ABCMdb

ABCC2 p.Trp1254Phe

Predicted by SNAP2:	A: D (95%), C: D (95%), D: D (95%), E: D (95%), F: D (80%), G: D (95%), H: D (95%), I: D (95%), K: D (95%), L: D (91%), M: D (91%), N: D (95%), P: D (95%), Q: D (95%), R: D (95%), S: D (95%), T: D (95%), V: D (95%), Y: D (91%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, Y: D,

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[hide] ABCC2/Abcc2: a multispecific transporter with domi... Drug Metab Rev. 2010 Aug;42(3):402-36. Jemnitz K, Heredi-Szabo K, Janossy J, Ioja E, Vereczkey L, Krajcsi P
ABCC2/Abcc2: a multispecific transporter with dominant excretory functions.
Drug Metab Rev. 2010 Aug;42(3):402-36., [PMID:20082599]

Abstract [show]
ABCC2/Abcc2 (MRP2/Mrp2) is expressed at major physiological barriers, such as the canalicular membrane of liver cells, kidney proximal tubule epithelial cells, enterocytes of the small and large intestine, and syncytiotrophoblast of the placenta. ABCC2/Abcc2 always localizes in the apical membranes. Although ABCC2/Abcc2 transports a variety of amphiphilic anions that belong to different classes of molecules, such as endogenous compounds (e.g., bilirubin-glucuronides), drugs, toxic chemicals, nutraceuticals, and their conjugates, it displays a preference for phase II conjugates. Phenotypically, the most obvious consequence of mutations in ABCC2 that lead to Dubin-Johnson syndrome is conjugate hyperbilirubinemia. ABCC2/Abcc2 harbors multiple binding sites and displays complex transport kinetics.

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No. Sentence Comment
97 Mutant Predicted location Substrate Activity changes Reference Human MRP2 Δ1-188 TMD0 LTC4 ↓ Fernandez et al., 2002 K316A JC, TM6 GMF ↔ Ryu et al., 2000 K324A TM6 GMF ↓ Ryu et al., 2000 K329A TM6 GMF ↔ Ryu et al., 2000 R412G DJ IC MTX ↓ Hulot et al., 2005 W417I IC, TM7-TM8 E2-17βG ↓ Hirouchi et al., 2004 LTC4 ↓ DNP-SG ↓ H439A TM8 GMF ↔ Ryu et al., 2000 K483A IC, JM, TM9 GMF ↓ Ryu et al., 2000 K590A JC, TM11 GMF ↔ Ryu et al., 2000 S789F NBD1 E2-17βG ↓ Hirouchi et al., 2004 LTC4 ↓ DNP-SG ↓↓ R1023A EC, JM, TM13 GMF ↔ Ryu et al., 2000 H1042A TM13 GMF ↔ Ryu et al., 2000 R1100A JC, TM14 GMF ↔ Ryu et al., 2000 P1158A IC, JM, TM15 LTC4 ↓↓ Letourneau et al., 2007 E2-17βG ↔ MTX ↔ Table 1. continued on next page Mutant Predicted location Substrate Activity changes Reference I1173F DJ IC, TM15-16 LTC4 No act Keitel et al., 2003 E2-17βG No act R1210A EC, JC, TM16 GMF ↓↓ Ryu et al., 2000 R1230A TM16 GMF ↔ R1257A JC, TM17 GMF ↓↓ W1254A JC, TM17 E2-17βG ↓↓ Ito et al., 2001a W1254C ↓↓ W1254F ↔ W1254Y ↔ W1254A JC, TM17 LTC4 ↓↓ Ito et al., 2001b W1254C ↓↓↓ W1254F ↓↓ W1254Y ↓↓ W1254A JC, TM17 MTX ↓↓ Ito et al., 2001a W1254C ↓↓ W1254F ↓↓ W1254Y ↓↓↓ A1450T NBD2 E2-17βG ↓↓ Hirouchi et al., 2004 LTC4 ↓↓ DNP-SG ↓↓ Rat Mrp2 K308M IC, JM, TM6 TLC-S ↔ Ito et al., 2001b DNP-G ↑ LTC4 ↓ E3040G ↔ K320M TM6 TLC-S ↑ DNP-G ↑ LTC4 ↓ E3040G ↑ K325M TM6 TLC-S ↓* DNP-G ↓↓↓* LTC4 ↓↓↓* E3040G ↓ D329N TM6 TLC-S ↔ DNP-G ↓ LTC4 ↓↓↓* E3040G ↓ R586L TM11 TLC-S ↓ DNP-G ↓↓* LTC4 ↓↓* E3040G ↔ R1019M IC, JM, TM13 TLC-S ↔ DNP-G ↑* LTC4 ↔ E3040G ↔ R1096L TM14 TLC-S ↑ DNP-G ↑ LTC4 ↔ E3040G ↔ EC, extracellular; IC, intracellular; JC, near the cytosol in the membrane; JM, juxtamembrane; TLC-S, tauro-litocholate-sulfate; GMF, glutathione- methyl-fluorescein; ↑, activity over control>1.2; ↔, 1.2>activity over control>0.8; ↓, 0.8>activity over control>0.5; ↓↓, 0.5>activity over control>0.1; ↓↓↓, 0.1>activity over control. Login to comment
105 The W1254F mutation did not affect estradiol-17β-glucuronide (E2-17βG) transport, but almost completely erased LTC4 and methotrexate (MTX) transport by the protein (Ito et al., 2001a). Login to comment

[hide] Estradiol 3-glucuronide is transported by the mult... Drug Metab Dispos. 2004 Oct;32(10):1139-45. Epub 2004 Jul 27. Gerk PM, Li W, Vore M
Estradiol 3-glucuronide is transported by the multidrug resistance-associated protein 2 but does not activate the allosteric site bound by estradiol 17-glucuronide.
Drug Metab Dispos. 2004 Oct;32(10):1139-45. Epub 2004 Jul 27., [PMID:15280218]

Abstract [show]
beta-estradiol 17-(beta-D-glucuronide) (E217G) is a well known cholestatic agent and substrate of multidrug resistance-associated protein 2 (Mrp2), whereas beta-estradiol 3-(beta-D-glucuronide) (E23G) is a noncholestatic regioisomer of E217G with unknown transport properties. The purpose of this study was to compare and contrast the Mrp2-mediated transport of E217G and E23G. The full coding region of rat Mrp2 was cloned into the baculovirus genome, the recombinant baculovirus used to infect Sf9 cells, and ATP-dependent transport of 3H-E23G and 3H-E217G in Sf9 cell membranes was characterized. Mrp2 transported E23G into an osmotically sensitive space, requiring ATP, with S50=55.7 microM, Vmax=326 pmol.mg(-1).min(-1), and a Hill coefficient of 0.88. ATP-dependent Mrp2-mediated E217G transport was markedly stimulated at high E217G concentrations, consistent with positive cooperativity (Hill coefficient 1.5). E217G (5-125 microM) increased S50 but not Vmax for E23G transport, consistent with competitive inhibition. E23G (0.4-400 microM) completely, potently (IC50=14.2 microM), and competitively inhibited E217G transport, but E217G (0.01-250 microM) inhibited only 53% of E23G transport (IC50=33.4 microM). Estriol 16alpha-(beta-D-glucuronide) potently and completely inhibited transport of E23G (IC50=2.23 microM), as did beta-estradiol 3-sulfate 17-(beta-D-glucuronide) (5-50 microM). In summary, E217G binds not only to an Mrp2 transport site, but also to an allosteric site that activates Mrp2 with positive cooperativity, thus activating its own transport and potentially that of other Mrp2 substrates, such as E23G. The noncholestatic E23G is an Mrp2 substrate and competes with E217G for transport, but does not activate the allosteric site.

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156 The W1254F MRP2 mutant retained E217G transport, but not leukotriene C4 or methotrexate transport. Login to comment

[hide] Single nucleotide polymorphisms in multidrug resis... Adv Drug Deliv Rev. 2002 Nov 18;54(10):1311-31. Suzuki H, Sugiyama Y
Single nucleotide polymorphisms in multidrug resistance associated protein 2 (MRP2/ABCC2): its impact on drug disposition.
Adv Drug Deliv Rev. 2002 Nov 18;54(10):1311-31., [PMID:12406647]

Abstract [show]
Multidrug resistance associated protein 2 (MRP2/ABCC2), expressed on the bile canalicular membrane, plays an important role in the biliary excretion of various kinds of substrates. In addition, MRP2 is also expressed on the apical membrane of epithelial cells such as enterocytes. It is possible that the inter-individual difference in the function of MRP2 affects the drug disposition. In the present article, we will summarize the physiological and pharmacological role of MRP2, particularly focusing on the factors affecting its transport function such as single nucleotide polymorphisms and/or the induction/down regulation of this transporter. Mutations found in patients suffering from the Dubin-Johnson syndrome, along with the amino acid residues which are involved in supporting the transport activity of MRP2, are also summarized.

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No. Sentence Comment
125 Conservatively and glutathione conjugates into the isolated mem- substituted Trp1254Phe transports E 17bG, but not2 brane vesicles from Sf9 cells expressing mutant rat LTC or methotrexate, whereas highly conservative-4 MRP2 [109]. Login to comment

[hide] Mutation of Trp1254 in the multispecific organic a... J Biol Chem. 2001 Oct 12;276(41):38108-14. Epub 2001 Aug 10. Ito K, Oleschuk CJ, Westlake C, Vasa MZ, Deeley RG, Cole SP
Mutation of Trp1254 in the multispecific organic anion transporter, multidrug resistance protein 2 (MRP2) (ABCC2), alters substrate specificity and results in loss of methotrexate transport activity.
J Biol Chem. 2001 Oct 12;276(41):38108-14. Epub 2001 Aug 10., [PMID:11500505]

Abstract [show]
The ATP-binding cassette (ABC) proteins comprise a large superfamily of transmembrane transporters that utilize the energy of ATP hydrolysis to translocate their substrates across biological membranes. Multidrug resistance protein (MRP) 2 (ABCC2) belongs to subfamily C of the ABC superfamily and, when overexpressed in tumor cells, confers resistance to a wide variety of anticancer chemotherapeutic agents. MRP2 is also an active transporter of organic anions such as methotrexate (MTX), estradiol glucuronide (E217betaG), and leukotriene C4 and is located on the apical membrane of polarized cells including hepatocytes where it acts as a biliary transporter. We recently identified a highly conserved tryptophan residue in the related MRP1 that is critical for the substrate specificity of this protein. In the present study, we have examined the effect of replacing the analogous tryptophan residue at position 1254 of MRP2. We found that only nonconservative substitutions (Ala and Cys) of Trp1254 eliminated [3H]E217betaG transport by MRP2, whereas more conservative substitutions (Phe and Tyr) had no effect. In addition, only the most conservatively substituted mutant (W1254Y) transported [3H]leukotriene C4, whereas all other substitutions eliminated transport of this substrate. On the other hand, all substitutions of Trp1254 eliminated transport of [3H]MTX. Finally, we found that sulfinpyrazone stimulated [3H]E217betaG transport by wild-type MRP2 4-fold, whereas transport by the Trp1254 substituted mutants was enhanced 6-10-fold. In contrast, sulfinpyrazone failed to stimulate [3H]MTX transport by either wild-type MRP2 or the MRP2-Trp1254 mutants. Taken together, our results demonstrate that Trp1254 plays an important role in the ability of MRP2 to transport conjugated organic anions and identify this amino acid in the putative last transmembrane segment (TM17) of this ABC protein as being critical for transport of MTX.

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47 Mutagenesis was carried out using the TransformerTM kit (CLONTECH, Palo Alto, CA) according to the manufacturer`s instructions with the following sense mutagenic primers (substituted nucleotides are underlined): W1254A (5Ј-C- ACAAACCCTGAACGCTTCTGGTGAGGATGAC-3Ј), W1254C (5Ј-CAC- AAACCCTGAACTGTCTGGTGAGGATGAC-3Ј), W1254F (5Ј-CACAAA- CCCTGAACTTTCTGGTGAGGATGAC-3Ј), and W1254Y (5Ј-CACAAA- CCCTGAACTATCTGGTGAGGATGAC-3Ј). Login to comment
74 These included nonconservative substitutions with a nonaromatic nonpolar amino acid (Ala; W1254A-MRP2) and a nonaromatic polar amino acid (Cys; W1254C-MRP2), as well as conservative substitutions with polar (Tyr; W1254Y-MRP2) and nonpolar (Phe; W1254F-MRP2) aromatic amino acids. Login to comment
76 As shown in Fig. 1B, wild-type MRP2 and its four mutants (W1254A, W1254C, W1254Y, and W1254F) were all expressed in the HEK293T cells at comparable levels. Login to comment
77 Mean values of expression levels from four to six independent transfections relative to wild-type MRP2 were W1254A, 1.1; W1254C, 0.9; W1254F, 0.9; W1254Y, 1.0, respectively (Fig. 1B). Login to comment
81 However, E217betaG transport by the W1254Y-MRP2 and W1254F-MRP2 mutants was similar to that of wild-type MRP2, in marked contrast to the comparable conservatively substituted MRP1-Trp1246 mutants, which were inactive for transport of this substrate (40). Login to comment
84 [3 H]E217betaG Transport by W1254F-MRP2 Is No Longer Inhibited by LTC4-It has been shown previously that LTC4 inhibits MRP2-mediated E217betaG transport (39, 44). Login to comment
85 Consequently, to determine whether E217betaG transport by a mutant MRP2 protein that no longer transports LTC4 could still be inhibited by this cysteinyl leukotriene, [3 H]E217betaG transport by the W1254F-MRP2 mutant was examined. Login to comment
87 In contrast, LTC4 had no significant effect on [3 H]E217betaG transport by W1254F-MRP2, indicating that the loss of LTC4 transport by this mutant is associated with a loss of binding of this substrate. Login to comment
97 B, immunoblot of membrane vesicles prepared from HEK293T cells transfected with empty vector (pcDNA3.1(-)), wild-type (WT-MRP2) and mutant (W1254A, W1254C, W1254F, and W1254Y) MRP2 cDNAs. Login to comment
103 A, [3 H]E217betaG uptake was measured in membrane vesicles prepared from HEK293T cells transfected with empty vector (pcDNA3.1(-); open bar), wild-type MRP2 (WT-MRP2; solid bar), and mutant (W1254A-MRP2, W1254C-MRP2, W1254F-MRP2, and W1254Y-MRP2; shaded bars) cDNA expression vectors. Login to comment
106 The results shown are the means Ϯ S.D. of triplicate determinations in a single experiment, and similar results were obtained in a second experiment. relative levels of [3 H]MTX transport by the W1254A, W1254C, W1254F, and W1254Y mutants were 14, 14, 11, and 1%, respectively, of wild-type MRP2. Login to comment
108 [3 H]E217betaG Transport by Wild-type MRP2, but Not W1254F-MRP2, Is Inhibited by MTX-It has previously been shown that MTX inhibits MRP2-mediated transport of the GSH-conjugated substrates, dinitrophenyl-glutathione and N-ethylma- leimide-glutathione (21, 24). Login to comment
111 To determine whether E217betaG transport by mutant MRP2 proteins that no longer transport MTX remained inhibitable by this antifolate, [3 H]E217betaG transport in membrane vesicles prepared from cells expressing W1254F-MRP2 and W1254Y-MRP2 was examined. Login to comment
112 In contrast to wild-type MRP2, MTX had no significant effect on [3 H]E217betaG transport by either W1254F-MRP2 or W1254Y-MRP2 at concentrations of either 500 or 1000 ␮M MTX (Fig. 4B). Login to comment
116 In addition, we observed that [3 H]E217betaG transport by the MRP2 mutants W1254A, W1254C, W1254F, and W1254Y was enhanced 9-, 6-, 9-, and 10-fold, respectively, by sulfinpyrazone compared with 4-fold for wild-type MRP2. Login to comment
129 Membrane vesicles prepared from HEK293T cells expressing wild-type (WT-MRP2) and mutant W1254F-MRP2 were incubated with [3 H]E217betaG in the absence (open bars) and presence (solid and shaded bars) of 1 ␮M LTC4 for 5 min as described under "Experimental Procedures." Login to comment
133 B, membrane vesicles (8 ␮g of protein) from cells expressing WT-MRP2 and mutants W1254F-MRP2 and W1254Y-MRP2 were incubated at 37 °C with 400 nM [3 H]E217betaG in transport buffer and other components for 5 min in the absence (open bars) or the presence of MTX (500 ␮M, shaded bar; 1000 ␮M, solid bar) as described under "Experimental Procedures." Login to comment
139 However, LTC4 transport by MRP2 was also markedly reduced when Trp1254 was replaced with Phe but less so when it was replaced with Tyr. Login to comment
141 The selective loss of LTC4 transport activity of the W1254F-MRP2 mutant was matched by the inability of the cysteinyl leukotriene to inhibit [3 H]E217betaG transport by this mutant, suggesting that loss of LTC4 transport is caused by a loss of binding of this substrate to the protein. Login to comment
151 However, although [3 H]E217betaG transport by the MRP2- W1254F and MRP2-W1254Y mutants appeared unchanged despite their inability to transport MTX, transport of the conjugated estrogen was no longer inhibited by the antifolate agent. Login to comment
156 Of the three MRP2 substrates examined in the present study, none were transported by the nonconservatively substituted W1254C and W1254A mutants, one was transported by the W1254F mutant, and two were transported by the W1254Y mutant. Login to comment
159 Effect of sulfinpyrazone on [3 H]E217betaG uptake and [3 H]MTX uptake by wild-type and Trp1254 mutant MRP2 proteins. A, [3 H]E217betaG uptake was measured in the absence (open bars) and the presence (solid and shaded bars) of sulfinpyrazone (100 ␮M) in membrane vesicles prepared from cells expressing wild-type (WT-MRP2) and mutant (W1254A, W1254C, W1254F, and W1254Y) MRP2 proteins as described under "Experimental Procedures." Login to comment