ABCMdb

ABCC1 p.Trp1246Phe

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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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
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
133 E217betaG transport by the W1246Y and W1246F mutants was also extremely low (ϳ10% of wild-type MRP1) (Fig. 3E). 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

[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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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

[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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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

[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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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

[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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29 In contrast, substitution of TM17-Trp1246 with Phe, Tyr, Ala, or Cys selectively eliminates E217betaG and NNAL-O-glucuronide transport and drug resistance but has little or no effect on LTC4 and GSH transport (Ito et al., 2001a; Leslie et al., 2001a). 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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No. Sentence Comment
763 In contrast, substitution of Trp1246 with Phe, Tyr, Ala, or Cys selectively eliminated E217 G and NNAL-O-glucuronide transport and drug resistance but showed little or no effect on LTC4 and GSH transport [166, 341, 371]. Login to comment