ABCMdb

ABCC1 p.Tyr1243Phe

Predicted by SNAP2:	A: D (53%), C: D (53%), D: D (85%), E: D (85%), F: D (59%), G: D (80%), H: D (85%), I: N (57%), K: D (85%), L: D (63%), M: D (63%), N: D (80%), P: D (91%), Q: D (85%), R: D (85%), S: D (63%), T: N (57%), V: N (53%), W: D (85%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: N,

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[hide] Determinants of the substrate specificity of multi... J Biol Chem. 2002 Jun 7;277(23):20934-41. Epub 2002 Mar 29. Zhang DW, Cole SP, Deeley RG
Determinants of the substrate specificity of multidrug resistance protein 1: role of amino acid residues with hydrogen bonding potential in predicted transmembrane helix 17.
J Biol Chem. 2002 Jun 7;277(23):20934-41. Epub 2002 Mar 29., 2002-06-07 [PMID:11925441]

Abstract [show]
Human multidrug resistance protein 1 (MRP1) confers resistance to many natural product chemotherapeutic agents and actively transports structurally diverse organic anion conjugates. We previously demonstrated that two hydrogen-bonding amino acid residues in the predicted transmembrane 17 (TM17) of MRP1, Thr(1242) and Trp(1246), were important for drug resistance and 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) transport. To determine whether other residues with hydrogen bonding potential within TM17 influence substrate specificity, we replaced Ser(1233), Ser(1235), Ser(1237), Gln(1239), Thr(1241), and Asn(1245) with Ala and Tyr(1236) and Tyr(1243) with Phe. Mutations S1233A, S1235A, S1237A, and Q1239A had no effect on any substrate tested. In contrast, mutations Y1236F and T1241A decreased resistance to vincristine but not to VP-16, doxorubicin, and epirubicin. Mutation Y1243F reduced resistance to all drugs tested by 2-3-fold. Replacement of Asn(1245) with Ala also decreased resistance to VP-16, doxorubicin, and epirubicin but increased resistance to vincristine. This mutation also decreased E(2)17betaG transport approximately 5-fold. Only mutation Y1243F altered the ability of MRP1 to transport both leukotriene 4 and E(2)17betaG. Together with our previous results, these findings suggest that residues with side chain hydrogen bonding potential, clustered in the cytoplasmic half of TM17, participate in the formation of a substrate binding site.

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No. Sentence Comment
2 To determine whether other residues with hydrogen bonding potential within TM17 influence substrate specificity, we replaced Ser1233 , Ser1235 , Ser1237 , Gln1239 , Thr1241 , and Asn1245 with Ala and Tyr1236 and Tyr1243 with Phe. Login to comment
5 Mutation Y1243F reduced resistance to all drugs tested by 2-3-fold. Login to comment
8 Only mutation Y1243F altered the ability of MRP1 to transport both leukotriene 4 and E217betaG. Login to comment
113 Mutation of one polar-aromatic residue, Y1243F, caused an approximately 2-3-fold reduction of resistance to all four drugs, whereas three mutations, Y1236F, T1241A, and N1245A, resulted in a 2-3-fold loss of resistance to only certain drugs. Login to comment
117 Subcellular Localization of Mutant and Wild Type MRP1 in Transfected HEK293 Cells-To determine whether effects of mutations Y1236F, T1241A, Y1243F, and N1245A on the drug FIG. 1. Login to comment
140 The only mutation that affected LTC4 transport was conversion of Tyr1243 to Phe, which decreased transport of LTC4 by ϳ30% (Fig. 4). Login to comment
142 However, mutations Y1243F and N1245A both decreased the levels of E217betaG transport ϳ5-6-fold (Fig. 5). Login to comment
144 Kinetic Parameters of [3 H]LTC4 and [3 H]E217betaG Transport-We have shown that mutation Y1243F affected the ability of the protein to transport both LTC4 and E217betaG and that mutation N1245A decreased the transport of only E217betaG. Login to comment
159 However, replacement of Tyr1243 with Phe decreased the normalized Vmax value for LTC4 transport ϳ30% relative to wild type MRP1 (Vmax ϭ 75 pmol/mg/min for mutation Y1243F). Login to comment
160 The apparent Km value for mutation Y1243F was 112 nM (Fig. 6 and Table II). Login to comment
161 Thus, the Y1243F mutation decreased the Vmax/Km ratio for LTC4 ϳ2-fold. Login to comment
162 For E217betaG transport, a nonlinear regression analysis of the data generated a Km value of 1.4 ␮M for wild type MRP1, consistent with previous estimates (10), compared with 5.4 and 10.9 ␮M for mutations Y1243F and N1245A, respectively (Fig. 6 and Table II). Login to comment
163 The normalized Vmax values for mutations Y1243F and N1245A were lower than that for wild type MRP1 (Vmax ϭ 403 pmol/mg/min for wild type MRP1 versus 316 pmol/ mg/min for mutation Y1243F and 288 pmol/mg/min for mutation N1245A) (Fig. 6 and Table II). Login to comment
164 Thus mutations Y1243F and N1245A decreased the Vmax/Km ratio for E217betaG ϳ5-and 11-fold, respectively. Login to comment
165 Effect of Mutations Y1243F and N1245A on the Inhibition of MRP1-mediated E217betaG/LTC4 Transport by LTC4/ E217betaG-As an alternative means of assessing the effects of the TM17 mutations on the interaction between LTC4 and the human protein, we examined the ability of LTC4 to inhibit transport of E217betaG. Login to comment
168 Converting Asn1245 to Ala reproducibly decreased the IC50 value (207 nM), whereas mutation of Tyr1243 to Phe resulted in a slight, reproducible increase (407 nM). Login to comment
170 For the wild type protein, the IC50 value for E217betaG was 3.6 ␮M compared with 11.8 ␮M for mutation Y1243F and 15.1 ␮M for mutation N1245A. Login to comment
171 Since these results are independent of protein expression levels, they provide additional evidence that the observed decrease in transport of mutations Y1243F and N1245A at nonsaturating concentrations of E217betaG is primarily attributable to changes in the affinity of the proteins for this substrate. Login to comment

[hide] Analysis of human multidrug resistance protein 1 (... Mol Pharmacol. 2005 Nov;68(5):1455-65. Epub 2005 Aug 16. Wu P, Oleschuk CJ, Mao Q, Keller BO, Deeley RG, Cole SP
Analysis of human multidrug resistance protein 1 (ABCC1) by matrix-assisted laser desorption ionization/time of flight mass spectrometry: toward identification of leukotriene C4 binding sites.
Mol Pharmacol. 2005 Nov;68(5):1455-65. Epub 2005 Aug 16., [PMID:16105987]

Abstract [show]
Multidrug resistance in tumor cells may be caused by reduced drug accumulation resulting from expression of one or more proteins belonging to the ATP-binding cassette (ABC) transporter superfamily. In addition to their drug efflux properties, certain ABC proteins such as multidrug resistance protein 1 (MRP1) (ABCC1) mediate the ATP-dependent transport of a broad array of organic anions. The intrinsically photoreactive glutathione-conjugated cysteinyl leukotriene C4 (LTC4) is a high-affinity physiological substrate of MRP1 and is widely regarded as a model compound for evaluating the substrate binding and transport properties of wild-type and mutant forms of the transporter. In the present study, we have optimized high-level expression of recombinant human MRP1 in Pichia pastoris and developed a two-step purification scheme that results in purification of the transporter to >90% homogeneity. Peptide mapping by matrix-assisted laser desorption ionization/time of flight mass spectrometry of the peptides generated by in-gel protease digestions of purified underglycosylated MRP1 identified 96.7% of the MRP1 sequence with >98% coverage of its 17 transmembrane helices. Subsequent comparisons with mass spectra of MRP1 photolabeled with LTC4 identified six candidate LTC4-modified peptide fragments that are consistent with the conclusion that the intracellular juxtamembrane positions of transmembrane helices 6, 7, 10, 17, and a COOH-proximal portion of the cytoplasmic loop that links the first and second membrane spanning domains are part of the LTC4 binding site of the transporter. Our studies confirm the usefulness of mass spectrometry for analysis of mammalian polytopic membrane proteins and for identification of substrate binding sites of human MRP1.

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No. Sentence Comment
262 Likewise, substitution of Tyr1243 with Phe causes a 70% reduction in 17beta-estradiol-D-17beta-glucuronide transport but has little effect on LTC4 transport (Zhang et al., 2002). 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
474 Other mutations either affected the transport of LTC4 and/or E217G (Y1243F) or reduced resistance to drugs (Y1236F, T1241A and Y1243F), suggesting that residues of the cytoplasmic half of TM17 with side chain hydrogen bonding potential participate in the formation of a substrate-binding site [218]. 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 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