ABCMdb

ABCC1 p.Trp1246Ala

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Publications

PMID: 11278867 [PubMed] Ito K et al: "Mutation of a single conserved tryptophan in multidrug resistance protein 1 (MRP1/ABCC1) results in loss of drug resistance and selective loss of organic anion transport."

No. Sentence Comment

3 A similar phenotype was observed when Trp1246 was replaced with Ala, Phe, and Tyr. Login to comment
47 Mutagenesis was then performed according to the manufacturer`s instructions with the following sense mutagenic primers (substituted nucleotides are underlined): W1246C, 5Ј-CCACGTACT- TGAACTGCCTGGTTCGGATGTC-3Ј; W1246A, 5Ј-CCACGTACTTGAA- CGCGCTGGTTCGGATGTC-3Ј; W1246F, 5Ј-CCACGTACTTGAACTTC- CTGGTTCGGATGTC-3Ј; and W1246Y, 5Ј-CCACGTACTTGAACTATCT- GGTTCGGATGTC-3Ј. Login to comment
54 Mutant MRP1-GFP fusion proteins were generated by replacing the 1.3-kb BsmBI/EcoRI fragment in the pcDNA3.1(-)-MRP1K-GFP construct with the comparable fragment containing either the W1246C or W1246A mutation generated above and designated pcDNA-3.1(-)- W1246C-MRP1-GFP and pcDNA3.1(-)-W1246A-MRP1-GFP, respectively. Login to comment
128 These included substitution with a nonpolar non-aromatic amino acid (Ala; W1246A-MRP1) as well as conservative substitutions with polar (Tyr; W1246Y-MRP1) and nonpolar (Phe; W1246F-MRP1) aromatic amino acids. Login to comment
131 The LTC4 transport levels of the W1246A-MRP1 mutant (Fig. 3B) and the W1246Y-MRP1 and W1246F-MRP1 mutants (Fig. 3C) were similar to those of wild-type MRP1 and the W1246C-MRP1 mutant. Login to comment
132 In contrast, like the W1246C mutant, the W1246A mutant did not transport E217betaG (Fig. 3D). Login to comment
146 B and C, time course of [3 H]LTC4 uptake by inside-out membrane vesicles prepared from HEK293T cells expressing MRP1 mutants W1246A (Ⅺ) and W1246C (f) (B) and W1246F (Œ) and W1246Y () (C). Login to comment
149 D and E, time course of [3 H]E217betaG uptake by inside-out membrane vesicles prepared from MRP1 mutants W1246A (Ⅺ) and W1246C (f) (D) and W1246F (Œ) and W1246Y () (E). Login to comment
156 Trp1246 Mutant MRP1 Molecules Are Correctly Routed to the Plasma Membrane-To ensure that the loss of transport activity in the Trp1246 MRP1 mutants was not caused by impaired trafficking of the mutant molecules to the plasma membrane, GFP-tagged constructs of wild-type MRP1 and mutants W1246C and W1246A were generated and transiently transfected into HEK293T cells. Login to comment
181 HEK293T cells were transfected with pcDNA3.1(-)-MRP1K-GFP (A, D, and G), pcDNA3.1(-)-W1246C-MRP1-GFP (B, E, and H), and pcDNA3.1(-)-W1246A-MRP1-GFP (C, F, and I); and 48 h later, cells were processed for confocal fluorescence microscopy as described under "Experimental Procedures." Login to comment
211 Additional mutants in which Trp1246 was substituted with Ala, Phe, and Tyr displayed transport characteristics similar to those of W1246C-MRP1, indicating that replacement of the tryptophan residue rather than the introduced cysteine was responsible for the phenotype. Login to comment
241 Our recent finding that wild-type MRP1, but not W1246A-MRP1, transports the O-glucuronide of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol indicates that Trp1246 is important for the transport of other glucuronide conjugates as well.5 It has been proposed (and there is considerable evidence to support the notion) that substrates for P-glycoprotein are taken up from the inner leaflet of the plasma membrane (39). Login to comment

PMID: 11375986 [PubMed] Leslie EM et al: "Transport of the beta -O-glucuronide conjugate of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) by the multidrug resistance protein 1 (MRP1). Requirement for glutathione or a non-sulfur-containing analog."

No. Sentence Comment

61 MRP1 and MRP2 Expression Vectors and Transfections in HEK293T cells-Construction of the expression vector pcDNA3.1(-)-MRP1K encoding wild-type MRP1 (WT-MRP1) and the vector encoding MRP1 with the substitution of Trp1246 3Ala (W1246A-MRP1) have been described previously (32). Login to comment
86 [3 H]NNAL-O-glucuronide transport assays with membrane vesicles prepared from HEK293T cells transiently transfected with empty vector, wild-type MRP1, mutant W1246A-MRP1, and wild-type MRP2 cDNAs were carried out under the same conditions as described above except incubations were for 15 min. Login to comment
93 [3 H]LTC4 Transport Studies-To measure LTC4 uptake, assays were carried out at 23 °C in a 50-␮l volume containing vesicle protein prepared from T5 HeLa cells or HEK293T cells transiently transfected with empty vector, wild-type, and W1246A-MRP1 cDNAs (2 ␮g), [3 H]LTC4 (50 nM; 40 nCi), NNK or NNAL or NNAL-O-glucuronide (0, 10, 100 ␮M, respectively) and components as described above for [3 H] NNAL-O-glucuronide transport. Login to comment
145 To determine whether Trp1246 was also essential for GSH-dependent transport of NNAL-O-glucuronide, transport assays were carried out using membrane vesicles prepared from HEK293T cells transfected with pcDNA3.1(-) (empty vector), WT-MRP1, and W1246A-MRP1 cDNAs. Login to comment
146 The W1246A-MRP1 mutant and WT-MRP1 proteins were expressed at similar levels in the HEK293T cells as determined by immunoblot analysis of the membrane vesicles (Fig. 8A). Login to comment
147 Moreover, [3 H]LTC4 uptake by the W1246A-MRP1 mutant was comparable with uptake by WT-MRP1 whereas [3 H]E217betaG uptake by the mutant protein was undetectable, as reported previously (Fig. 8B) (32). Login to comment
148 In contrast, whereas the addition of GSH markedly stimulated uptake of [3 H]NNAL-O-glucuronide by WT-MRP1, uptake by the MRP1-W1246A mu- FIG. 5. Login to comment
169 As expected, [3 H]NNAL-O-glucuronide uptake in membrane vesicles from the wild-type, W1246A mutant and vector control-transfected cells in the absence of GSH was extremely low and indistinguishable from one another. Login to comment
179 GSH-dependent uptake of [3 H]NNAL-O-glucuronide by wild-type and mutant W1246A-MRP1. Login to comment
180 A, membrane vesicles prepared from HEK293T cells transfected with wild-type MRP1 (WT-MRP1), mutant MRP1 (W1246A-MRP1), or empty vector (pcDNA3.1(-)) were immunoblotted with the MRP1-specific MAb QCRL-1 as described in "Experimental Procedures." Login to comment
214 However, a commonality of at least some binding determinants is suggested by the observation that neither E217betaG nor NNAL-O-glucuronide (ϩGSH) were transported by the mutant W1246A-MRP1 (Fig. 8) (32). Login to comment
221 Consistent with this conclusion is the observation that the W1246A mutant of MRP1 retains its ability to transport LTC4 but does not transport the tobacco-derived glucuronide (Fig. 8) (32). Login to comment

PMID: 12034727 [PubMed] Mao Q et al: "GSH-dependent photolabeling of multidrug resistance protein MRP1 (ABCC1) by [125I]LY475776. Evidence of a major binding site in the COOH-proximal membrane spanning domain."

No. Sentence Comment

56 Cell Culture and Membrane Protein Preparation-The doxorubicin-selected, multidrug-resistant H69AR small cell lung cancer cell line that expresses high levels of MRP1, and the transfected HeLa cell lines that express recombinant wild-type MRP1 (T5 or WT-MRP1), and mutant MRP1 bearing substitutions of Trp1246 (W1246F-MRP1, W1246Y-MRP1, W1245C-MRP1, and W1246A-MRP1) were maintained as described previously (15, 37). Login to comment
154 After normalizing expression levels of the mutant MRP1 molecules relative to wild-type MRP1 (Fig. 8B), it was estimated that [125 I]LY475776 labeling of HeLa cell membrane proteins containing the W1246F-MRP1 mutant was ϳ23% of wild-type MRP1 membrane proteins, whereas labeling of membranes containing the W1246Y-MRP1, W1246C-MRP1, and W1246A-MRP1 mutants was less than 10% of wild-type MRP1 levels. Login to comment
163 Membrane vesicles (50 ␮g of protein) were prepared from stably transfected HeLa cells expressing wild-type (WT-MRP1) and mutant MRP1 molecules in which Trp1246 has been replaced with Ala (W1246A), Phe (W1246F), Cys (W1246C), or Tyr (W1246Y). Login to comment

PMID: 12388549 [PubMed] Koike K et al: "Multiple membrane-associated tryptophan residues contribute to the transport activity and substrate specificity of the human multidrug resistance protein, MRP1."

No. Sentence Comment

196 However, the specific environment of the two Trp residues appears to differ, since Tyr and Phe substitutions restore the transport activity of the W1198A but not W1246A mutant (39). Login to comment

PMID: 14965249 [PubMed] Haimeur A et al: "The MRP-related and BCRP/ABCG2 multidrug resistance proteins: biology, substrate specificity and regulation."

No. Sentence Comment

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

PMID: 16816140 [PubMed] Deeley RG et al: "Transmembrane transport of endo- and xenobiotics by mammalian ATP-binding cassette multidrug resistance proteins."

No. Sentence Comment

798 For example, mutation of Trp1246 to Cys, Ala, Phe, or Tyr eliminated E217betaG and NNAL-O-glucuronide transport, resistance to natural product drugs, and binding of the GSH-dependent inhibitor LY475776, but had little effect or no effect on LTC4 transport (207, 281, 327). Login to comment
797 For example, mutation of Trp1246 to Cys, Ala, Phe, or Tyr eliminated E217betaG and NNAL-O-glucuronide transport, resistance to natural product drugs, and binding of the GSH-dependent inhibitor LY475776, but had little effect or no effect on LTC4 transport (207, 281, 327). Login to comment
799 For example, mutation of Trp1246 to Cys, Ala, Phe, or Tyr eliminated E217betaG and NNAL-O-glucuronide transport, resistance to natural product drugs, and binding of the GSH-dependent inhibitor LY475776, but had little effect or no effect on LTC4 transport (207, 281, 327). Login to comment

PMID: 17257580 [PubMed] Vincent M et al: "The predicted transmembrane fragment 17 of the human multidrug resistance protein 1 (MRP1) behaves as an interfacial helix in membrane mimics."

No. Sentence Comment

32 In particular, mutations in which Trp1246 was replaced by Cys, Ala, Phe, or Tyr abolished the transport of estradiol 17-(β-D-glucuronide), an endogenous estradiol metabolite formed in the liver and excreted into bile, and prevented the drug resistance mediated by MRP1 [10]. Login to comment

PMID: 17295059 [PubMed] Chang XB et al: "A molecular understanding of ATP-dependent solute transport by multidrug resistance-associated protein MRP1."

No. Sentence Comment

117 Many mutations in TM17, such as Y1236F, T1241A, T1242A, T1242C, T1242S, T1242L, Y1243F, N1245A, W1246C, W1246A, W1246F, W1246Y, or R1249K, significantly affect MRP1 function [83-86]. Login to comment

PMID: 19949927 [PubMed] Chang XB et al: "Molecular mechanism of ATP-dependent solute transport by multidrug resistance-associated protein 1."

No. Sentence Comment

104 Mutations of C43S in TM1 (112); P343A, K332L and K332D in TM6 (113, 114); W445A and P448A in TM8 (113, 115); T550A, T556A and P557A in TM10 (113, 116); N590A, F594A, P595A, N597A, S604A and S605A in TM11 (113, 117, 118); E1089Q, E1089A, E1089L, E1089N, K1092, S1097 and N1100 in TM14 (119, 120); R1197K in TM16 (121); Y1236F, T1241A, T1242A, T1242C, T1242S, T1242L, Y1243F, N1245A, W1246C, W1246A, W1246F, W1246Y or R1249K in TM17 (121-124) significantly affect MRP1 function. Login to comment

PMID: 12388190 [PubMed] Oleschuk CJ et al: "Substitution of Trp1242 of TM17 alters substrate specificity of human multidrug resistance protein 3."

No. Sentence Comment

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

PMID: 24157718 [PubMed] Abel S et al: "Bindings of hMRP1 transmembrane peptides with dodecylphosphocholine and dodecyl-beta-d-maltoside micelles: a molecular dynamics simulation study."

No. Sentence Comment

39 Indeed, mutations of the ionizable residues (for example, R1197E, R1202(G,L) and E1204L) have impact on protein expression, substrate binding and/or transport [37], whereas the mutation of a single tryptophan W1246A in TM17 affects the estradiol 17-b2;-D-glucuronide transport [36]. Login to comment