ABCMdb

ABCC2 p.Trp1254Ala

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] The MRP-related and BCRP/ABCG2 multidrug resistanc... Curr Drug Metab. 2004 Feb;5(1):21-53. Haimeur A, Conseil G, Deeley RG, Cole SP
The MRP-related and BCRP/ABCG2 multidrug resistance proteins: biology, substrate specificity and regulation.
Curr Drug Metab. 2004 Feb;5(1):21-53., [PMID:14965249]

Abstract [show]
Several members of different families of the ATP-binding cassette (ABC) superfamily of transport proteins are capable of transporting an extraordinarily structurally diverse array of endo- and xenobiotics and their metabolites across cell membranes. Together, these transporters play an important role in the absorption, disposition and elimination of these chemicals in the body. In tumor cells, increased expression of these drug transporters is associated with resistance to multiple chemotherapeutic agents. In this review, current knowledge of the biochemical, physiological and pharmacological properties of nine members of the multidrug resistance protein (MRP)-related ABCC family (MRP1, MRP2, MRP3, MRP4, MRP5, MRP6, MRP7, ABCC11 and ABCC12) as well as the G family member, ABCG2/BCRP, are summarized. A focus is placed on the structural similarities and differences of these drug transporters as well as the molecular determinants of their substrate specificities and transport activities. Factors that regulate expression of the MRP-related proteins and ABCG2/BCRP are also reviewed.

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402 Effects of Non-conservative and Conservative Substitutions of MRP1-Trp1246, MRP2-Trp1254 and MRP3-Trp1242 on Transport of Common Substrates MRP-Related Amino Acid Substitution % Wild-type MRP Transport Activity* Transporter LTC4 E217βG MTX MRP1-Trp 1246 Ala Tyr 100 100 < 10 < 10 < 10 10 MRP2-Trp 1254 Ala Tyr < 10 30 < 10 100 14 1 MRP3-Trp 1242 Ala Tyr 70 65 250 700 20 20 * data are from References [125, 284, 324] directly involved in forming the binding site for this modulator [284]. Login to comment

[hide] Functional characterization of protein variants of... Hum Mutat. 2012 Apr;33(4):750-62. doi: 10.1002/humu.22041. Epub 2012 Feb 28. Arlanov R, Porter A, Strand D, Brough R, Karpova D, Kerb R, Wojnowski L, Schwab M, Lang T
Functional characterization of protein variants of the human multidrug transporter ABCC2 by a novel targeted expression system in fibrosarcoma cells.
Hum Mutat. 2012 Apr;33(4):750-62. doi: 10.1002/humu.22041. Epub 2012 Feb 28., [PMID:22290738]

Abstract [show]
The multidrug resistance-associated protein 2 (MRP2/ABCC2) is involved in the efflux of endogenous and xenobiotic substrates, including several anticancer and antiviral drugs. The functional consequences of ABCC2 protein variants remain inconsistent, which may be due to shortcomings of the in vitro assays used. To study systematically the functional consequences of nonsynonymous ABCC2 variants, we used a novel "Screen and Insert" (ScIn) technology to achieve stable and highly reproducible expression of 13 ABCC2 variants in HT1080 cells. Western blotting revealed lower (30-65%) ABCC2 expression for D333G, R1174H, and R1181L as compared with wild type (WT; 100%), whereas the linked variant V1188E/C1515Y resulted in higher expression (150%). R1174H caused mislocalization of ABCC2 to the cytoplasm with an endoplasmic reticulum-like distribution. Variants N1244K and R1174H decreased transport of glutathione-methylfluorescein (GS-MF) and glutathione-monochlorobimane (GS-MCB) by 80% and 50%, respectively, whereas R1181L and P1291L reduced only GS-MCB transport by 50% as compared with WT. Contrary to protein data, the double variant V1188E/C1515Y decreased specific transport activity for GS-MF and GS-MCB by 40%. The ScIn approach is a feasible and reliable method to functionally characterize systematically ABCC2 variants. D333G, R1174H, R1181L, N1244K, P1291L, and double variant V1188E/C1515Y have been identified as most promising for further clinical evaluation.

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277 Moreover, a substitution of W1254 by conserved and nonconserved amino acids (e.g., W1254A, W1254Y) in TM17 seems to be critical for substrate binding and transport activity [Ito et al., 2001b]. Login to comment

[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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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

[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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124 membrane domains conserved in organic anion Non-conservatively substituted Trp1254Ala and transporting MRP 1, 2 and 3, and examined their Trp1254Cys lost the ability to transport these three function by determining the transport of glucuronide types of substrates (Fig. 2) [112]. 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
80 Like the nonconservatively substituted MRP1-Trp1246 mutants, the W1254A-MRP2 and W1254C-MRP2 mutants either did not transport or only very poorly transported [3 H]E217betaG (Fig. 2B). 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
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
138 Aromatic interactions also appear critical for LTC4 transport activity by MRP2, again in contrast to MRP1, because transport of this conjugated organic anion was eliminated when Trp1254 was replaced with Ala and Cys. 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

[hide] Substitution of Trp1242 of TM17 alters substrate s... Am J Physiol Gastrointest Liver Physiol. 2003 Feb;284(2):G280-9. Epub 2002 Oct 9. Oleschuk CJ, Deeley RG, Cole SP
Substitution of Trp1242 of TM17 alters substrate specificity of human multidrug resistance protein 3.
Am J Physiol Gastrointest Liver Physiol. 2003 Feb;284(2):G280-9. Epub 2002 Oct 9., [PMID:12388190]

Abstract [show]
Multidrug resistance protein 3 (MRP3) is an ATP-dependent transporter of 17beta-estradiol 17beta(d-glucuronide) (E(2)17betaG), leukotriene C(4) (LTC(4)), methotrexate, and the bile salts taurocholate and glycocholate. In the present study, the role of a highly conserved Trp residue at position 1242 on MRP3 transport function was examined by expressing wild-type MRP3 and Ala-, Cys-, Phe-, Tyr-, and Pro-substituted mutants in human embryonic kidney 293T cells. Four MRP3-Trp(1242) mutants showed significantly increased E(2)17betaG uptake, whereas transport by the Pro mutant was undetectable. Similarly, the Pro mutant did not transport LTC(4). By comparison, LTC(4) transport by the Ala, Cys, Phe, and Tyr mutants was reduced by approximately 35%. The Ala, Cys, Phe, and Tyr mutants all showed greatly reduced methotrexate and leucovorin transport, except the Tyr mutant, which transported leucovorin at levels comparable with wild-type MRP3. In contrast, the MRP3-Trp(1242) substitutions did not significantly affect taurocholate transport or taurocholate and glycocholate inhibition of E(2)17betaG uptake. Thus Trp(1242) substitutions markedly alter the substrate specificity of MRP3 but leave bile salt binding and transport intact.

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193 Effects of nonconservative (Ala) and conservative (Tyr) substitutions of MRP1-Trp1246 , MRP2-Trp1254 , and MRP3-Trp1242 on transport activity of common substrates Transporter Substitution %Wild-type MRP Transport Activity LTC4 E217betaG MTX MRP1-Trp1246 Ala 100 Ͻ10 Ͻ10* Tyr 100 Ͻ10 10* MRP2-Trp1254 Ala Ͻ10 Ͻ10 14 Tyr 30 100 1 MRP3-Trp1242 Ala 70 250 20 Tyr 65 700 20 Data are from Ito et al. (17, 18) and the present study, with exceptions (*) noted (I. Letouneau, C. J. Oleschuk, R. G. Deeley, and S. P. C. Cole, unpublished observations). Login to comment