanalog, the concentration of the [125I]azidoAG-A was varied from 1 to 10 µM (Fig. 6B). As shown in Fig. 6B, the labeling of the native form of MRP1 and the C1 fragment were saturable. When the concentration of [125I]azidoAG-A was greater than 5 µM, a weakly photolabeled 120-kDa having the same size as the N-terminal fragment appeared. We think this band is nonspecific and not the N-terminal half of MRP1 since a large excess of AG-A almost completely inhibited the labeling of intact MRP1 but not of this 120-kDa band (Fig. 6C).


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Fig. 6.   Photoaffinity labeling of trypsinized MRP1 with [125I]azidoAG-A. Photolabeling of MRP1 with [125I]azidoAG-A was performed in the presence of 2 mM GSH as described under "Experimental Procedures." Membrane vesicles from KB/MRP cells (100 µg of protein/lane) were treated as follows. A, photolabeling of MRP1 was carried out with 5 µM [125I]azidoAG-A followed by MRP1 cleavage with the indicated concentrations of trypsin. B, photolabeling of MRP1 was carried out with 1-10 µM [125I]azidoAG-A followed by treatment for 30 min at 37 °C with 10 µg/ml trypsin. C, vesicles were pretreated for 30 min at 37 °C with 10 µg/ml trypsin before photolabeling MRP1 with 7 µM [125I]azidoAG-A in the presence or absence of 100 µM AG-A. Trypsin reactions were stopped by adding 2 mM phenylmethylsulfonyl fluoride and 0.5 TIU (trypsin inhibitory unit)/ml aprotinin, and the samples were subjected to 7.5% SDS-PAGE and autoradiography.

A baculovirus expression system was used as an alternative approach to study the binding site of AG-A on MRP1. Truncated, co-expressed, and mutated MRP1s were summarized in Fig. 7. It was reported that, although it was underglycosylated, MRP1 expressed in Sf21 insect cells transported LTC4 with kinetic parameters similar to those of MRP1 expressed in transfected mammalian cells, and that a functional transporter could be reconstituted when both halves of MRP1 were co-expressed (43). Gao et al. reported that reconstitution of a functional transporter in cells infected with a dual expression vector was extremely efficient compared with co-infection with two recombinant baculoviruses (44). cDNAs encoding N1-932 (N-proximal molecule of MRP1) and/or C932-1531 (the C-proximal molecule of MRP1) were cloned in a baculovirus dual expression vector. Spodoptera frugiperda Sf21 insect cells were infected with a recombinant baculovirus encoding wild type MRP1, N1-932, or C932-1531 alone, or with both N1-932 and C932-1531 from a dual expression vector with a multiplicity of infection of 5-10. MRPr1 and MRPm6 antibodies were used to determine the expression levels of the proteins in membrane vesicles prepared from the cells. As shown in Fig. 8A, both N1-932 and C932-1531 were efficiently expressed as 104- and 67-kDa peptides, respectively. Under the conditions used for the infection, the levels of the half-molecules were comparable both when they were expressed alone or co-expressed from a dual vector. The ATP-dependent LTC4 transport by membrane vesicles prepared from cells infected with a dual expression vector encoding both N1-932 and C932-1531 was almost the same as that with vesicles prepared from cells infected with a wild type MRP1 virus (data not shown). In membrane vesicles expressing N1-932 or C932-1531 alone, C932-1531 was only weakly photolabeled by the photoanalog of AG-A, and the labeling was not stimulated by GSH. However, when C932-1531 was co-expressed with N1-932, the binding of AG-A to C932-1531 was greatly enhanced in the presence of the indicated concentrations of GSH (Fig. 8, B and C). These results indicate that GSH stimulates AG-A binding to C932-1531, and that N1-932 is required for this binding.


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Fig. 7.   Membrane topology model of MRP1 and the various constructs expressed in Sf21 insect cells. The topology of MRP1 is characterized by the presence of an extra N-terminal domain (TMD0) with five transmembrane segments, which is connected to the P-gp-like core by a cytoplasmic linker (L0). Positions of nucleotide binding domains (NBD1 and NBD2) are also shown. A schematic diagram depicting Sf21-expressed MRP1 constructs is aligned and compared with the full-length MRP1. N1-932+C932-1531, TMD0L0+Delta MRP, and CoreN+C932-1531 represent two fragments of MRP1 co-expressed by the dual expression vector.


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Fig. 8.   GSH stimulates AG-A binding to C932-1531 in membrane vesicles from Sf21 insect cells co-expressing half-molecules of MRP1. A, membrane vesicles were prepared from Sf21 insect cells infected with recombinant baculovirus encoding full-length MRP1 or N1-932 and/or C932-1531 half-molecules. Membrane proteins (20 µg) of each sample were subjected to 7.5% SDS-PAGE and immunoblotted with MRPr1 and MRPm6. Left, detection of N1-932 by MRPr1; right, detection of C932-1531 by MRPm6. B, membrane vesicles containing full-length MRP1 or N1-932 and/or C932-1531 of MRP1 (50 µg of protein/lane) were photolabeled with 5 µM [125I]azidoAG-A in the absence (-) or presence of the indicated concentrations of GSH. Samples were subjected to 7.5% SDS-PAGE, and autoradiograms were developed after 10 h of exposure. Membrane vesicles from Sf21 insect cells without infection were used as a control. C, the data in B were quantified as described in the legend to Fig. 2. Open triangles, N1-932 alone; filled circles, N1-932 in membrane vesicles co-expressing both half-molecules; open squares, C932-1531 alone; filled squares, C932-1531 in membrane vesicles co-expressing both half-molecules.

GSH Is Not Required for Photolabeling of the P-gp-like Core Region of MRP1 with [125I]AzidoAG-A-- GSH is required for the binding of AG-A to C932-1531. To investigate the site of interaction of GSH on MRP1, TMD0L0 region of MRP1 (aa 1-281) (8) was co-expressed with Delta MRP (aa 281-1531), the P-gp-like core region of MRP1, from a dual vector in Sf21 insect cells. The expression levels of TMD0L0 and Delta MRP were determined by immunoblotting with the MRP1 N- and C-terminal specific mAbs, MRPr1 and MRPm6, respectively. Delta MRP was detected as a 139-kDa band by MRPm6, but not by MRPr1. TMD0L0 was detected as a band of about 32 kDa by MRPr1 but not by MRPm6 (Fig. 9A). ATP-dependent LTC4 transport was examined using membrane vesicles expressing Delta MRP alone or both Delta MRP and TMD0L0. The LTC4 transport activity in the membrane vesicles expressing both Delta MRP and TMD0L0 was similar to that in membrane vesicles expressing wild type MRP1, while it was almost abrogated in membrane vesicles expressing Delta MRP alone (data not shown). These data are in accordance with previous reports (8, 45). Membrane vesicles expressing Delta MRP alone or co-expressing both Delta MRP and TMD0L0 were photolabeled with [125I]azidoAG-A in the absence or presence of the indicated concentrations of GSH. In membrane vesicles co-expressing both Delta MRP and TMD0L0, the Delta MRP was weakly photolabeled by AG-A in the absence of GSH, and the labeling was enhanced 13-fold when the concentration of GSH was increased to 10 mM. In the absence of GSH, Delta MRP in membrane vesicles expressing Delta MRP alone was photolabeled by [125I]azidoAG-A more efficiently than that in membrane vesicles co-expressing both Delta MRP and TMD0L0. However, this labeling was not enhanced by GSH (Fig. 9, B and C).


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Fig. 9.   Comparison of GSH-dependent photolabeling of membrane vesicles expressing P-gp-like core region of MRP1 (Delta MRP) alone or both Delta MRP and TMD0L0. A, immunoblots of membrane proteins prepared from Sf21 insect cells expressing P-gp-like core region of MRP1 (Delta MRP) alone or together with TMD0L0. Membrane proteins (20 µg) of each sample were subjected to 7.5% and 12% SDS-PAGE and immunoblotted with MRPm6 and MRPr1, respectively. Membrane vesicles (20 µg) expressing full-length MRP1 were applied to 7.5% gel as a control. Left, detection of TMD0L0 by MRPr1; right, detection of core MRP by MRPm6. B, membrane vesicles expressing either Delta MRP alone or both Delta MRP and TMD0L0 (50 µg of protein/lane) were incubated with 5 µM [125I]azidoAG-A in the absence or presence of GSH (0.2-10 mM), the samples were then separated by 7.5% SDS-PAGE. Autoradiograms were developed after 12 h of exposure at room temperature. C, radioactive bands corresponding to the core region of MRP1 were excised and bound [125I]azidoAG-A was quantified as described in the legend to Fig. 2. Filled circles, Delta MRP; filled squares, Delta MRP and TMD0L0.

In order to investigate the GSH-independent binding site of AG-A on Delta MRP, CoreN (amino acids 281-932), which contains the TMD1 and NBD1 domains of MRP1, was co-expressed with C932-1531 using a dual expression vector. A myc tag was introduced to the C terminus of core N for the detection of the expressed protein. As shown in Fig. 10A, CoreN was detected as a band of 72 kDa by the anti-Myc antibody whereas C932-1531 was detected as a 67-kDa band by MRPm6. Fig. 10B shows the GSH-independent binding of [125I]azidoAG-A to both halves of the core region of MRP1. Inhibition studies using ADM and LTC4 as inhibitors showed that the GSH-independent binding of AG-A to the core region of MRP1 was not inhibited by either ADM or LTC4 (Fig. 10C). Based on the finding that truncation of the MRP-specific TMD0L0 abolished the specific GSH-dependent binding of AG-A to MRP1, it seems probable that the GSH interaction site on MRP1 is located on the truncated region.


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Fig. 10.   Photolabeling of both halves of the P-gp-like core region of MRP1 without GSH. A, membrane proteins from Sf21 cells expressing both the N-proximal molecule (core N) and the C932-1531 of MRP1 from a dual vector (CoreN+C932-1531) and both halves of MRP1 (N1-932+C932-1531) as a control were separated by 7.5% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Left and right, detection of CoreN by myc-specific antibody but not MRP1-specific mAb MRPr1; middle, detection of C932-1531 by MRP1-specific MRPm6 antibody. B, membrane proteins (50 µg/lane) isolated from Sf21 insect cells infected with a recombinant virus encoding both CoreN and C932-1531 were photolabeled with 5 µM [125I]azidoAG-A in the absence or presence of the indicated concentrations of GSH. Autoradiograms were developed after 20 h of exposure. C, membrane vesicles co-expressing both halves of the core region of MRP1were incubated with 5 µM [125I]azidoAG-A in the absence or presence of the indicated concentrations of ADM or LTC4. Photolabeling was conducted in the absence of GSH. Autoradiograms were developed after 20 h of exposure.

L0 Is the Site of GSH Interaction on MRP1-- Since GSH is hydrophilic, the GSH binding site on MRP1 may be located in a hydrophilic domain. To determine if L0 is the site of GSH-MRP1 interaction, we constructed a plasmid encoding L0Delta MRP (aa 203-1531), a polypeptide lacking the first transmembrane domain TMD0. L0Delta MRP expressed in membrane vesicles reacted with both MRPr1 and MRPm6 antibodies and the expression levels were similar to that of full-length MRP1. LTC4 transporting activity was retained in this truncated MRP1 (data not shown), which is in accordance with a previous report (8). Photolabeling was performed in the absence or presence of increasing concentrations of GSH. GSH stimulated the binding of AG-A to L0Delta MRP in a dose-dependent manner. Together with the data showing that TMD0L0 is required for GSH-dependent binding of AG-A to MRP1, it seems likely that L0 is the site of GSH interaction on MRP1.

Glutathione S-transferases (EC 2.5.1.18) (GSTs) are a family of isoenzymes that conjugate GSH to a variety of organic compounds that contain an electrophilic center (46). They are dimeric enzymes composed of two identical subunits, each of which contains one binding site for GSH (G-site) and a second binding site for hydrophobic substrates (H-site). Despite the low amino acid sequence homologies among GSTs belonging to different classes, all GST isoenzymes have a conserved G-site (47). The G-site of the human placental GST (pi GST), which has been studied in three dimensions, is formed by an irregular alpha -helix (helix 2) which, through Trp38 and Lys44, provides important electrostatic interaction with the carboxylate group of the glycine moiety of GSH (48, 49). We examined the L0 region of MRP1 for the presence of conserved residues that might interact with GSH. There are four tryptophan residues in the L0 region of MRP1, but only two of them are followed by lysine. Furthermore, hydropathy analysis of the region indicated that only Trp261 and Lys267 exist in a predicted alpha -helix. These residues were thus likely candidates for GSH binding. This was tested by substituting Trp261 and Lys267 with Ala and Met, respectively, and the subsequent mutated L0 region tested in binding studies. The mutated L0 (ML0) region was linked to the N terminus of Delta MRP to construct ML0Delta MRP. As shown in Fig. 11A, ML0Delta MRP protein, which has a level of expression comparable to that of wild type L0Delta MRP, reacted with both MRPr1 and MRPm6 antibodies. Photolabeling was performed using ML0Delta MRP membrane vesicles in the absence or presence of increasing concentrations of GSH. Fig. 11 (B and C) shows that the binding of AG-A to ML0Delta MRP was increased only 2.7-fold in the presence of 10 mM GSH compared with a 7.5-fold increase in the binding of AG-A to L0Delta MRP. This observation suggests that the L0 region of MRP1 may indeed form part of the GSH binding site.


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Fig. 11.   Comparison of GSH-dependent photolabeling of L0Delta MRP and ML0Delta MRP by [125I]azidoAG-A. A, membrane proteins (20 µg/lane) from Sf21 cells expressing L0Delta MRP or ML0Delta MRP were separated by 7.5% SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and immunoblotted with MRP1-specific mAb MRPr1. B, the L0Delta MRP or mutant L0Delta MRP (ML0Delta MRP) proteins were photolabeled as described under "Experimental Procedures." The two proteins were photolabeled at the same time to compare labeling efficiency. Autoradiograms were developed after 25 h of exposure at room temperature. C, the labeled bands were excised, radioactivities were determined. Filled circles, L0Delta MRP; filled squares, ML0Delta MRP.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

There is considerable experimental evidence showing that P-gp mediates MDR by directly binding and then transporting natural chemotherapeutic agents in an ATP-dependent manner (12, 13). It has been shown that MRP1 is an organic anion transporter, which can also transport non-anionic agents, such as VCR, only in the presence of GSH (16, 50). However, the role of GSH in facilitating drug transport is unclear. As an organic anion transporter, it was speculated that MRP1 confers resistance by eliminating drugs conjugated with GSH or other small molecules (24). However, there is some evidence against this idea. First, conjugated forms of some anti-cancer drugs, such as ADM and VCR, were not found in the culture medium of MRP1-transfected drug-resistant cells exposed to these agents (15). Second, phase I and II biotransformation reactions that form the GSH conjugates are known to occur primarily in liver, but it seems unlikely that these reactions occur at the same rate in all of the MRP1-overexpressing cells from other organs. Alternative speculations concerning the role of GSH are that GSH is either required as an activator, facilitating substrate binding and/or transport, or as a cotransported substrate (4). In order to clarify these issues, we synthesized a photoaffinity analog of AG-A that reversed MRP1-mediated MDR, and demonstrated that GSH is required for the binding of AG-A to MRP1.

Loe et al. (16) found that, in addition to GSH, S-methyl-GSH, but not dipeptides such as cysteinylglycine and gamma -glutamylcysteine, could also stimulate VCR transport by MRP1, suggesting that the stimulation of drug transport requires the complete GSH tripeptide but is not dependent on a free thiol. The free thiol of GSH was also not needed for the binding of AG-A to MRP1, since S-methyl-GSH stimulated AG-A binding to MRP1 with the same efficiency as GSH and a number of sulfhydryl reducing agents could not substitute for GSH in binding assays. It has been reported that S-decyl-GSH inhibited LTC4 uptake with a Ki of 116 nM, indicating that alkylated GSH is a competitive inhibitor of LTC4 uptake by MRP1-enriched membrane vesicles (14). We found that S-octyl-GSH did not stimulate AG-A binding to MRP1. On the contrary, it inhibited the GSH-dependent AG-A binding to MRP1 in a dose-dependent manner (data not shown). This long S-alkyl-GSH derivative may have bound to the GSH binding site on MRP1 and competitively inhibited the interaction of GSH with the site. Our findings suggest that GSH may be required for the binding of VCR to the drug binding site of MRP1.

MRP1 and P-gp belong to the same ATP-binding cassette transporter superfamily. However, MRP1 is characterized by an extra ~200 amino acid residues, TMD0, which is connected to the P-gp-like core region by a linker region L0. The precise functions of these two MRP1 specific domains are not known. In the present study, when the MRP1-specific TMD0L0 was truncated, AG-A bound to both halves of the core region, and GSH was not required for this binding. However, when L0 was introduced into the P-gp-like core region, GSH-dependent binding was recovered. Mutations of two amino acids in L0 considerably abrogated the GSH-dependent binding of AG-A to L0Delta MRP. These findings strongly suggest that L0 is the site of GSH interaction on MRP1.

Concerning the function of TMD0, a previous study suggested that TMD0 is not required for LTC4 transport by MRP1 (8). When TMD0 alone was introduced into the core region of MRP1, AG-A did not bind to the resultant polypeptide, even in the presence of GSH (data not shown). The photolabeling study suggested that TMD0 is not required for and inhibits drug binding to MRP1.

The drug binding site(s) on MRP1 is unknown. Photoaffinity labeling studies using photoaffinity analogs revealed that P-gp contains at least two drug-binding regions situated within the transmembrane segments 5 and 6 in its N-terminal half, and 11and 12 in its C-terminal half. These sites may form one large drug interaction site, or may represent two or more distinct drug binding sites (23, 51). Our data suggest that the labeling site of [125I]azidoAG-A on MRP1 is within C932-1531. However N1-932 is required for the labeling, since C932-1531 expressed alone was not efficiently photolabeled with the photoanalog of AG-A. It is still not clear why AG-A photolabeled Delta MRP more efficiently than intact MRP1 in the absence of GSH, and why this labeling was not inhibited by ADM, VCR, and LTC4. Delta MRP may form a tertiary structure different from that of MRP1. As a result, nonphysiological drug binding site(s) may be formed on Delta MRP.

It has been demonstrated that murine mrp1 confers MDR when expressed in human cells (52). However, the mouse protein did not confer resistance to anthracycline. Recent studies with murine/human hybrid proteins demonstrated that amino acids 959-1187, which are in the C-terminal half of MRP1, are critical for anthracycline resistance (53). Our finding that the AG-A labeling site on MRP1 is in C932-1531 is consistent with this previous report.

Our findings also suggest that the site of AG-A binding on MRP1 is similar to or partially overlaps the binding sites of LTC4, E217beta G, ADM, VCR, VP-16, PAK-104P, MK-571, and ONO-1078. It has been shown that MRP1 is an organic anion transporter, with LTC4 being one of the highest affinity MRP1 substrates identified to date (14). In accordance with this, LTC4 had the highest inhibitory activity among the agents we used in our inhibition study.

KB/MRP cells were 30.4-, 24.5-, and 39-fold more resistant to VCR, ADM, and VP16, respectively, than KB/CV cells, but were not resistant to cisplatin (28, 11). The present study showed that VCR and ADM considerably inhibited the photolabeling of MRP1 by AG-A, while cisplatin did not inhibit, even at 200 µM. We recently demonstrated that AG-A completely reversed the resistance to VCR, ADM, and VP16 of KB/MRP cells and found that AG-A, a compound with a chemical structure different from known MDR reversing agents, reversed MRP1-mediated MDR by two means. One was by directly interacting with MRP1 and the other by reducing the cellular concentration of GSH (28). In the present photolabeling study, the inhibitory activity of VP-16 was considerably lower than that of VCR and ADM, although resistance to VP-16 in KB/MRP was high (28). AG-A may reverse resistance to VP-16 mainly by lowering the cellular concentration of GSH. Alternatively, metabolite(s) of VP-16, but not VP-16 itself, may be a substrate for MRP1, or more than one drug binding site may exist on MRP1, and the binding site of VP-16 on MRP1 may partially overlap but be different from that of AG-A.

Previous studies showed that MRP1 is photolabeled by LTC4 (25, 26). However, it is impossible to investigate the role of GSH in drug binding using LTC4 since LTC4 itself is a GSH conjugate. Recently, some photoactive quinoline-based drugs (IAAQ, IACI) have been shown to bind directly to MRP1 in the absence of GSH (17-19). These authors demonstrated that MRP1-overexpressing cells are less sensitive to these quinoline-based drugs than the parental cells, and that these drugs are substrates of MRP1 on the basis of the results of MTT assays and drug accumulation studies. Daoud et al. (19) reported that IACI photolabeled three small peptides located in the N- and C-halves of MRP1 in the absence of GSH. We found that AG-A bound to the C-terminal half of MRP1 only in the presence of GSH, and that AG-A bound to both halves of Delta MRP in the absence of GSH. The GSH-dependent binding of AG-A to the C-terminal half of intact MRP1, but not the GSH-independent binding of AG-A to Delta MRP, was inhibited by LTC4, ADM, and VCR. Therefore, we consider that the GSH-independent binding of AG-A to Delta MRP, the P-gp-like core region of MRP1, differs from the physiological drug binding site on MRP1. The reason why the photoactive quinoline-based drugs can photolabel MRP1 in the absence of GSH is unknown. The photoanalog of AG-A is different from the photoactive quinoline-based drugs in its requirement for GSH for binding to the specific drug binding site(s) of MRP1. Quinoline-based drugs may bind to other sites of MRP1 besides the drug binding site in the C-terminal half, and that may enable them to be transported by MRP1.

To investigate whether AG-A is a substrate for MRP1, we examined the accumulation of the photoanalog of AG-A in KB/MRP and KB/CV cells in the dark. The accumulation of the photoanalog in KB/MRP cells was similar to that in KB/CV cells, suggesting that [125I]azidoAG-A bound to MRP1 but was not transported by it. The affinity of [125I]azidoAG-A to the drug binding site of MRP1 may be too high for it to be dissociated.

Borst et al. (54) recently proposed a model for MRP1/2 with two drug binding sites. He proposed that one site may have a relatively high affinity for GSH and a low affinity for drug (G-site), and the other site may have a relatively high affinity for drug and a low affinity for GSH (D-site). Our findings suggest that the G- and the D-sites of this model are located in the L0 and C-terminal half of MRP1, respectively. GSH may bind to L0 and cause a conformational change (55), which facilitates the specific binding of AG-A to MRP1. LTC4 and compounds conjugated with glutathione, glucuronide, or sulfate do not need free GSH to facilitate their binding and (or) transport by MRP1. Borst et al. (54) suggested that such compounds have a relatively high affinity for both the G- and D-sites, and therefore are transported efficiently in the absence of GSH. Both this report and the previous reports indicate that mutated MRP1 lacking the entire TMD0 but containing L0 can transport LTC4, whereas a mutant MRP1 lacking both TMD0 and L0 cannot. The role of L0 in LTC4 transport has been unclear. We now know that the lack of LTC4 transporting activity in membrane vesicles overexpressing the P-gp-like core region of MRP1 is probably due to the loss of the GSH binding site. Elucidation of the topology and interaction of GSH- and drug-binding sites should contribute to understanding the molecular basis for the transport of unmodified drugs by MRP1. The agents that compete with GSH for binding to L0 may reverse MRP1-mediated drug resistance.

In conclusion, our results indicate that GSH is required for the binding of an unconjugated agent to MRP1 and suggested that GSH interacts with L0 of MRP1. The photoanalog of AG-A will be useful for identifying the drug binding site within MRP1, and the role of GSH in transporting substrates by MRP1.

    ACKNOWLEDGEMENTS

We thank Dr. Piet Borst (Netherlands Cancer Institute) for the MRP1 cDNA, Dr. Susan P. C. Cole (Queen's University) for the QCRL-3 antibody, Dr. Kazumitsu Ueda (Kyoto University) for KB/MRP cells, Dr. Noriyuki Suzuki (AGENE Research Institute) for Sf21 cells and technical advice, Drs. Tatsuya Maeda (University of Tokyo), Kazutake Tsujikawa (Osaka University) and Yutaka Hoshikawa (Tokyo Metropolitan Institute of Medical Science) for technical advice and useful discussion, and Dr. Pauline O'Grady for her critical reading the manuscript. We also thank Hiromi Kakura for excellent secretarial assistance, and Etsuko Sudo, Hui Gao, Kana Kamimura, and Minae Tada for excellent technical assistance.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by a research fellowship from the Japan Society for the Promotion of Science.

|| To whom correspondence should be addressed. Tel.: 81-99-275-5490; Fax: 81-99-265-9687; E-mail: akiyamas@m3.kufm.kagoshima-u.ac.jp.

Published, JBC Papers in Press, April 11, 2001, DOI 10.1074/jbc.M101554200

    ABBREVIATIONS

The abbreviations used are: MDR, multidrug resistance; P-gp, P-glycoprotein; MRP1, human multidrug resistance protein; TMD, transmembrane domain; NBD, nucleotide-binding domain; GSH, glutathione; VCR, vincristine; ADM, adriamycin; VP-16, etoposide; LTC4, leukotriene C4; E217beta G, 17-beta -estradiol-17 (beta -D-glucuronate); AG-A, agosterol A; MK571, 3-([{3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl}-{(3-dimethylamino-3-oxopropyl)-thio} -methyl]thio)propanoic acid; ONO-1078, 4-oxo-8-[p-(4-phenylbutyl-oxy)benzoylamino]-2-(tetrazol-5-yl)-4H-1-benzopyran hemihydrate; PAK-104P, 2-[4-(diphenylmethyl)-1-piperazinyl]-5-(trans-4,6-dimethyl-1,3,2-dioxaphosphorinan-2-yl)-2,6-dimethyl-4-(3-nitrophenyl)-3-pyridinecarboxylate P-oxide; IAAQ, N-{4-[1-hydroxy-2-(dibutylamino)ethyl] quinolin-8'-yl}-4-azidosalicylamide; IACI, N-(hydrocinchonidin-8-yl)-4-azido-2-hydroxybenzamide; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; L, linker domain.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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