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.
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
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