ABCMdb

ABCC1 p.Phe594Ala

Predicted by SNAP2:	A: D (59%), C: D (63%), D: D (85%), E: D (80%), G: D (75%), H: D (75%), I: D (53%), K: D (80%), L: D (59%), M: N (66%), N: D (63%), P: D (85%), Q: D (66%), R: D (75%), S: D (71%), T: D (59%), V: D (71%), W: D (85%), Y: N (53%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: 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, W: D, Y: N,

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[hide] Molecular modeling correctly predicts the function... J Biol Chem. 2004 Jan 2;279(1):463-8. Epub 2003 Oct 15. Campbell JD, Koike K, Moreau C, Sansom MS, Deeley RG, Cole SP
Molecular modeling correctly predicts the functional importance of Phe594 in transmembrane helix 11 of the multidrug resistance protein, MRP1 (ABCC1).
J Biol Chem. 2004 Jan 2;279(1):463-8. Epub 2003 Oct 15., 2004-01-02 [PMID:14561746]

Abstract [show]
The human ATP-binding cassette (ABC) transporter, multidrug resistance protein 1 (MRP1/ABCC1), confers resistance to a broad range of anti-cancer agents and transports a variety of organic anions. At present, essentially no structural data exists for MRP1 that might be used to elucidate its mechanism of transport. Consequently, we have applied a modeling strategy incorporating crystal and indirect structural data from other ABC transporters to construct a model of the transmembrane domains of the core region of MRP1 that includes the amino acid side chains. Three conserved Trp residues and one non-conserved Tyr residue, shown previously to be of functional importance (Koike, K., Oleschuk, C. J., Haimeur, A., Olsen, S. L., Deeley, R. G., and Cole, S. P. C. (2002) J. Biol. Chem. 277, 49495-49503), were found to line the "pore" in our model proximal to the membrane cytosol interface. A fifth aromatic residue (Phe594) was identified that, with the Trp and Tyr residues, completed a ring or "basket" of aromatic amino acids and, accordingly, we postulated that it would also be of functional importance. To test this idea, MRP1-Phe594 mutants were expressed in human embryonic kidney cells, and their properties were examined using membrane vesicles. Substitution of Phe594 with Ala substantially reduced or eliminated the transport of five organic anion substrates by MRP1 and abrogated the binding of leukotriene C4. On the other hand, the conservatively substituted F594W and F594Y mutants remained transport competent, although significant substrate- and substitution-specific changes were observed. These studies provide some structural insight into a possible substrate binding/transport site of MRP1 at the beginning of a putative substrate translocation pathway and demonstrate the usefulness of modeling for directing structure-function analyses of this transporter.

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No. Sentence Comment
6 Substitution of Phe594 with Ala substantially reduced or eliminated the transport of five organic anion substrates by MRP1 and abrogated the binding of leukotriene C4. Login to comment
51 To test this hypothesis, we used site-directed mutagenesis to replace MRP1-Phe594 with Ala, Trp, and Tyr and examined the transport and organic anion binding properties of these mutants. Login to comment
69 Phe594 substitutions were generated in the pBluescriptSK(ϩ) plasmid above according to the manufacturer`s instructions with the following mutagenic primers (substituted nucleotides are underlined): F594A, 5Ј-G TTC AAC ATC CTC CGG GCT CCC CTG AAC ATT CTC C-3Ј; F594W, 5Ј-C TTG TTC AAC ATC CTC CGC TGG CCC CTG AAC ATT CTC CCC-3Ј; and F594Y, 5Ј-G TTC AAC ATC CTC CGC TAT CCC CTG AAC ATT CTC C-3Ј. Login to comment
99 LTC4 Transport and Photo-labeling Is Eliminated by Ala Substitution of Phe594 -As shown in Fig. 2A, all three mutants generated (F594A, F594W, and F594Y) were expressed at levels 60-100% of those of wild-type MRP1 in transfected HEK cells. Login to comment
102 After correcting for differences in MRP1 protein expression levels, ATP-dependent LTC4 uptake by the F594A mutant was reduced by more than 90%, whereas uptake by the F594W and F594Y mutants was comparable with that by wild-type MRP1. Login to comment
103 To further investigate the loss of LTC4 transport by the F594A mutant, protein-labeling experiments were carried out with this intrinsically photoactivatable arachidonic acid derivative. Login to comment
111 A, representative immunoblot of membrane vesicles prepared from HEK293T cells transfected with empty vector (pcDNA3.1(-)), wild-type (WT-MRP1), and mutant (F594A, F594W, and F594Y) MRP1 cDNAs. Login to comment
121 In contrast, no such band is detectable in photolabeled vesicles from cells expressing comparable levels of the F594A mutant. Login to comment
122 This indicates that this non-conservative substitution of Phe594 abrogates photolabeling by LTC4 and, hence, binding of this substrate to MRP1, a finding that is consistent with the complete loss of LTC4 transport activity by the F594A mutant. Login to comment
162 Thus, although the aromatic properties of the residue at position 594 are critical for retaining overall activity (i.e. F594 (wild-type), F594W, and F594Y are active but F594A is not), the addition of hydrogen bonding capacity to the amino acid side chain (as in F594W and F594Y) also influences substrate specificity, as shown previously for the polar aromatic residues at positions 553, 1198, 1243, and 1246. Login to comment

[hide] Transmembrane helix 11 of multidrug resistance pro... Biochemistry. 2004 Jul 27;43(29):9413-25. Zhang DW, Nunoya K, Vasa M, Gu HM, Theis A, Cole SP, Deeley RG
Transmembrane helix 11 of multidrug resistance protein 1 (MRP1/ABCC1): identification of polar amino acids important for substrate specificity and binding of ATP at nucleotide binding domain 1.
Biochemistry. 2004 Jul 27;43(29):9413-25., 2004-07-27 [PMID:15260484]

Abstract [show]
Human multidrug resistance protein 1 (MRP1) is an ATP binding cassette (ABC) transporter that confers resistance to many natural product chemotherapeutic agents and can transport structurally diverse conjugated organic anions. MRP1 has three polytopic transmembrane domains (TMDs) and a total of 17 TM helices. Photolabeling and mutagenesis studies of MRP1 indicate that TM11, the last helix in the second TMD, may form part of the protein's substrate binding pocket. We have demonstrated that certain polar residues within a number of TM helices, including Arg(593) in TM11, are determinants of MRP1 substrate specificity or overall activity. We have now extended these analyses to assess the functional consequences of mutating the remaining seven polar residues within and near TM11. Mutations Q580A, T581A, and S585A in the predicted outer leaflet region of the helix had no detectable effect on function, while mutation of three residues close to the membrane/cytoplasm interface altered substrate specificity. Two of these mutations affected only drug resistance. N597A increased and decreased resistance to vincristine and VP-16, respectively, while S605A decreased resistance to vincristine, VP-16 and doxorubicin. The third, S604A, selectively increased 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) transport. In contrast, elimination of the polar character of the residue at position 590 (Asn in the wild-type protein) uniformly impaired the ability of MRP1 to transport potential physiological substrates and to confer resistance to three different classes of natural product drugs. Kinetic and photolabeling studies revealed that mutation N590A not only decreased the affinity of MRP1 for cysteinyl leukotriene 4 (LTC(4)) but also substantially reduced the binding of ATP to nucleotide binding domain 1 (NBD1). Thus, polar interactions involving residues in TM11 influence not only the substrate specificity of MRP1 but also an early step in the proposed catalytic cycle of the protein.

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Sentences [show]
No. Sentence Comment
136 Nonconservative mutation of Phe594 to Ala resulted in decreased transport of all substrates tested and loss of photolabeling with LTC4. Login to comment
138 Like the F594A mutation, nonconservative substitution of the two adjacent residues Arg593 and Pro595 also decreased transport of all organic anion substrates tested (35, 49). Login to comment

[hide] Substrate recognition and transport by multidrug r... FEBS Lett. 2006 Feb 13;580(4):1103-11. Epub 2005 Dec 21. Deeley RG, Cole SP
Substrate recognition and transport by multidrug resistance protein 1 (ABCC1).
FEBS Lett. 2006 Feb 13;580(4):1103-11. Epub 2005 Dec 21., 2006-02-13 [PMID:16387301]

Abstract [show]
Multidrug resistance protein (MRP) 1 belongs to the 'C' branch of the ABC transporter superfamily. MRP1 is a high-affinity transporter of the cysteinyl leukotriene C(4) and is responsible for the systemic release of this cytokine in response to an inflammatory stimulus. However, the substrate specificity of MRP1 is extremely broad and includes many organic anion conjugates of structurally unrelated endo- and xenobiotics. In addition, MRP1 transports unmodified hydrophobic compounds, such as natural product type chemotherapeutic agents and mutagens, such as aflatoxin B(1). Transport of several of these compounds has been shown to be dependent on the presence of reduced glutathione (GSH). More recently, GSH has also been shown to stimulate the transport of some conjugated compounds, including sulfates and glucuronides. Here, we summarize current knowledge of the substrate specificity and modes of transport of MRP1 and discuss how the protein may recognize its structurally diverse substrates.

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No. Sentence Comment
147 On the other hand, while alanine substitution of Phe594 eliminates LTC4 binding and overall transport activity, conservative mutations have differential effects on substrate specificity suggesting that this residue may interact directly with substrate [61]. Login to comment

[hide] Insight in eukaryotic ABC transporter function by ... FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19. Frelet A, Klein M
Insight in eukaryotic ABC transporter function by mutation analysis.
FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19., 2006-02-13 [PMID:16442101]

Abstract [show]
With regard to structure-function relations of ATP-binding cassette (ABC) transporters several intriguing questions are in the spotlight of active research: Why do functional ABC transporters possess two ATP binding and hydrolysis domains together with two ABC signatures and to what extent are the individual nucleotide-binding domains independent or interacting? Where is the substrate-binding site and how is ATP hydrolysis functionally coupled to the transport process itself? Although much progress has been made in the elucidation of the three-dimensional structures of ABC transporters in the last years by several crystallographic studies including novel models for the nucleotide hydrolysis and translocation catalysis, site-directed mutagenesis as well as the identification of natural mutations is still a major tool to evaluate effects of individual amino acids on the overall function of ABC transporters. Apart from alterations in characteristic sequence such as Walker A, Walker B and the ABC signature other parts of ABC proteins were subject to detailed mutagenesis studies including the substrate-binding site or the regulatory domain of CFTR. In this review, we will give a detailed overview of the mutation analysis reported for selected ABC transporters of the ABCB and ABCC subfamilies, namely HsCFTR/ABCC7, HsSUR/ABCC8,9, HsMRP1/ABCC1, HsMRP2/ABCC2, ScYCF1 and P-glycoprotein (Pgp)/MDR1/ABCB1 and their effects on the function of each protein.

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No. Sentence Comment
465 F594A or substitution in P595 resulted in a global loss of organic anion transport and LTC4 binding [229,232]. 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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Sentences [show]
No. Sentence Comment
910 Indeed, mutation of Phe594 to Ala drastically reduced transport of four different substrates tested and eliminated photolabeling by LTC4 (52). Login to comment
909 Indeed, mutation of Phe594 to Ala drastically reduced transport of four different substrates tested and eliminated photolabeling by LTC4 (52). Login to comment
911 Indeed, mutation of Phe594 to Ala drastically reduced transport of four different substrates tested and eliminated photolabeling by LTC4 (52). Login to comment

[hide] Transport of glutathione and glutathione conjugate... Trends Pharmacol Sci. 2006 Aug;27(8):438-46. Epub 2006 Jul 3. Cole SP, Deeley RG
Transport of glutathione and glutathione conjugates by MRP1.
Trends Pharmacol Sci. 2006 Aug;27(8):438-46. Epub 2006 Jul 3., [PMID:16820223]

Abstract [show]
Glutathione (GSH)-conjugated xenobiotics and GSH-conjugated metabolites (e.g. the cysteinyl leukotriene C4) must be exported from the cells in which they are formed before they can be eliminated from the body or act on their cellular targets. This efflux is often mediated by the multidrug resistance protein 1 (MRP1) transporter, which also confers drug resistance to tumour cells and can protect normal cells from toxic insults. In addition to drugs and GSH conjugates, MRP1 exports GSH and GSH disulfide, and might thus have a role in cellular responses to oxidative stress. The transport of several drugs and conjugated organic anions by MRP1 requires the presence of GSH, but it is not well understood how GSH (and its analogues) enhances transport. Site-directed mutagenesis studies and biophysical analyses have provided important insights into the structural determinants of MRP1 that influence GSH and GSH conjugate binding and transport.

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No. Sentence Comment
86 Accordingly, it was correctly predicted that an Ala substitution of Phe594 would adversely affect MRP1 function [38]. 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
112 Many mutations in TM11, such as N590A, F594A, N597A, S604A and S605A, also modulate the drug resistance profile of MRP1 [79, 80]. 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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Sentences [show]
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