ABCMdb

ABCC1 p.Trp1246Cys

Predicted by SNAP2:	A: D (91%), C: D (91%), D: D (95%), E: D (95%), F: D (85%), 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 (91%), Y: D (85%),
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] Mutation of a single conserved tryptophan in multi... J Biol Chem. 2001 May 11;276(19):15616-24. Epub 2001 Feb 21. Ito K, Olsen SL, Qiu W, Deeley RG, Cole SP
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.
J Biol Chem. 2001 May 11;276(19):15616-24. Epub 2001 Feb 21., 2001-05-11 [PMID:11278867]

Abstract [show]
Multidrug resistance protein 1 (MRP1/ABCC1) belongs to the ATP-binding cassette transporter superfamily and is capable of conferring resistance to a broad range of chemotherapeutic agents and transporting structurally diverse conjugated organic anions. In this study, we found that substitution of a highly conserved tryptophan at position 1246 with cysteine (W1246C-MRP1) in the putative last transmembrane segment (TM17) of MRP1 eliminated 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) transport by membrane vesicles prepared from transiently transfected human embryonic kidney cells while leaving the capacity for leukotriene C(4)- and verapamil-stimulated glutathione transport intact. In addition, in contrast to wild-type MRP1, leukotriene C(4) transport by the W1246C-MRP1 protein was no longer inhibitable by E(2)17betaG, indicating that the mutant protein had lost the ability to bind the glucuronide. A similar phenotype was observed when Trp(1246) was replaced with Ala, Phe, and Tyr. Confocal microscopy of cells expressing Trp(1246) mutant MRP1 molecules fused at the C terminus with green fluorescent protein showed that they were correctly routed to the plasma membrane. In addition to the loss of E(2)17betaG transport, HeLa cells stably transfected with W1246C-MRP1 cDNA were not resistant to the Vinca alkaloid vincristine and accumulated levels of [(3)H]vincristine comparable to those in vector control-transfected cells. Cells expressing W1246C-MRP1 were also not resistant to cationic anthracyclines (doxorubicin, daunorubicin) or the electroneutral epipodophyllotoxin VP-16. In contrast, resistance to sodium arsenite was only partially diminished, and resistance to potassium antimony tartrate remained comparable to that of cells expressing wild-type MRP1. This suggests that the structural determinants required for transport of heavy metal oxyanions differ from those for chemotherapeutic agents. Our results provide the first example of a tryptophan residue being so critically important for substrate specificity in a eukaryotic ATP-binding cassette transporter.

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1 In this study, we found that substitution of a highly conserved tryptophan at position 1246 with cysteine (W1246C-MRP1) in the putative last transmembrane segment (TM17) of MRP1 eliminated 17beta-estradiol 17-(beta-D-glucuronide) (E217betaG) transport by membrane vesicles prepared from transiently transfected human embryonic kidney cells while leaving the capacity for leukotriene C4- and verapamil-stimulated glutathione transport intact. Login to comment
2 In addition, in contrast to wild-type MRP1, leukotriene C4 transport by the W1246C-MRP1 protein was no longer inhibitable by E217betaG, indicating that the mutant protein had lost the ability to bind the glucuronide. Login to comment
5 In addition to the loss of E217betaG transport, HeLa cells stably transfected with W1246C-MRP1 cDNA were not resistant to the Vinca alkaloid vincristine and accumulated levels of [3 H]vincristine comparable to those in vector control-transfected cells. Login to comment
6 Cells expressing W1246C-MRP1 were also not resistant to cationic anthracyclines (doxorubicin, daunorubicin) or the electroneutral epipodophyllotoxin VP-16. 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
106 The DNA fragments obtained were of the size expected, and when sequenced, it was discovered that, in addition to the P196A mutation, the transfected copies of MRP1 cDNA in the first cell mutant line with an altered phenotype contained a G 3 C nucleotide mutation, resulting in substitution of cysteine for tryptophan at position 1246. Login to comment
108 A W1246C Substitution Causes Complete Loss of E217betaG Transport Activity in Membrane Vesicles from Transiently Transfected HEK293T Cells-As a first step to confirming the importance of Trp1246 , this amino acid was substituted with cysteine in pcDNA3.1(-)-MRP1K by site-directed mutagenesis, and the resulting construct was then transiently transfected into HEK293T cells. Login to comment
111 For the experiments shown in Fig. 2, the levels of W1246C-MRP1 in the transient transfectants were ϳ70% those of wild-type MRP1 (Fig. 2A). Login to comment
112 A time course of LTC4 uptake was performed, and the levels of uptake by the W1246C-MRP1 mutant relative to wild-type MRP1 were2 K. Ito, R. G. Deeley, and S. P. C. Cole, unpublished observations. FIG. 1. Login to comment
122 Subsequent kinetic analyses of LTC4 transport by the W1246C-MRP1 mutant indicated that its affinity for this substrate was reduced compared with wild-type MRP1 (Km ϭ 189 nM versus 79 nM), but the Vmax values for the two proteins were similar (40.6 versus 35.1 pmol/mg/min).3 These results indicate that substitution of Trp1246 with Cys reduces the affinity of MRP1 for this substrate, but that the transport capacities of the mutant and wild-type proteins are comparable. Login to comment
123 In contrast, uptake of E217betaG by the W1246C-MRP1 mutant was dramatically reduced and was comparable to that of the vector-transfected controls (Fig. 2C). Login to comment
125 Thus, the ability of E217betaG to inhibit LTC4 transport by the W1246C-MRP1 mutant was examined, and the results are shown in Fig. 2D. E217betaG (25 ␮M) inhibited LTC4 transport by wild-type MRP1 by 54%, as expected. Login to comment
126 In contrast, E217betaG had no effect on LTC4 transport by W1246C-MRP1, indicating that the loss of E217betaG transport by the mutant protein is associated with a loss of binding of this substrate. 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
136 Transport activity of wild-type MRP1 and mutant W1246C-MRP1 in transiently transfected HEK293T cells. Login to comment
139 B, shown is the time course of LTC4 uptake in membrane vesicles prepared from HEK293T cells transiently transfected with wild-type MRP1 (q), mutant W1246C-MRP1 (f), and empty control (E) cDNA expression vectors. Membrane vesicles were incubated at 23 °C with 50 nM [3 H]LTC4 in transport buffer for the times indicated. Results shown are means Ϯ S.D. of triplicate determinations in a single experiment. Similar results were found in three additional independent experiments. Login to comment
140 C, shown is the time course of E217betaG uptake in membrane vesicles prepared from HEK293T cells transiently transfected with wild-type MRP1 (q), mutant W1246C-MRP1 (f), and control empty (E) cDNA expression vectors. Membrane vesicles were incubated at 37 °C with 400 nM [3 H]E217betaG in transport buffer for the times indicated. Results shown are means Ϯ S.D. of triplicate determinations in a single experiment. Similar results were found in three additional independent experiments. Login to comment
141 D, membrane vesicles prepared from wild-type MRP1and W1246C-MRP1-transfected cells (A) were incubated for 2 min with [3 H]LTC4 at 23 °C in the absence (white bars) and presence (black bars) of 25 ␮M E217betaG. Login to comment
144 A, immunoblots of membrane vesicles prepared from HEK293T cells transiently transfected with pcDNA3.1(-)-MRP1K (wild-type MRP1 (WT-MRP1)), pcDNA3.1(-)-W1236A-MRP1, pcDNA3.1(-)-W1246C-MRP1, pcDNA3.1(-)-W1246F-MRP1, pcDNA3.1(-)-W1246Y-MRP1, and pcDNA3.1(-) alone as a control. 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
159 To examine whether Trp1246 plays a role in conferring drug resistance, stably transfected cell lines were established by transfection of HeLa cells with pcDNA3.1(-)-MRP1K and pcDNA3.1(-)-W1246C-MRP1. Login to comment
163 In contrast, the IC50 of the W1246C-MRP1 mutant for vincristine was similar to that of the vector control-transfected cell line. Login to comment
164 When accumulation of [3 H]vincristine was measured, steady-state concentrations of the drug in the W1246C-MRP1-transfected cells were comparable to those in vector control-transfected cells (33.83 Ϯ 1.94 versus 34.25 Ϯ 2.24 pmol/106 cells/h) (Fig. 6B). Login to comment
166 The drug resistance phenotype of the W1246C-MRP1-transfected cells was further characterized by determining the sensitivity of these cells to the cationic anthracyclines doxorubicin and daunorubicin as well as the electroneutral epipodophyllotoxin VP-16 (etoposide). Login to comment
167 As shown in Fig. 7, the IC50 values of the W1246C-MRP1 mutant cells for all three drugs were similar to those of the vector control-transfected cells. Login to comment
168 Thus, in addition to eliminating the ability to bind and transport the conjugated estrogen E217betaG, the W1246C substitution in MRP1 results in loss of resistance to all classes of natural product drugs tested. Login to comment
169 Resistance to Potassium Antimony Tartrate Is Retained, but Resistance to Sodium Arsenite Is Reduced, in W1246C-MRP1-expressing Cells-We have previously shown that in addition to conferring resistance to anticancer drugs, both human and murine MRP1 can confer low level resistance to arsenical and antimonial oxyanions (4, 28). Login to comment
170 When tested for sensitivity to potassium antimony tartrate, the IC50 of the W1246C mutant HeLa cells was comparable to that of cells expressing wild-type MRP1 (40 versus 30 ␮g/ml) (Fig. 8A). Login to comment
172 In contrast, W1246C mutant cells displayed reduced resistance to sodium arsenite (Fig. 8B). Login to comment
173 Thus, the IC50 of W1246C mutant HeLa cells for sodium arsenite was 1 ␮g/ml 4 W. Qiu, A. Haimeur, R. G. Deeley, and S. P. C. Cole, unpublished observations. FIG. 4. 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
195 A, vincristine sensitivity of HeLa cell lines stably transfected with the pcDNA3.1(-) vector (⅜), pcDNA3.1(-)-MRP1K (ⅷ), and pcDNA3.1(-)-W1246C-MRP1 (f) was determined using a tetrazolium salt-based chemosensitivity assay as described under "Experimental Procedures." Login to comment
197 B, [3 H]vincristine (VCR) accumulation in HeLa cell lines stably transfected with the pcDNA3.1(-) vector (white bar), pcDNA3.1(-)-MRP1K (wild-type MRP1 (WT-MRP1); black bar), and pcDNA3.1(-)-W1246C-MRP1 (gray bar). Login to comment
202 HeLa cell lines stably transfected with the pcDNA3.1(-) vector (⅜), pcDNA3.1(-)-MRP1K (ⅷ), and pcDNA3.1(-)-W1246C-MRP1 (f) were exposed to doxorubicin (A), daunorubicin (B), and VP-16 (C) for 72 h at 37 °C, and then cell viability was measured as described under "Experimental Procedures." Login to comment
205 In contrast, a second independently derived transfected cell line also expressing an MRP1 protein with the same P196A mutation displayed a phenotype similar to that of cells expressing wild-type MRP1.2 It was subsequently discovered that the transfected MRP1 in the first P196A mutant cell line had acquired a second non-engineered mutation causing the substitution of Cys for Trp at position 1246. Login to comment
206 Consequently, we generated a single W1246C mutant of MRP1 so that its phenotype could be compared with that of the double P196A/W1246C mutant. Login to comment
207 When transiently transfected into HEK293T cells, W1246C-MRP1 could be expressed at levels comparable to those of wild-type MRP1 and also transported LTC4 with a similar efficiency. Login to comment
209 However, as observed for the double P196A/ W1246C mutant, transport of E217betaG by W1246C-MRP1 membrane vesicles was not detectably different from that obtained with vesicles from vector control-transfected cells, and although LTC4 transport appeared unchanged in the W1246C mutants, it could no longer be inhibited by E217betaG. Login to comment
210 Taken together, these results allowed us to conclude that the altered phenotype of the P196A/W1246C-transfected cells was attributable to the single tryptophan substitution at position 1246 in MSD3 and did not require the additional proline substitution at position 196 in the cytoplasmic loop between MSD1 and MSD2. 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
212 To examine the drug resistance phenotype of the W1246C mutation, stably transfected HeLa cells were generated. Login to comment
213 In addition to the selective loss of E217betaG transport, we found that W1246C-MRP1-expressing cells were no longer resistant to natural product chemotherapeutic agents, including the electroneutral VP-16 and the cationic vincristine and anthracyclines. Login to comment
214 Consistent with this loss of drug resistance, vincristine accumulation in intact cells expressing W1246C-MRP1 was comparable to that in vector control-transfected cells, which was ϳ2-fold higher than vincristine accumulation in HeLa cells expressing wild-type MRP1. Login to comment
215 On the other hand, the W1246C-MRP1-expressing cells were still resistant to antimony tartrate and partially resistant to sodium arsenite. Login to comment
230 HeLa cell lines stably transfected with the pcDNA3.1(-) vector (⅜), pcDNA3.1(-)-MRP1K (q), and pcDNA3.1(-)- W1246C-MRP1 (f) were exposed to potassium antimony tartrate (A) or sodium arsenite (B) for 72 h at 37 °C, and then cell viability was measured as described under "Experimental Procedures." Login to comment
234 Thus, the absence of E217betaG transport activity and drug resistance in the W1246C-MRP1 mutant can be explained by the fact that although cysteine can participate in hydrogen bonding, it is considerably smaller than tryptophan and lacks aromaticity. Login to comment

[hide] GSH-dependent photolabeling of multidrug resistanc... J Biol Chem. 2002 Aug 9;277(32):28690-9. Epub 2002 May 28. Mao Q, Qiu W, Weigl KE, Lander PA, Tabas LB, Shepard RL, Dantzig AH, Deeley RG, Cole SP
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.
J Biol Chem. 2002 Aug 9;277(32):28690-9. Epub 2002 May 28., 2002-08-09 [PMID:12034727]

Abstract [show]
Substrates transported by the 190-kDa multidrug resistance protein 1 (MRP1) (ABCC1) include endogenous organic anions such as the cysteinyl leukotriene C(4). In addition, MRP1 confers resistance against various anticancer drugs by reducing intracellular accumulation by co-export of drug with reduced GSH. We have examined the properties of LY475776, an intrinsically photoactivable MRP1-specific tricyclic isoxazole modulator that inhibits leukotriene C(4) transport by this protein in a GSH-dependent manner. We show that [125I]LY475776 photolabeling of MRP1 requires GSH but is also supported by several non-reducing GSH derivatives and peptide analogs. Limited proteolysis revealed that [(125)I]LY475776 labeling was confined to the 75-kDa COOH-proximal half of MRP1. More extensive proteolysis generated two major 125I-labeled fragments of approximately 56 and approximately 41 kDa, and immunoblotting with regionally directed antibodies showed that these fragments correspond to amino acids approximately 1045-1531 and approximately 1150-1531, respectively. However, an approximately 33-kDa COOH-terminal immunoreactive fragment was not labeled, inferring that the major [125I]LY475776-labeling site resides approximately between amino acids 1150-1250. This region encompasses transmembrane (TM) segments 16 and 17 at the COOH-proximal end of the third membrane spanning domain of the protein. [125I]LY475776 labeling of mutant MRP1 molecules with substitutions of Trp(1246) in TM17 were reduced >80% compared with wild-type MRP1, confirming that TM17 is important for LY475776 binding. Finally, vanadate-induced trapping of ADP inhibited [125I]LY475776 labeling, suggesting that ATP hydrolysis causes a conformational change in MRP1 that reduces the affinity of the protein for this inhibitor.

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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

[hide] Functional studies on the MRP1 multidrug transport... Anticancer Res. 2004 Mar-Apr;24(2A):449-55. Szentpetery Z, Sarkadi B, Bakos E, Varadi A
Functional studies on the MRP1 multidrug transporter: characterization of ABC-signature mutant variants.
Anticancer Res. 2004 Mar-Apr;24(2A):449-55., [PMID:15152943]

Abstract [show]
BACKGROUND: MRP1 is a key multidrug resistance ATP-binding Cassette (ABC) transporter in tumor cells. A functionally important signature motif is conserved within all ABC domains. Our current studies aimed to elucidate the role of these motifs in the cooperation of MRP1 ABC domains. MATERIALS AND METHODS: We designed human MRP1 mutants based on a bacterial ABC structure. Conserved leucines (Leu) were replaced by arginines (Arg), while glycines (Gly) were substituted for aspartic acids (Asp). The activity of these mutants was assayed by measuring ATPase activity and vesicular transport. ATP-binding and transition-state formation were studied by a photoreactive ATP analog. RESULTS: The Leu to Arg mutants retained both ATPase and transport activity, while the Gly to Asp mutants were inactive in all functional assays, while showing normal ATP-binding. CONCLUSION: Our results reinforce the notion that a single mutation in one of the ABC-signature regions affects the function of the whole protein. The relative role of the conservative leucines and glycines in MRP1 indicates a similar three-dimensional structure within the catalytic center of various ABC proteins.

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35 Substitution of a highly conserved tryptophan at position 1246 with cysteine in TM17 was also shown to modulate the substrate specificity of MRP1 (21). Login to comment

[hide] Transmembrane transport of endo- and xenobiotics b... Physiol Rev. 2006 Jul;86(3):849-99. Deeley RG, Westlake C, Cole SP
Transmembrane transport of endo- and xenobiotics by mammalian ATP-binding cassette multidrug resistance proteins.
Physiol Rev. 2006 Jul;86(3):849-99., [PMID:16816140]

Abstract [show]
Multidrug Resistance Proteins (MRPs), together with the cystic fibrosis conductance regulator (CFTR/ABCC7) and the sulfonylurea receptors (SUR1/ABCC8 and SUR2/ABCC9) comprise the 13 members of the human "C" branch of the ATP binding cassette (ABC) superfamily. All C branch proteins share conserved structural features in their nucleotide binding domains (NBDs) that distinguish them from other ABC proteins. The MRPs can be further divided into two subfamilies "long" (MRP1, -2, -3, -6, and -7) and "short" (MRP4, -5, -8, -9, and -10). The short MRPs have a typical ABC transporter structure with two polytropic membrane spanning domains (MSDs) and two NBDs, while the long MRPs have an additional NH2-terminal MSD. In vitro, the MRPs can collectively confer resistance to natural product drugs and their conjugated metabolites, platinum compounds, folate antimetabolites, nucleoside and nucleotide analogs, arsenical and antimonial oxyanions, peptide-based agents, and, under certain circumstances, alkylating agents. The MRPs are also primary active transporters of other structurally diverse compounds, including glutathione, glucuronide, and sulfate conjugates of a large number of xeno- and endobiotics. In vivo, several MRPs are major contributors to the distribution and elimination of a wide range of both anticancer and non-anticancer drugs and metabolites. In this review, we describe what is known of the structure of the MRPs and the mechanisms by which they recognize and transport their diverse substrates. We also summarize knowledge of their possible physiological functions and evidence that they may be involved in the clinical drug resistance of various forms of cancer.

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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

[hide] The predicted transmembrane fragment 17 of the hum... Biochim Biophys Acta. 2007 Mar;1768(3):538-52. Epub 2006 Dec 16. Vincent M, Gallay J, Jamin N, Garrigos M, de Foresta B
The predicted transmembrane fragment 17 of the human multidrug resistance protein 1 (MRP1) behaves as an interfacial helix in membrane mimics.
Biochim Biophys Acta. 2007 Mar;1768(3):538-52. Epub 2006 Dec 16., [PMID:17257580]

Abstract [show]
The human multidrug resistance protein MRP1 (or ABCC1) is one of the most important members of the large ABC transporter family, in terms of both its biological (tissue defense) and pharmacological functions. Many studies have investigated the function of MRP1, but structural data remain scarce for this protein. We investigated the structure and dynamics of predicted transmembrane fragment 17 (TM17, from Ala(1227) to Ser(1251)), which contains a single Trp residue (W(1246)) involved in MRP1 substrate specificity and transport function. We synthesized TM17 and a modified peptide in which Ala(1227) was replaced by a charged Lys residue. Both peptides were readily solubilized in dodecylmaltoside (DM) or dodecylphosphocholine (DPC) micelles, as membrane mimics. The interaction of these peptides with DM or DPC micelles was studied by steady-state and time-resolved Trp fluorescence spectroscopy, including experiments in which Trp was quenched by acrylamide or by two brominated analogs of DM. The secondary structure of these peptides was determined by circular dichroism. Overall, the results obtained indicated significant structuring ( approximately 50% alpha-helix) of TM17 in the presence of either DM or DPC micelles as compared to buffer. A main interfacial location of TM17 is proposed, based on significant accessibility of Trp(1246) to brominated alkyl chains of DM and/or acrylamide. The comparison of various fluorescence parameters including lambda(max), lifetime distributions and Trp rotational mobility with those determined for model fluorescent transmembrane helices in the same detergents is also consistent with the interfacial location of TM17. We therefore suggest that TM17 intrinsic properties may be insufficient for its transmembrane insertion as proposed by the MRP1 consensus topological model. This insertion may also be controlled by additional constraints such as interactions with other TM domains and its position in the protein sequence. The particular pattern of behavior of this predicted transmembrane peptide may be the hallmark of a fragment involved in substrate transport.

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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

[hide] A molecular understanding of ATP-dependent solute ... Cancer Metastasis Rev. 2007 Mar;26(1):15-37. Chang XB
A molecular understanding of ATP-dependent solute transport by multidrug resistance-associated protein MRP1.
Cancer Metastasis Rev. 2007 Mar;26(1):15-37., [PMID:17295059]

Abstract [show]
Over a million new cases of cancers are diagnosed each year in the United States and over half of these patients die from these devastating diseases. Thus, cancers cause a major public health problem in the United States and worldwide. Chemotherapy remains the principal mode to treat many metastatic cancers. However, occurrence of cellular multidrug resistance (MDR) prevents efficient killing of cancer cells, leading to chemotherapeutic treatment failure. Numerous mechanisms of MDR exist in cancer cells, such as intrinsic or acquired MDR. Overexpression of ATP-binding cassette (ABC) drug transporters, such as P-glycoprotein (P-gp or ABCB1), breast cancer resistance protein (BCRP or ABCG2) and/or multidrug resistance-associated protein (MRP1 or ABCC1), confers an acquired MDR due to their capabilities of transporting a broad range of chemically diverse anticancer drugs. In addition to their roles in MDR, there is substantial evidence suggesting that these drug transporters have functions in tissue defense. Basically, these drug transporters are expressed in tissues important for absorption, such as in lung and gut, and for metabolism and elimination, such as in liver and kidney. In addition, these drug transporters play an important role in maintaining the barrier function of many tissues including blood-brain barrier, blood-cerebral spinal fluid barrier, blood-testis barrier and the maternal-fetal barrier. Thus, these ATP-dependent drug transporters play an important role in the absorption, disposition and elimination of the structurally diverse array of the endobiotics and xenobiotics. In this review, the molecular mechanism of ATP-dependent solute transport by MRP1 will be addressed.

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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
157 Hela cells transfected with W1246C mutated MRP1 are not resistant to vincristine, doxorubicin, daunorubicin, or VP-16 [84]. Login to comment
158 In contrast, the resistance to sodium arsenite of Hela cells transfected with W1246C mutated MRP1 is partially diminished whereas resistance to potassium antimony tartrate remained comparable to that of cells expressing wild-type MRP1 [84]. Login to comment
160 W1246C-mutated MRP1 eliminated E217βG transport while leaving the capacity for LTC4 and verapamil-stimulated GSH transport intact [84]. Login to comment
161 In addition, in contrast to wild-type MRP1, LTC4 transport by W1246C-mutated MRP1 was no longer inhibited by E217βG, indicating that the mutant MRP1 had lost the ability to bind the E217βG whole molecule or the glucuronide portion. Login to comment

[hide] Molecular basis for reduced estrone sulfate transp... Drug Metab Dispos. 2009 Jul;37(7):1411-20. Epub 2009 Apr 27. Maeno K, Nakajima A, Conseil G, Rothnie A, Deeley RG, Cole SP
Molecular basis for reduced estrone sulfate transport and altered modulator sensitivity of transmembrane helix (TM) 6 and TM17 mutants of multidrug resistance protein 1 (ABCC1).
Drug Metab Dispos. 2009 Jul;37(7):1411-20. Epub 2009 Apr 27., [PMID:19398503]

Abstract [show]
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 Lys(332) in transmembrane helix (TM) 6 and Trp(1246) 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-Trp(1246) 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)-cyclohexy lmethyl]-benzamide (LY465803). In contrast, TM6-Lys(332) is important for enabling GSH and GSH-containing compounds to serve as substrates (e.g., leukotriene C(4)) 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 C(4), D(4), and F(4) to inhibit estradiol glucuronide transport by wild-type and K332L mutant MRP1 provide further evidence that TM6-Lys(332) is involved in the recognition of the gamma-Glu portion of substrates and modulators containing GSH or GSH-like moieties.

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3 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. Login to comment
36 B-E, ATP-dependent uptake of 3 H-labeled organic anions was measured in membrane vesicles prepared from HEK293T (HEK) cells transfected with wild-type MRP1 (shaded bar) and K332L and W1246C mutant (open bars) cDNAs as described under Materials and Methods. Login to comment
57 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). Login to comment
85 Equilibrium binding of [3 H]estrone 3-sulfate to wild-type MRP1 and the K332L and W1246C mutants was determined as described previously (Rothnie et al., 2006, 2008). Login to comment
91 To investigate the effect of estrone 3-sulfate concentration on [3 H]estrone 3-sulfate binding, 10 ␮g of membrane protein from wild-type and mutant MRP1-transfected cells were incubated with various concentrations of [3 H]estrone 3-sulfate (50 nM to 10 ␮M) and 3 mM S-MeGSH in the presence or absence of the competitive substrates E217betaG (1 mM) in the case of the K332L mutant and LTC4 (10 ␮M) in the case of the W1246C mutant. Login to comment
95 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. Login to comment
96 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). Login to comment
98 As shown in Fig. 1B, uptake of [3 H]methotrexate by the K332L mutant was comparable with wild-type MRP1, whereas uptake by W1246C was reduced by Ͼ75%. Login to comment
100 As reported previously (Ito et al., 2001a; Haimeur et al., 2002), [3 H]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). Login to comment
101 On the other hand, the K332L mutant transported [3 H]E217betaG at 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). Login to comment
102 S-MeGSH Fails to Increase Estrone 3-Sulfate Binding to the K332L and W1246C Mutants. Login to comment
103 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. Login to comment
107 In contrast, S-MeGSH had little or no effect on estrone 3-sulfate binding to the K332L (Fig. 2A) or W1246C (Fig. 2B) mutants. Login to comment
108 The EC50 for S-MeGSH of wild-type MRP1 was 1.24 Ϯ 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. Login to comment
109 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). Login to comment
110 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. Login to comment
112 K332L Decreases Ability of S-MeGSH to Stimulate Estrone 3-Sulfate Uptake, Whereas W1246C Decreases Affinity for Estrone 3-Sulfate. Login to comment
114 S-MeGSH stimulated binding of [3 H]estrone 3-sulfate by MRP1 mutants K332L and W1246C. Login to comment
117 B, binding of [3 H]estrone 3-sulfate to wild-type MRP1 (f), W1246C mutant (Œ), and control HEK (E) membranes (10 ␮g of protein) measured in the presence of S-MeGSH (0.01-18 mM) as described in A. Login to comment
119 C, specific binding of [3 H]estrone 3-sulfate to wild-type MRP1 (f), W1246C (Œ), K332L (F), and control HEK (E) membranes (10 ␮g of protein) was measured at estrone 3-sulfate concentrations ranging from 0.01 to 10 mM in the presence of 3 mM S-MeGSH. Login to comment
123 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. Login to comment
125 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. Login to comment
128 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). Login to comment
138 Effect of MRP1 Modulators on the Activity of MRP1 Mutants K332L and W1246C. Login to comment
139 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. Login to comment
141 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. Login to comment
148 In contrast to its more potent inhibitory effect on E217betaG transport by the K332L mutant, MK571 was a less potent inhibitor (ϳ3-fold) of LTC4 transport by the W1246C mutant than by wild-type MRP1 (Table 2; Fig. 6A). Login to comment
149 However, similar to K332L, the inhibitory potency of BAY u9773 for LTC4 transport by wild-type and W1246C was comparable (Table 2; Fig. 6B). Login to comment
150 Also similar to K332L, LY465803 (ϩGSH) showed a far greater (Ͼ250-fold) potency to inhibit wild-type MRP1 than the W1246C mutant (Table 2; Fig. 6C). Login to comment
151 Finally, in contrast to K332L, the sensitivity of W1246C and wild-type MRP1 to FIG. 3. Login to comment
152 [3 H]Estrone 3-sulfate and [3 H]E217betaG vesicular uptake by K332L and W1246C mutant and wild-type MRP1 proteins. Login to comment
153 A, ATP-dependent [3 H]estrone 3-sulfate uptake by membrane vesicles prepared from untransfected control cells (HEK) (solid bar) and cells transfected with cDNA vectors containing wild-type MRP1 (shaded bar), and K332L and W1246C mutant MRP1 (open bars) was measured in the absence of S-MeGSH. Login to comment
154 Inset, [3 H]estrone 3-sulfate uptake by membrane vesicles prepared from untransfected HEK cells (solid bars) and HEK cells expressing comparable levels of wild-type MRP1 (shaded bars), and MRP1 mutants K332L and W1246C (open bars) was measured in the absence and presence of S-MeGSH as described under Materials and Methods. Login to comment
162 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. Login to comment
180 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. Login to comment
194 TABLE 2 Effect of modulators on LTC4 uptake by wild-type and W1246C mutant MRP1 The values shown represent the means (ϮS.D.) of IC50 values obtained in three independent experiments. Login to comment
195 Modulator LTC4 Uptake IC50 Wild-type W1246C ␮M ␮M MK571 0.50 Ϯ 0.16 1.53 Ϯ 0.67a BAY u9773 0.4 Ϯ 0.1 0.57 Ϯ 0.35 LY465803 (ϩGSH) 0.046 Ϯ 0.007 13.2 Ϯ 9.1a S-Decyl-GSH 0.09 Ϯ 0.02 0.09 Ϯ 0.06 a Significantly different from IC50 in wild-type MRP1 (p Ͻ 0.01). Login to comment
198 However, this was not the case for membranes prepared from transfected HEK cells expressing either the K332L or W1246C mutants. Login to comment
200 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. Login to comment
202 In contrast, impaired transport by the W1246C mutant is caused by a loss of binding of the sulfated estrogen. Login to comment
207 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. Login to comment
208 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. Login to comment
211 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 ␮M versus 46 nM). Login to comment
212 This observation is consistent with our earlier finding that radiolabeling of the COOH-proximal half of the W1246C mutant by [125 I]LY475776, a photoactivateable derivative of LY465803, was substantially reduced relative to the same region of wild-type MRP1 (Mao et al., 2002). Login to comment
214 Effect of small molecule modulators and S-decyl-GSH on [3 H]LTC4 transport by wild-type and W1246C mutant MRP1. ATP-dependent vesicular uptake of [3 H]LTC4 by wild-type MRP1 (open symbols, dotted lines) and W1246C mutant MRP1 (closed symbols, solid lines) was measured in the presence of the indicated concentrations of modulators and S-decyl-GSH. Login to comment
229 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. Login to comment

[hide] Molecular mechanism of ATP-dependent solute transp... Methods Mol Biol. 2010;596:223-49. Chang XB
Molecular mechanism of ATP-dependent solute transport by multidrug resistance-associated protein 1.
Methods Mol Biol. 2010;596:223-49., [PMID:19949927]

Abstract [show]
Millions of new cancer patients are diagnosed each year and over half of these patients die from this devastating disease. Thus, cancer causes a major public health problem worldwide. Chemotherapy remains the principal mode to treat many metastatic cancers. However, occurrence of cellular multidrug resistance (MDR) prevents efficient killing of cancer cells, leading to chemotherapeutic treatment failure. Over-expression of ATP-binding cassette transporters, such as P-glycoprotein, breast cancer resistance protein and/or multidrug resistance-associated protein 1 (MRP1), confers an acquired MDR due to their capabilities of transporting a broad range of chemically diverse anticancer drugs across the cell membrane barrier. In this review, the molecular mechanism of ATP-dependent solute transport by MRP1 will be addressed.

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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

[hide] Structural and functional properties of human mult... Curr Med Chem. 2011;18(3):439-81. He SM, Li R, Kanwar JR, Zhou SF
Structural and functional properties of human multidrug resistance protein 1 (MRP1/ABCC1).
Curr Med Chem. 2011;18(3):439-81., [PMID:21143116]

Abstract [show]
Multidrug ABC transporters such as P-glycoprotein (P-gp/MDR1/ABCB1) and multidrug resistance protein 1 (MRP1/ABCC1) play an important role in the extrusion of drugs from the cell and their overexpression can be a cause of failure of anticancer and antimicrobial chemotherapy. Recently, the mouse P-gp/Abcb1a structure has been determined and this has significantly enhanced our understanding of the structure-activity relationship (SAR) of mammalian ABC transporters. This paper highlights our current knowledge on the structural and functional properties and the SAR of human MRP1/ABCC1. Although the crystal structure of MRP1/ABCC1 has yet to be resolved, the current topological model of MRP1/ABCC1 contains two transmembrane domains (TMD1 and TMD2) each followed by a nucleotide binding domain (NBD) plus a third NH2-terminal TMD0. MRP1/ABCC1 is expressed in the liver, kidney, intestine, brain and other tissues. MRP1/ABCC1 transports a structurally diverse array of important endogenous substances (e.g. leukotrienes and estrogen conjugates) and xenobiotics and their metabolites, including various conjugates, anticancer drugs, heavy metals, organic anions and lipids. Cells that highly express MRP1/ABCC1 confer resistance to a variety of natural product anticancer drugs such as vinca alkaloids (e.g. vincristine), anthracyclines (e.g. etoposide) and epipodophyllotoxins (e.g. doxorubicin and mitoxantrone). MRP1/ABCC1 is associated with tumor resistance which is often caused by an increased efflux and decreased intracellular accumulation of natural product anticancer drugs and other anticancer agents. However, most compounds that efficiently reverse P-gp/ABCB1-mediated multidrug resistance have only low affinity for MRP1/ABCC1 and there are only a few effective and relatively specific MRP1/ABCC1 inhibitors available. A number of site-directed mutagenesis studies, biophysical and photolabeling studies, SAR and QSAR, molecular docking and homology modeling studies have documented the role of multiple residues in determining the substrate specificity and inhibitor selectivity of MRP1/ABCC1. Most of these residues are located in the TMs of TMD1 and TMD2, in particular TMs 4, 6, 7, 8, 10, 11, 14, 16, and 17, or in close proximity to the membrane/cytosol interface of MRP1/ABCC1. The exact transporting mechanism of MRP1/ABCC1 is unclear. MRP1/ABCC1 and other multidrug transporters are front-line mediators of drug resistance in cancers and represent important therapeutic targets in future chemotherapy. The crystal structure of human MRP1/ABCC1 is expected to be resolved in the near future and this will provide an insight into the SAR of MRP1/ABCC1 and allow for rational design of anticancer drugs and potent and selective MRP1/ABCC1 inhibitors.

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765 Both Lys332Leu and Trp1246Cys mutants were as sensitive as wild-type MRP1/ABCC1 to MK-571, LY171883, and the potent MRP1/ABCC1 inhibitor 6-[4 -carboxyphenylthio]-5[S]- hydroxy-7[E], 11[Z]14[Z]-eicosatetrenoic acid (BAY u9773, a leukotriene-like dual antagonist that acts on both CysLT1 and CysLT2 receptors) [341]. Login to comment
768 In contrast to its more potent inhibitory effect on E217 G transport by the Lys332Leu mutant, MK-571 was a less potent inhibitor ( 3-fold) of LTC4 transport by the Trp1246Cys mutant than by wild-type MRP1. Login to comment
769 However, similar to Lys332Leu, the inhibitory potency of BAY u9773 for LTC4 transport by wild-type and Trp1246Cys was comparable. Login to comment