ABCMdb

ABCC1 p.Trp1246Ala

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
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

[hide] Transport of the beta -O-glucuronide conjugate of ... J Biol Chem. 2001 Jul 27;276(30):27846-54. Epub 2001 May 25. Leslie EM, Ito K, Upadhyaya P, Hecht SS, Deeley RG, Cole SP
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.
J Biol Chem. 2001 Jul 27;276(30):27846-54. Epub 2001 May 25., 2001-07-27 [PMID:11375986]

Abstract [show]
Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) play a crucial role in the induction of lung cancer, and NNAL-O-glucuronide formation and elimination are important steps in detoxification of these compounds. In the present study, we investigated the ATP-binding cassette (ABC) protein, MRP1 (ABCC1), as a candidate transporter responsible for NNAL-O-glucuronide export. MRP1 mediates the active transport of numerous GSH-, sulfate-, and glucuronide-conjugated organic anions and can transport certain xenobiotics by a mechanism that may involve co-transport with GSH. Using membrane vesicles prepared from transfected cells, we found that MRP1 transports [3H]NNAL-O-glucuronide but is dependent on the presence of GSH (Km 39 microm, Vmax 48 pmol x mg(-1) x min(-1)). We also found that the sulfur atom in GSH was dispensable because transport was supported by the GSH analog, gamma-glutamyl-alpha-aminobutyryl-glycine. Despite stimulation of NNAL-O-glucuronide transport by GSH, there was no detectable reciprocal stimulation of [3H]GSH transport. Moreover, whereas the MRP1 substrates leukotriene C4 (LTC4) and 17beta-estradiol 17beta-(d-glucuronide) (E(2)17betaG) inhibited GSH-dependent uptake of [3H]NNAL-O-glucuronide, only [3H]LTC4 transport was inhibited by NNAL-O-glucuronide (+GSH) and the kinetics of inhibition were complex. A mutant form of MRP1, which transports LTC4 but not E(2)17betaG, also did not transport NNAL-O-glucuronide suggesting a commonality in the binding elements for these two glucuronidated substrates, despite their lack of reciprocal transport inhibition. Finally, the related MRP2 transported NNAL-O-glucuronide with higher efficiency than MRP1 and unexpectedly, GSH inhibited rather than stimulated uptake. These studies provide further insight into the complex interactions of the MRP-related proteins with GSH and their conjugated organic anion substrates, and extend the range of xenotoxins transported by MRP1 and MRP2 to include metabolites of known carcinogens involved in the etiology of lung and other cancers.

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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

[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] Multiple membrane-associated tryptophan residues c... J Biol Chem. 2002 Dec 20;277(51):49495-503. Epub 2002 Oct 17. Koike K, Oleschuk CJ, Haimeur A, Olsen SL, Deeley RG, Cole SP
Multiple membrane-associated tryptophan residues contribute to the transport activity and substrate specificity of the human multidrug resistance protein, MRP1.
J Biol Chem. 2002 Dec 20;277(51):49495-503. Epub 2002 Oct 17., 2002-12-20 [PMID:12388549]

Abstract [show]
The multidrug resistance protein, MRP1, is a clinically important ATP-binding cassette transporter in which the three membrane-spanning domains (MSDs), which contain up to 17 transmembrane (TM) helices, and two nucleotide binding domains (NBDs) are configured MSD1-MSD2-NBD1-MSD3-NBD2. In tumor cells, MRP1 confers resistance to a broad spectrum of drugs, but in normal cells, it functions as a primary active transporter of organic anions such as leukotriene C(4) and 17beta-estradiol 17beta-(D-glucuronide). We have previously shown that mutation of TM17-Trp(1246) eliminates 17beta-estradiol 17beta-(D-glucuronide) transport and drug resistance conferred by MRP1 while leaving leukotriene C(4) transport intact. By mutating the 11 remaining Trp residues that are in predicted TM segments of MRP1, we have now determined that five of them are also major determinants of MRP1 function. Ala substitution of three of these residues, Trp(445) (TM8), Trp(553) (TM10), and Trp(1198) (TM16), eliminated or substantially reduced transport levels of five organic anion substrates of MRP1. In contrast, Ala substitutions of Trp(361) (TM7) and Trp(459) (TM9) caused a more moderate and substrate-selective reduction in MRP1 function. More conservative substitutions (Tyr and Phe) of the Trp(445), Trp(553), and Trp(1198) mutants resulted in substrate selective retention of transport in some cases (Trp(445) and Trp(1198)) but not others (Trp(553)). Our findings suggest that the bulky polar aromatic indole side chain of each of these five Trp residues contributes significantly to the transport activity and substrate specificity of MRP1.

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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

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

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

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

[hide] 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

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

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

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

[hide] Bindings of hMRP1 transmembrane peptides with dode... Biochim Biophys Acta. 2014 Jan;1838(1 Pt B):493-509. doi: 10.1016/j.bbamem.2013.10.012. Epub 2013 Oct 21. Abel S, Lorieau A, de Foresta B, Dupradeau FY, Marchi M
Bindings of hMRP1 transmembrane peptides with dodecylphosphocholine and dodecyl-beta-d-maltoside micelles: a molecular dynamics simulation study.
Biochim Biophys Acta. 2014 Jan;1838(1 Pt B):493-509. doi: 10.1016/j.bbamem.2013.10.012. Epub 2013 Oct 21., [PMID:24157718]

Abstract [show]
In this paper, we describe molecular dynamics simulation results of the interactions between four peptides (mTM10, mTM16, TM17 and KTM17) with micelles of dodecylphosphocholine (DPC) and dodecyl-beta-d-maltoside (DDM). These peptides represent three transmembrane fragments (TM10, 16 and 17) from the MSD1 and MSD2 membrane-spanning domains of an ABC membrane protein (hMRP1), which play roles in the protein functions. The peptide-micelle complex structures, including the tryptophan accessibility and dynamics were compared to circular dichroism and fluorescence studies obtained in water, trifluoroethanol and with micelles. Our work provides additional results not directly accessible by experiments that give further support to the fact that these peptides adopt an interfacial conformation within the micelles. We also show that the peptides are more buried in DDM than in DPC, and consequently, that they have a larger surface exposure to water in DPC than in DDM. As noted previously by simulations and experiments we have also observed formation of cation-pi bonds between the phosphocholine DPC headgroup and Trp peptide residue. Concerning the peptide secondary structures (SS), we find that in TFE their initial helical conformations are maintained during the simulation, whereas in water their initial SS are lost after few nanoseconds of simulation. An intermediate situation is observed with micelles, where the peptides remain partially folded and more structured in DDM than in DPC. Finally, our results show no sign of beta-strand structure formation as invoked by far-UV CD experiments even when three identical peptides are simulated either in water or with micelles.

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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