Abstract
Multidrug resistance protein 1 (MRP1) confers drug resistance and also mediates cellular efflux of many organic anions. MRP1 also transports glutathione (GSH); furthermore, this tripeptide stimulates transport of several substrates, including estrone 3-sulfate. We have previously shown that mutations of Lys332 in transmembrane helix (TM) 6 and Trp1246 in TM17 cause different substrate-selective losses in MRP1 transport activity. Here we have extended our characterization of mutants K332L and W1246C to further define the different roles these two residues play in determining the substrate and inhibitor specificity of MRP1. Thus, we have shown that TM17-Trp1246 is crucial for conferring drug resistance and for binding and transport of methotrexate, estradiol glucuronide, and estrone 3-sulfate, as well as for binding of the tricyclic isoxazole inhibitor N-[3-(9-chloro-3-methyl-4-oxo-4H-isoxazolo-[4,3-c]quinolin-5-yl)-cyclohexylmethyl]-benzamide (LY465803). In contrast, TM6-Lys332 is important for enabling GSH and GSH-containing compounds to serve as substrates (e.g., leukotriene C4) or modulators (e.g., S-decyl-GSH, GSH disulfide) of MRP1 and, further, for enabling GSH (or S-methyl-GSH) to enhance the transport of estrone 3-sulfate and increase the inhibitory potency of LY465803. On the other hand, both mutants are as sensitive as wild-type MRP1 to the non–GSH-containing inhibitors (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid (MK571), 1-[2-hydroxy-3-propyl-4-[4-(1H-tetrazol-5-yl)butoxy]phenyl]-ethanone (LY171883), and highly potent 6-[4′-carboxyphenylthio]-5[S]-hydroxy-7[E], 11[Z]14[Z]-eicosatetrenoic acid (BAY u9773). Finally, the differing abilities of the cysteinyl leukotriene derivatives leukotriene C4, D4, and F4 to inhibit estradiol glucuronide transport by wild-type and K332L mutant MRP1 provide further evidence that TM6-Lys332 is involved in the recognition of the γ-Glu portion of substrates and modulators containing GSH or GSH-like moieties.
Multidrug resistance protein 1 (MRP1/ABCC1) is a member of the “C” branch of the ATP-binding cassette superfamily and was originally identified in a multidrug-resistant lung cancer cell line (Cole et al., 1992). The 1531-amino acid MRP1 comprises five domains: three membrane-spanning domains (MSDs), containing five, six, and six transmembrane (TM) α-helices, respectively, and two cytoplasmic nucleotide binding domains (NBDs) (Fig. 1A) (Leslie et al., 2005). By mediating their ATP-dependent cellular efflux, MRP1 confers resistance to several classes of structurally unrelated anticancer drugs, many of which are natural products or their derivatives (Leslie et al., 2005). Glutathione (GSH), a ubiquitous tripeptide antioxidant that plays a critical role in many essential cellular processes, is required for efflux of at least some of these agents by a cotransport or cross-stimulated mechanism (Loe et al., 1996, 1998; Cole and Deeley, 2006). GSH may also be conjugated to reactive electrophilic chemical species generated during normal metabolism or during oxidative stress to form organic anions that are then effluxed by MRP1 (Cole and Deeley, 2006). Thus, in addition to anticancer drugs, MRP1 can transport many GSH-conjugated organic anions, including the endogenous metabolites cysteinyl leukotriene C4 (LTC4) and 4-hydroxynonenal-SG, as well as xenobiotic metabolites such as the GSH conjugates of aflatoxin B1 (Cole and Deeley, 2006).
Certain glucuronide- and sulfate-conjugated organic anions are also transported by MRP1, and in the case of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL)-O-glucuronide and estrone 3-sulfate, this transport is dependent on, or markedly stimulated by, GSH or a nonreducing analog such as S-methyl-GSH (S-MeGSH). However, in contrast to natural product anticancer drugs, NNAL-O-glucuronide and estrone 3-sulfate do not reciprocally stimulate GSH transport by MRP1 (Leslie et al., 2001a; Qian et al., 2001). Finally, GSH alone is poorly transported by MRP1, but its transport is markedly stimulated by apigenin (and several other bioflavonoids), as well as verapamil (Loe et al., 2000; Leslie et al., 2001b, 2003b). The oxidized form of GSH, glutathione disulfide (GSSG), is a much better substrate for MRP1 (Leier et al., 1996); however, unlike GSH, it does not stimulate the transport of other substrates but rather inhibits it.
MRP1 function has been often evaluated by measuring ATP-dependent uptake of a radiolabeled organic anion substrate into inside-out membrane vesicles prepared from cells expressing wild-type or mutant forms of the transporter (Loe et al., 1996; Deeley and Cole, 2006). In this way, a number of amino acids important for the stable plasma membrane expression of MRP1, or for its overall activity, have been identified (Haimeur et al., 2002, 2004; Ren et al., 2002; Koike et al., 2004; Situ et al., 2004; Conseil et al., 2006, 2009; Deeley and Cole, 2006; Chang 2007). Amino acids important for the substrate specificity of MRP1 have also been identified, two of which are Lys332, located in TM6 in the second MSD, and Trp1246, located in TM17 in the third MSD (Fig. 1A) (Ito et al., 2001a; Haimeur et al., 2002, 2004). Thus, both nonconservative (Asp, Leu) and conservative (Arg) substitutions of TM6-Lys332 result in a selective loss of LTC4 and GSH transport, whereas 17β-estradiol 17-(β-d-glucuronide) (E217βG) transport remains comparable with wild-type MRP1 (Haimeur et al., 2002, 2004). In contrast, substitution of TM17-Trp1246 with Phe, Tyr, Ala, or Cys selectively eliminates E217βG and NNAL-O-glucuronide transport and drug resistance but has little or no effect on LTC4 and GSH transport (Ito et al., 2001a; Leslie et al., 2001a). These and other observations indicate that TM6 and TM17 have distinct roles in determining the substrate specificity of MRP1 (Bao et al., 2005).
A number of compounds have been reported to chemosensitize multidrug resistant tumor cells that express MRP1 both in vitro and in xenograft model systems (Boumendjel et al., 2005; Shukla et al., 2008; Zhou et al., 2008). In addition to being potential novel chemotherapeutic agents (or prototypes for developing such agents), these compounds are valuable experimental tools for elucidating the functional and structural properties of MRP1. GSH, in addition to stimulating substrate transport as described above, markedly enhances the potency of several of these modulators. For example, several bioflavonoids (e.g., apigenin) and agosterol A, as well as several tricyclic isoxazole derivatives (e.g., LY465803 and LY475776), all inhibit MRP1-mediated transport in a manner that is dependent on, or stimulated by, GSH (Loe et al., 2000; Leslie et al., 2001b, 2003b; Ren et al., 2001; Mao et al., 2002; Norman et al., 2002; Dantzig et al., 2004). However, unlike the organic anion substrates such as LTC4 and E217βG, almost nothing is known about the domains and/or amino acids involved in determining the inhibitor specificity of MRP1.
In the present study, we have used vesicular transport and equilibrium binding assays to further investigate the effects of the TM6-Lys332 and TM17-Trp1246 mutations on the substrate specificity of MRP1. We have also examined the effects of these mutations on the sensitivity of MRP1 to a series of structurally diverse small molecule modulators and leukotriene derivatives.
Materials and Methods
Materials. [14,15,19,20-3H]LTC4 (158 Ci mmol–1), [6,7-3H]estrone 3-sulfate (57.3 Ci mmol–1), and [6,7-3H]E217βG (45 Ci mmol–1) were purchased from PerkinElmer Life and Analytical Sciences (Woodbridge, ON, Canada), and [3′,5′,7′-3H(n)]methotrexate sodium salt (33.5 Ci mmol–1) was from Moravek Biochemicals (Brea, CA). LTC4 and methotrexate sodium salt were purchased from Calbiochem (San Diego, CA) and Affinity Labeling Technologies Inc. (Lexington, KY), respectively. S-MeGSH, S-decyl-GSH, GSSG, dithiothreitol, AMP, ATP, apigenin, estrone 3-sulfate, E217βG, and BAY u9773 were from Sigma-Aldrich (Oakville, ON, Canada). LTD4, LTF4, LY171883, and MK571 were from Cayman Chemical (Ann Arbor, MI). LY465803 was a gift from Eli Lilly & Co. (Indianapolis, IN) (Dantzig et al., 2004).
Transfection of MRP1 Expression Vectors in Human Embryonic Kidney 293T Cells. The generation of wild-type, K332L, and W1246C mutant MRP1 pcDNA3.1 expression constructs has been described previously (Ito et al., 2001a; Haimeur et al., 2002). The wild-type and mutant expression vectors were transfected into human embryonic kidney (HEK293T) cells as before (Conseil et al., 2006). For membrane vesicle preparations, 18 × 106 cells were seeded per 15-cm dish and transfected 24 h later with 20 μg of plasmid DNA using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. After 48 h, the HEK293T cells were collected, resuspended in buffer 1 (50 mM Tris, pH 7.4, 250 mM sucrose) containing 250 mM CaCl2 and protease inhibitors, and then stored as cell pellets at –70°C until needed.
Preparation of Membranes and Membrane Vesicles. Transfected and untransfected (control) HEK293T cells were resuspended in buffer 1 and subjected to argon cavitation (300 psi, 5 min, 4°C) (Loe et al., 1996; Conseil et al., 2006). Unexploded cells and debris were removed by low-speed centrifugation (1900g, 10 min, 4°C), and the supernatant was layered onto a 35% sucrose cushion. After centrifugation (250,000g, 1 h, 4°C), membranes were collected from the sucrose interface, diluted with buffer 2 (50 mM Tris, pH 7.4, 25 mM sucrose), centrifuged again, and membranes were frozen until required. Alternatively, to form membrane vesicles, the membrane pellet was resuspended in buffer 1 at ∼5 μg of protein/μl and passed 10 times through a 27-gauge needle to form vesicles. Membrane vesicle preparations were then stored at –70°C until required. Protein concentrations were measured using a Bio-Rad (Mississauga, ON, Canada) Bradford assay with bovine serum albumin as a standard.
Measurement of MRP1 Protein Expression Levels. Relative levels of wild-type and mutant MRP1 proteins were determined by immunoblot analysis of membrane protein fractions from transfected cells essentially as described using the human MRP1-specific murine monoclonal antibody QCRL-1 (diluted 1:10,000) (Ito et al., 2001a; Conseil et al., 2006). Relative levels of wild-type and mutant MRP1 proteins were estimated by densitometric analysis of films using Image J software (http://rsb.info.nih.gov/ij/). To confirm equal protein loading of the gel lanes, the blots were stained with Amido Black.
MRP1-Mediated ATP-Dependent Transport of 3H-Labeled Substrates by Inside-Out Membrane Vesicles. ATP-dependent uptake of 3H-labeled substrates by MRP1-enriched membrane vesicles was measured using a rapid filtration technique adapted to a microplate format as described previously (Létourneau et al., 2005; Conseil et al., 2006). In brief, 2 μg of membrane vesicle protein was incubated with, respectively, 50 nM/20 nCi [3H]LTC4 for 1 min at 23°C, 400 nM/40 nCi [3H]E217βG for 1 min at 37°C, or 300 nM/25 nCi [3H]estrone 3-sulfate with 3 mM GSH (+10 mM dithiothreitol) or 3 mM S-MeGSH alone for 1 min at 37°C in a 50-μl reaction mixture containing 4 mM AMP or ATP, 10 mM MgCl2, and an ATP-regenerating system. Uptake of [3H]methotrexate was measured using 10 μg of membrane vesicle protein incubated with 100 μM/250 nCi [3H]methotrexate and other reaction components for 20 min at 37°C. The incubation mixture was filtered through a PerkinElmer Life and Analytical Sciences Unifilter-96 GF/B plate using a Packard (Waltham, MA) Filtermate Harvester and tritium-bound to the filter quantitated. Uptake in the presence of AMP was subtracted from uptake in the presence of ATP to determine ATP-dependent uptake values. Results were expressed as means (±S.D.) after correcting for differences in relative levels of MRP1 expression if necessary.
When transport assays were carried out in the presence of modulators (including GSH derivatives and cysteinyl leukotriene derivatives), vesicles were preincubated with the modulators for 15 min on ice before proceeding with the transport assays. Stock solutions of the modulators were prepared in buffer 1 except as follows: MK571 and LTF4 in methanol; apigenin, BAY u9773, and LY465803 in dimethyl sulfoxide; LY171883 and LTD4 in ethanol; and S-decyl-GSH and GSSG in 1 N NH4OH. The final concentration of vehicle never exceeded 1% of the final reaction volume. Data were expressed as a percentage of control uptake levels in the absence of modulator and fitted to a sigmoidal dose-response curve by nonlinear regression analysis using Graph-Pad Prism 3.0 software (GraphPad Software Inc., San Diego, CA). Statistically significant differences (p < 0.05) between IC50 values for the mutant and wild-type MRP1s were determined using a paired Student's t test.
[3H]Estrone 3-Sulfate Binding Assays. Equilibrium binding of [3H]estrone 3-sulfate to wild-type MRP1 and the K332L and W1246C mutants was determined as described previously (Rothnie et al., 2006, 2008). In brief, membrane protein (10 μg) was allowed to equilibrate with [3H]estrone 3-sulfate (50 nM; 50 nCi) in the presence of various concentrations of S-MeGSH in the absence of nucleotide in a total volume of 50 μl of hypotonic buffer (50 mM HEPES, pH 7.4) at 23°C for 1 h. After equilibration, ice-cold buffer (20 mM Tris, pH 7.4, 20 mM MgCl2) was added, and samples were filtered immediately and washed. Radioactivity was quantitated and data were fitted to a sigmoidal dose-response curve by nonlinear regression analysis as before, and the EC50 values for S-MeGSH were determined as the concentration of S-MeGSH at which half-maximal estrone sulfate binding occurred. Data points are expressed as means (±S.E.) of three or more independent experiments.
Saturation Binding Isotherms. To investigate the effect of estrone 3-sulfate concentration on [3H]estrone 3-sulfate binding, 10 μg of membrane protein from wild-type and mutant MRP1-transfected cells were incubated with various concentrations of [3H]estrone 3-sulfate (50 nM to 10 μM) and 3 mM S-MeGSH in the presence or absence of the competitive substrates E217βG (1 mM) in the case of the K332L mutant and LTC4 (10 μM) in the case of the W1246C mutant. Nonspecific binding (in the presence of E217βG or LTC4) was subtracted from total binding (in the absence of E217βGor LTC4) to determine specific binding, and the data were fitted with a one-site binding hyperbola as described previously (Rothnie et al., 2006). The commercially available [3H]estrone 3-sulfate could not be used to generate concentrations greater than 3 μM, and this was therefore achieved by supplementing with unlabeled estrone 3-sulfate as described previously (Rothnie et al., 2006).
Results
Effect of Lys332 and Trp1246 Mutations on 3H-Labeled Organic Anion Transport by MRP1. In the first series of experiments, the ability of the K332L and W1246C mutants to transport methotrexate and estrone 3-sulfate (in the presence of GSH and S-MeGSH) was measured. After transfecting HEK293T cells with the wild-type and mutant constructs, membrane vesicles were prepared, and relative expression levels of the K332L and W1246C mutants were determined by immunoblotting and found to be similar to wild-type MRP1 (data not shown), as reported previously (Ito et al., 2001a; Haimeur et al., 2002). The membrane vesicles were then used to measure ATP-dependent uptake of 3H-labeled organic anion substrates. As shown in Fig. 1B, uptake of [3H]methotrexate by the K332L mutant was comparable with wild-type MRP1, whereas uptake by W1246C was reduced by >75%. When [3H]estrone 3-sulfate transport (in the presence of GSH or S-MeGSH; 3 mM) was measured, both mutants displayed levels of uptake that were comparable with the HEK293T negative control (Fig. 1C). As reported previously (Ito et al., 2001a; Haimeur et al., 2002), [3H]LTC4 transport by the K332L mutant was also comparable with the untransfected HEK293T negative control, whereas that of the W1246C mutant was similar to wild-type MRP1 (Fig. 1D). On the other hand, the K332L mutant transported [3H]E217βGat levels comparable with wild-type MRP1, whereas the W1246C mutant showed no detectable transport of this conjugated estrogen, as expected from previous studies (Ito et al., 2001a) (Fig. 1E).
S-MeGSH Fails to Increase Estrone 3-Sulfate Binding to the K332L and W1246C Mutants. To determine whether changes in the ability of S-MeGSH to stimulate binding might contribute to the low levels of estrone 3-sulfate transport displayed by the K332L and W1246C mutants, equilibrium binding assays with MRP1 in the nucleotide-free state were carried out. Because of the low affinity of MRP1 for S-MeGSH (and GSH) and the lack of commercially available radiolabeled S-MeGSH (and the relatively low specific activity of commercially available [3H]GSH), it is not possible to measure directly the dissociation constant for S-MeGSH/GSH using equilibrium binding assays. Instead, we measured the EC50 of S-MeGSH on the equilibrium binding of estrone 3-sulfate in the presence of various concentrations of S-MeGSH as an indirect estimate of S-MeGSH affinity for MRP1. As expected and reported previously (Rothnie et al., 2006), estrone 3-sulfate binding to wild-type MRP1 increased in the presence of S-MeGSH in a concentration-dependent manner (Fig. 2, A and B). In contrast, S-MeGSH had little or no effect on estrone 3-sulfate binding to the K332L (Fig. 2A) or W1246C (Fig. 2B) mutants. The EC50 for S-MeGSH of wild-type MRP1 was 1.24 ± 0.07 mM, whereas the binding curves for the K332L and W1246C mutants were almost flat, precluding reliable estimations of their EC50s for this tripeptide. Estrone 3-sulfate binding values for both the K332L and W1246C mutants at apparent saturation were just 20% that of wild-type MRP1 (after subtraction of nonspecific binding).
To exclude the possibility that S-MeGSH-stimulated estrone 3-sulfate binding to the K332L and W1246C mutants might be underestimated in the above experiments because the initial concentration of estrone 3-sulfate used (50 nM) was too low, the binding assays were repeated at higher concentrations of this conjugated estrogen. However, as shown in Fig. 2C, performing the binding assays in the presence of 3 mM S-MeGSH and initial concentrations of estrone 3-sulfate as high as 10 μM did not substantially increase binding to the mutants relative to wild-type MRP1.
K332L Decreases Ability ofS-MeGSH to Stimulate Estrone 3-Sulfate Uptake, Whereas W1246C Decreases Affinity for Estrone 3-Sulfate. The lack of detectable estrone 3-sulfate binding by the mutants as described above could be because of either a loss of S-MeGSH (or GSH) binding to MRP1 or a loss of binding of estrone 3-sulfate. To help distinguish between these possibilities, ATP-dependent estrone 3-sulfate transport by the K332L and W1246C mutants was measured in the absence of S-MeGSH (or GSH) and compared with transport by wild-type MRP1. As shown in Fig. 3A, [3H]estrone 3-sulfate uptake in the absence of S-MeGSH (or GSH) by K332L was similar to that of wild-type MRP1. In contrast, ATP-dependent uptake of estrone 3-sulfate by W1246C was very low and similar to that of negative control HEK293T membrane vesicles, and this did not change when S-MeGSH was present (Figs. 1C and 3A, inset). These observations indicate that like wild-type MRP1, the K332L mutant can still bind and transport estrone 3-sulfate in the absence of S-MeGSH (or GSH), but unlike wild-type MRP1, estrone 3-sulfate uptake by the K332L mutant is no longer stimulated by S-MeGSH. Conversely, unlike wild-type and K332L mutant MRP1, W1246C apparently no longer binds estrone 3-sulfate; thus, no transport is observed in either the presence or absence of S-MeGSH (or GSH).
Loss of Synergistic Inhibition of E217βG Uptake by GSH and Apigenin for the K332L Mutant. We have previously shown that noninhibitory concentrations of apigenin (10 μM) could markedly enhance the ability of GSH to inhibit LTC4 transport by wild-type MRP1 (Leslie et al., 2001b). Therefore, we reasoned that if the K332L mutant could no longer bind GSH (or S-MeGSH), then GSH (or S-MeGSH) would no longer be expected to act synergistically with apigenin to inhibit uptake of LTC4 or other organic anion substrates. Accordingly, ATP-dependent E217βG uptake by K332L-enriched membrane vesicles was measured in the absence or presence of GSH (3 mM) and apigenin (10 μM). As shown in Fig. 3B, apigenin alone at this concentration had no effect on [3H]E217βG uptake, whereas GSH alone decreased uptake by just 35%. Together, however, apigenin and GSH decreased uptake by 70%. In contrast, neither GSH or apigenin (10 μM) alone nor the combination of GSH and apigenin had any effect on E217βG uptake by the K332L mutant. Similar results were obtained when S-MeGSH was used instead of GSH (data not shown). Because higher concentrations of apigenin (30 μM) inhibited E217βG transport by wild-type and K332L MRP1 similarly (data not shown), these observations support the conclusion that GSH (and S-MeGSH) binding to the K332L mutant is severely impaired.
Effect of MRP1 Modulators on the Activity of MRP1 Mutants K332L and W1246C. Because of the marked differences in the substrate specificity of the K332L and W1246C mutants, we next investigated whether the two mutants differed in their sensitivity to compounds reported previously to be modulators of MRP1 transport activity. Thus, the effect of four small molecule inhibitors (MK571, LY465803, LY171883, and BAY u9773) (Fig. 4A) and two GSH derivatives (S-decyl-GSH and GSSG) (Fig. 4B) were tested for their ability to inhibit E217βG transport by the K332L mutant. Three of the small molecules (MK571, BAY u9773, and LY465803) and S-decyl-GSH were also examined for their ability to inhibit LTC4 transport by the W1246C mutant. MK571 and LY171883 are leukotriene D4 antagonists that act on the cysteinyl leukotriene receptor 1 (CysLT1) (Fleisch et al., 1985; Jones et al., 1989), whereas BAY u9773 is a leukotriene-like dual antagonist that acts on both CysLT1 and CysLT2 receptors (Tudhope et al., 1994) and competitively inhibits MRP1-mediated LTC4 transport with an apparent Ki of ∼0.4 μM (D. W. Loe and S. P. C. Cole, unpublished observations). LY465803 is a tricyclic isoxazole molecule that inhibits transport by MRP1 in a GSH-dependent manner (Dantzig et al., 2004), whereas S-decyl-GSH and GSSG are GSH derivatives reported previously to be potent competitive inhibitors of MRP1 (Loe et al., 1996).
As summarized in Table 1 and shown in Fig. 5, MK571 and LY171883 were moderately (2-fold) less potent inhibitors of E217βG uptake by wild-type MRP1 than by the K332L mutant, whereas the inhibitory potency of BAYu9773 was not significantly different for the mutant and wild-type transporters. In contrast, LY465803 (+GSH) showed a far greater potency (16-fold) to inhibit wild-type compared with K332L mutant MRP1. In a similar manner, the GSH derivative S-decyl-GSH and GSSG inhibited wild-type MRP1 activity with IC50s >100-fold and >30-fold lower than for K332L, respectively.
In contrast to its more potent inhibitory effect on E217βG transport by the K332L mutant, MK571 was a less potent inhibitor (∼3-fold) of LTC4 transport by the W1246C mutant than by wild-type MRP1 (Table 2; Fig. 6A). However, similar to K332L, the inhibitory potency of BAY u9773 for LTC4 transport by wild-type and W1246C was comparable (Table 2; Fig. 6B). Also similar to K332L, LY465803 (+GSH) showed a far greater (>250-fold) potency to inhibit wild-type MRP1 than the W1246C mutant (Table 2; Fig. 6C). Finally, in contrast to K332L, the sensitivity of W1246C and wild-type MRP1 to inhibition of LTC4 transport by S-decyl-GSH was the same (Table 2; Fig. 6C).
Effect of Leukotriene Derivatives on E217βG Transport Activity by K332L. The structure of BAY u9773 is identical to that of LTC4 except the GSH substituent of the latter has been replaced with an S-linked benzoic acid moiety. Thus, the equal sensitivity of the transport activities of the K332L and W1246C mutants to inhibition by BAY u9773, despite their profoundly different abilities to transport LTC4, suggests that the K332L mutant has lost its ability to bind to the GSH moiety of LTC4. To determine whether this loss of binding involves one or more of the three amino acids that comprise the GSH moiety of LTC4, E217βG transport assays with wild-type MRP1 and the K332L mutant were carried out in the presence of various concentrations of the cysteinyl leukotriene derivatives LTC4, LTD4, and LTF4. As expected from previous studies (Haimeur et al., 2002), the IC50 of LTC4 (containing γ-Glu, Cys, and Gly) of the K332L mutant was significantly increased (∼25-fold) compared with that of wild-type MRP1 (Table 3; Fig. 7A). When E217βG transport was carried out in the presence of LTD4 (lacking a γ-Glu moiety but containing Gly and Cys), the IC50s of wild-type MRP1 and the K332L mutant were the same, although the inhibitor potency of LTD4 compared with LTC4 was ∼40-fold less for wild-type MRP1 but just ∼2-fold less for K332L (Table 3; Fig. 7B). The IC50s of LTF4 (containing a γ-Glu moiety but lacking Gly) for K332L and wild-type MRP1 differed by 4-fold, with the inhibitor potency of LTF4 comparable with LTD4 (Table 3; Fig. 7C). Thus, the K332L mutant protein is considerably less sensitive than wild-type MRP1 with respect to inhibition by the γ-Glu-containing LTC4 and LTF4 but not the γ-Glu-less LTD4, suggesting that Lys332 is critical for recognizing the γ-Glu moiety of GSH.
Discussion
Site-directed mutagenesis and vesicular transport assays have been used extensively to identify particular domains or amino acids involved in the substrate selectivity and transport mechanism of MRP1 (Ren et al., 2002; Conseil et al., 2006, 2009; Deeley and Cole, 2006; Chang 2007). Evidence gathered to date supports a model in which each substrate of MRP1 establishes its own distinct and often mutually exclusive interactions with amino acids in at least one substrate binding pocket of the transporter. Several amino acids have been identified that when mutated cause a substrate-selective elimination or substantial reduction in the transport activity of MRP1 (Cole and Deeley, 2006; Deeley and Cole, 2006). In the present study, we have further characterized the substrate specificity of two of these, the TM6 mutant K332L and the TM17 mutant W1246C, and have also determined their inhibitor profiles.
We have found that whereas mutation of TM6-Lys332 selectively eliminates LTC4 (and GSH) transport, methotrexate transport remains intact. On the other hand, the TM17-Trp1246 mutant no longer transports this folate antimetabolite, in addition to no longer conferring drug resistance or transporting E217βG. Thus, TM6 and TM17 play distinct roles in the transport of methotrexate, as well as LTC4 and E217βG. In contrast to their selective effects on the aforementioned organic anions, both mutations adversely affected GSH (and S-MeGSH)-stimulated estrone 3-sulfate transport.
Vesicular substrate transport assays such as those used to characterize the Lys332 and Trp1246 mutants give an overall measure of a multistep process that involves substrate binding to a site (or sites) accessible from the cytoplasmic side of the membrane, translocation through the membrane, and substrate release on the opposite side of the membrane followed by “resetting” of the transport protein, so that another round of transmembrane transport can take place (Tanford, 1983). As such, transport assays are limited in the amount of detailed mechanistic information they can provide regarding how a mutation affects the individual steps of the transport process. For this reason, we have recently developed equilibrium binding assays to understand better why GSH (or S-MeGSH) stimulates estrone 3-sulfate transport by MRP1, but the converse (i.e., estrone 3-sulfate-stimulated GSH transport) is not observed (Qian et al., 2001; Rothnie et al., 2006, 2008). Using these assays, we showed that GSH (and S-MeGSH) increase estrone 3-sulfate binding to wild-type MRP1 in membranes prepared from the MRP1-overexpressing drug-selected cell line H69AR in a concentration-dependent manner and that estrone 3-sulfate binding to MRP1 is greater in the presence of S-MeGSH than GSH despite the fact that the affinity for these two tripeptides is the same (Rothnie et al., 2006). In the present study, we have now shown that estrone 3-sulfate binding is also substantially greater in the presence of S-MeGSH (and GSH) using membranes prepared from HEK cells transfected with wild-type MRP1 cDNA. However, this was not the case for membranes prepared from transfected HEK cells expressing either the K332L or W1246C mutants.
Because the observed lack of estrone 3-sulfate binding could be the result of either reduced S-MeGSH (or GSH) binding to MRP1 or reduced binding of estrone 3-sulfate, ATP-dependent estrone 3-sulfate transport by the mutants was measured in the absence of GSH or S-MeGSH (Fig. 3A). We found that GSH-independent estrone 3-sulfate transport by W1246C was similar to negative controls, whereas transport by K332L was comparable with wild-type MRP1. Thus, in the case of the K332L mutant, impaired GSH-stimulated estrone 3-sulfate transport seems to be caused by a loss of GSH binding. In contrast, impaired transport by the W1246C mutant is caused by a loss of binding of the sulfated estrogen. Furthermore, unlike wild-type MRP1, GSH no longer acted synergistically with apigenin to inhibit E217βG uptake by the K332L mutant. Taken together, these data strongly support the conclusion that TM17-Trp1246 is critical for recognition of methotrexate, E217βG, and estrone 3-sulfate by MRP1, whereas TM6-Lys332 is critical for recognition of GSH and compounds such as LTC4 that contain a GSH moiety.
The latter conclusion is also consistent with the inhibitor profile displayed by the K332L mutant (Table 1; Fig. 5). The inhibitory potencies of modulators such as MK571, LY171883, and BAY u9773, which do not contain a GSH moiety and are not GSH-dependent, on the transport activities of wild-type and K332L mutant MRP1 were much more similar (1.5–2-fold difference in IC50s) than those of modulators that contain a GSH or GSH-like moiety (S-decyl-GSH, GSSG) or are dependent on GSH (LY465803) for their activity (15–30-fold difference in IC50s). On the other hand, as might be expected because mutation of Trp1246 has little or no effect on transport of either GSH or the GSH-containing LTC4 (Table 2; Fig. 6), the inhibitory potency of S-decyl-GSH was the same for the W1246C mutant and wild-type MRP1. The inhibitory potencies of the non–GSH-containing modulators MK571 and BAY u9773 on LTC4 transport by wild-type and W1246C mutant MRP1 were also comparable. Together, these observations indicate that neither TM17-Trp1246 nor TM6-Lys332 is critical for recognition of the non–GSH-containing modulators MK571, BAY u9773, and LY171883. This in turn suggests that these modulators bind to a site(s) on MRP1 that is different, or at least does not overlap extensively, with the binding site(s) for the organic anion substrates tested (estrone 3-sulfate, methotrexate, GSH, LTC4, E217βG). However, TM17-Trp1246 seems to be important for recognition of the tricyclic isoxazole derivative LY465803 because the IC50 for this modulator (in the presence of GSH) was approximately 280-fold greater for the W1246C mutant than for wild-type MRP1 (13.2 μM versus 46 nM). This observation is consistent with our earlier finding that radiolabeling of the COOH-proximal half of the W1246C mutant by [125I]LY475776, a photoactivateable derivative of LY465803, was substantially reduced relative to the same region of wild-type MRP1 (Mao et al., 2002).
When transport assays were carried out in the presence of three cysteinyl leukotriene derivatives to determine whether reduced binding of compounds that contain GSH-like moieties by the K332L mutant involves one or more of the three amino acids that comprise this tripeptide, the results obtained suggest that the presence of Lys332 is most important for the recognition of the γ-Glu residue in the GSH moiety. Thus, E217βG transport by the K332L mutant was considerably less sensitive than wild-type MRP1 to inhibition by the γ-Glu-containing LTC4 and LTF4 (25- and 4-fold differences in IC50s, respectively) than for the γ-Glu-less LTD4, for which the IC50s were the same (Fig. 7; Table 3). The conclusion that Lys332 is involved in the recognition of the γ-Glu portion of substrates and modulators containing GSH or GSH-like moieties is also consistent with previous reports showing that the γ-Glu residue in GSH analogs is crucial for stimulating estrone 3-sulfate transport by MRP1 (Leslie et al., 2003a), as well as for stimulating photolabeling of MRP1 by [125I]LY475776 (Mao et al., 2002). Also relevant is the study of Burg et al. (2002) showing that the potent inhibition of MRP1 transport activity by GSH conjugates and peptidomimetic GSH conjugate analogs is dependent on the presence of a γ-Glu moiety.
It is conceivable that the negatively charged γ-Glu moiety interacts directly with the positively charged Lys332, and in recently derived atomic homology models of the core structure of MRP1, Lys332 projects into the putative substrate translocation pathway of the transporter, suggesting the opportunity for direct bonding interactions (DeGorter et al., 2008). However, the possibility that Lys332 is only indirectly involved in the recognition of γ-Glu-containing substrates and modulators cannot be excluded. It has been previously reported that mutation of the amino acid analogous to human MRP1 TM6-Lys332 in rat Mrp2 (Lys325) similarly selectively reduces GSH conjugate transport but does not affect the inhibitory potency of the cysteinyl leukotriene metabolites (Ito et al., 2001b). However, it seems likely that this is because of differences in the amino acid residues of human MRP1 and rat Mrp2 that comprise the cysteinyl leukotriene binding site(s) that are also presumably responsible for the well documented differences in affinity that MRP1 and Mrp2/MRP2 have for these metabolites (Cui et al., 1999).
In conclusion, we have extended our characterization of the MRP1 mutants K332L and W1246C and further defined the different roles of these two amino acids in determining the substrate and inhibitor specificity of MRP1. Together, our previous and present data suggest that MRP1 contains at least three classes of substrate/modulator binding sites: one that requires TM17-Trp1246, one that requires TM6-Lys332, and a third that requires neither of these residues. Thus, TM17-Trp1246 is important for conferring drug resistance and for transport of methotrexate, E217βG, and estrone 3-sulfate, as well as for binding of tricyclic isoxazole inhibitors such as LY465803. The presence of TM6-Lys332, on the other hand, is crucial for enabling GSH and GSH-containing compounds to serve as substrates or modulators of MRP1 and, further, for enabling GSH (or S-MeGSH) to enhance the transport of estrone 3-sulfate and increase the inhibitory potency of LY465803. Nevertheless, neither Trp1246 nor Lys332 is important with respect to the activity of the non–GSH-containing small molecule inhibitors MK571 and LY171833 or the highly potent BAY u9773. Finally, we have presented convincing evidence that Lys332 is involved in the recognition of the γ-Glu portion of substrates and modulators containing GSH or GSH-like moieties. Structural information on the GSH-coordinating residues in enzymes involved in GSH conjugation reactions and GSH metabolism has recently become available, including for LTC4 synthase, a member of the integral membrane enzymes of the membrane-associated proteins in the eicosanoid and glutathione metabolism family (Martinez Molina et al., 2008). Extensive polar interactions exist between GSH and LTC4 synthase, including two salt bridges between the carboxylate ends of GSH with two basic residues in the enzyme. Whether salt bridges are formed when GSH is bound to MRP1 and/or whether TM6-Lys332 is involved in these or other ionic bonding interactions between MRP1 and GSH-containing substrates and modulators awaits similar high-resolution structural information on this ATP-binding cassette transporter.
Acknowledgments
We thank Kathy Sparks for excellent technical assistance, Steven Molinski for advice, and Maureen Hobbs for help in preparation of the manuscript.
Footnotes
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This work was supported by the Canadian Institutes of Health Research [Grant MOP-10519].
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K.M. and A.N. contributed equally to this work.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.109.026633.
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ABBREVIATIONS: MRP, multidrug resistance protein; MSD, membrane-spanning domain; TM, transmembrane helix; NBD, nucleotide binding domain; GSH, glutathione; LTC4, leukotriene C4; NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; S-MeGSH, S-methyl-GSH; GSSG, glutathione disulfide; E217βG, estradiol glucuronide; LY465803, N-[3-(9-chloro-3-methyl-4-oxo-4H-isoxazolo[4,3-c]quinolin-5-yl)-cyclohexylmethyl]-benzamide; BAY u9773, 6-[4′-carboxyphenylthio]-5[S]-hydroxy-7[E], 11[Z]14[Z]-eicosatetrenoic acid; MK571, (E)-3-[[[3-[2-(7-chloro-2-quinolinyl) ethenyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid; LY171883, 1-[2-hydroxy-3-propyl-4-[4-(1H-tetrazol-5-yl)butoxy] phenyl]-ethanone; HEK, human embryonic kidney; CysLT1, cysteinyl leukotriene receptor 1; LY475776, N-(4-azido-3-iodo-phenyl)-2-[3-(9-chloro-3-methyl-4-oxo-4H-isoxazole[4,3-c]quinolin-5-yl)-cyclohexyl]acetamide.
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↵1 Current affiliation: Department of Surgery II, Shinshu University School of Medicine, Matsumoto, Nagano, Japan.
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↵2 Current affiliation: Department of Surgery, Toyooka Public Hospital, Toyooka City, Japan.
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↵3 Current affiliation: University of Warwick, Department of Biological Sciences, Gibbet Hill Campus, Coventry, United Kingdom.
- Accepted April 23, 2009.
- Received January 8, 2009.
- The American Society for Pharmacology and Experimental Therapeutics