ABCMdb

ABCC1 p.Thr1242Ala

Predicted by SNAP2:	A: N (82%), C: N (66%), D: N (72%), E: N (66%), F: N (78%), G: N (82%), H: N (82%), I: N (72%), K: N (53%), L: N (72%), M: N (78%), N: N (87%), P: N (53%), Q: N (78%), R: N (53%), S: N (87%), V: N (72%), W: N (61%), Y: N (78%),
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, V: N, W: N, Y: N,

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[hide] Identification of a nonconserved amino acid residu... J Biol Chem. 2001 Sep 14;276(37):34966-74. Epub 2001 Jun 27. Zhang DW, Cole SP, Deeley RG
Identification of a nonconserved amino acid residue in multidrug resistance protein 1 important for determining substrate specificity: evidence for functional interaction between transmembrane helices 14 and 17.
J Biol Chem. 2001 Sep 14;276(37):34966-74. Epub 2001 Jun 27., 2001-09-14 [PMID:11429411]

Abstract [show]
Murine multidrug resistance protein 1 (mrp1), differs from its human ortholog (MRP1) in that it fails to confer anthracycline resistance and transports the MRP1 substrate, 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG), very poorly. By mutating variant residues in mrp1 to those present in MRP1, we identified Glu(1089) of MRP1 as being critical for anthracycline resistance. However, Glu(1089) mutations had no effect on E(2)17betaG transport. We have now identified a nonconserved amino acid within the highly conserved COOH-proximal transmembrane helix of MRP1/mrp1 that is important for transport of the conjugated estrogen. Converting Ala(1239) in mrp1 to Thr, as in the corresponding position (1242) in MRP1, increased E(2)17betaG transport 3-fold. Any mutation of mrp1 Ala(1239), including substitution with Thr, decreased resistance to vincristine and VP-16 without altering anthracycline resistance. However, introduction of a second murine to human mutation, Q1086E, which alone selectively increases anthracycline resistance, into mrp1A1239T restored resistance to both vincristine and VP-16. To confirm the importance of MRP1 Thr(1242) for E(2)17betaG transport and drug resistance, we mutated this residue to Ala, Cys, Ser, Leu, and Lys. These mutations decreased E(2)17betaG transport 2-fold. Conversion to Asp eliminated transport of the estrogen conjugate and also decreased leukotriene C(4) transport approximately 2-fold. The mutations also reduced the ability of MRP1 to confer resistance to all drugs tested. As with mrp1, introduction of a second mutation based on the murine sequence to create MRP1E1089Q/T1242A restored resistance to vincristine and VP-16, but not anthracyclines, without affecting transport of leukotriene C(4) and E(2)17betaG. These results demonstrate the important role of Thr(1242) for E(2)17betaG transport. They also reveal a highly specific functional relationship between nonconserved amino acids in TM helices 14 and 17 of both mrp1 and MRP1 that enables both proteins to confer similar levels of resistance to vincristine and VP-16.

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11 As with mrp1, introduction of a second mutation based on the murine sequence to create MRP1E1089Q/ T1242A restored resistance to vincristine and VP-16, but not anthracyclines, without affecting transport of leukotriene C4 and E217betaG. Login to comment
66 To generate a MRP1E1089Q/T1242A double mutation, pGEM-MRP1-(2730-4832) containing mutation MRP1E1089Q was digested with StuI and XbaI to yield a 4.1-kilobase pair fragment comprised of nucleotides 2730-3758 of MRP1E1089Q attached to the vector fragment, and this fragment was then ligated to a 1.1-kilobase pair StuI-XbaI fragment encompassing nucleotides 3758-4832 of MRP1T1242A. Login to comment
134 Thus, Thr1242 was mutated to Ala, Cys, Ser, Leu, Lys, and Asp. Login to comment
137 After normalization for differences in expression levels, substitution of Thr1242 with Ala, Cys, Ser, Leu, and Lys decreased the ability of MRP1 to transport E217betaG by more than 2-fold relative to the wild type protein with no significant effect on LTC4 FIG. 2. Time course of ATP-dependent [3 H]LTC4 and [3 H]E217betaG uptake by membrane vesicles prepared from HEK293 stable transfectants expressing wild type mrp1 or A1239 mutant proteins. Login to comment
161 Consistent with the results obtained with the mrp1 mutant, substitution of Thr1242 with Ala in MRP1 decreased the normalized Vmax value for the mutant ϳ2-fold relative to wild type MRP1. Login to comment
167 In MRP1, substitution of Thr1242 with Ala increased the IC50 value from 15 to 134 ␮M. These results are independent of protein expression levels and provide strong evidence that the increase or decrease in E217betaG transport by mrp1A1239T and MRP1T1242A, respectively, is at least partially attributable to changes in the affinity of the proteins for this substrate. Login to comment
196 Consequently, a double mutation of MRP1 was also made, in which Glu1089 was replaced with Gln and Thr1242 was substituted with Ala (E1089Q/T1242A). Login to comment
228 Replacement of Thr1242 with Ala, Ser, Cys, Leu, and Lys decreased the ability of the protein to transport E217betaG 2-3-fold without significantly affecting LTC4 transport. Login to comment
234 The values shown represent the means Ϯ S.D. of relative resistance factors determined from 3-6 independent experiments. Resistance factors normalized for differences in the levels of mutant proteins expressed in the transfectant populations used are shown in parentheses. Transfectant Drug (relative resistance factor) Vincristine VP-16 Doxorubicin Epirubicin HEKMRP1 18.6 Ϯ 3.1 (18.6) 18.9 Ϯ 2.0 (18.9) 6.7 Ϯ 0.9 (6.7) 9.3 Ϯ 0.4 (9.3) n ϭ 6 n ϭ 6 n ϭ 6 n ϭ 6 HEKMRP1T1242A 14.5 Ϯ 0.9 (10.8) 13.6 Ϯ 1.2 (10.1) 2.6 Ϯ 0.2 (2.0) 3.2 Ϯ 0.3 (2.4) n ϭ 5 n ϭ 5 n ϭ 5 n ϭ 5 HEKMRP1T1242C 9.7 Ϯ 0.3 (9.7) 10.7 Ϯ 0.3 (10.7) 3.1 Ϯ 0.2 (3.1) 3.3 Ϯ 0.2 (3.3) n ϭ 3 n ϭ 3 n ϭ 3 n ϭ 3 HEKMRP1T1242S 9.1 Ϯ 0.5 (11.4) 9.6 Ϯ 0.7 (11.9) 2.5 Ϯ 0.5 (3.1) 2.6 Ϯ 0.4 (3.2) n ϭ 3 n ϭ 3 n ϭ 3 n ϭ 3 HEKMRP1T1242L 10.7 Ϯ 3.3 (11.9) 10.4 Ϯ 1.2 (10.7) 2.8 Ϯ 0.9 (2.9) 2.9 Ϯ 3.9 (3.0) n ϭ 3 n ϭ 3 n ϭ 3 n ϭ 3 HEKMRP1T1242D 6.5 Ϯ 1.2 (9.3) 7.0 Ϯ 0.5 (10.0) 2.3 Ϯ 0.2 (2.5) 2.4 Ϯ 0.1 (2.6) n ϭ 3 n ϭ 3 n ϭ 3 n ϭ 3 HEKMRP1T1242K 5.4 Ϯ 0.3 (11.5) 4.8 Ϯ 0.3 (12.0) 1.5 Ϯ 0.2 (3.7) 2.1 Ϯ 0.5 (4.1) n ϭ 3 n ϭ 3 n ϭ 3 n ϭ 3 HEKMRP1E1089Q/T1242A 18.3 Ϯ 5.3 (26.9) 17.8 Ϯ 1.5 (26.2) Ͻ1 Ͻ1 n ϭ 5 n ϭ 5 n ϭ 3 n ϭ 3 transport. Login to comment
252 In contrast, the IC50 for LTC4 transport by MRP1 T1242A was ϳ130 ␮M, a value similar to that obtained for wild type mrp1, while the IC50 of mrp1A1239T decreased to ϳ30 ␮M. It has been proposed that the transport of anionic/cationic substrates by MRP1 is facilitated by cationic/anionic acid residues present in the transmembrane helices (39). Login to comment
264 Transfectants tested were HEKmrp1 (f), HEKmrp1A1239T (Œ), and HEKmrp1Q1086E/A1239T (q) (C and D) and HEKMRP1 (Ⅺ), HEKMRP1T1242A (‚), and HEKMRP1E1089Q/T1242A (E) (E and F). Login to comment
268 Having found previously that mutation of Glu1089 in TM14 of MRP1 to Gln not only markedly reduced that ability of the protein to confer resistance to anthracyclines but also, to a lesser extent, to vincristine and VP-16 (29), we investigated the effect of combining this mutation, and the reciprocal mutation in the murine protein, with the A1239T and T1242A mutations of mrp1 and MRP1, respectively. Login to comment

[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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136 In contrast, the T1242A mutation had no significant effect on LTC4 transport, and the effect of the Trp1246 mutations was relatively minor (a 2-fold increase in Km) (38, 39).2 To determine whether any of the other mutations in TM17 of MRP1 altered the efficiency with which the protein transported either LTC4 or E217betaG, we examined ATP-dependent uptake of these compounds by membrane vesicles prepared from HEK transfectants expressing each of these mutant proteins (Figs. Login to comment

[hide] Photolabeling of human and murine multidrug resist... J Biol Chem. 2002 Sep 20;277(38):35225-31. Epub 2002 Jul 22. Qian YM, Grant CE, Westlake CJ, Zhang DW, Lander PA, Shepard RL, Dantzig AH, Cole SP, Deeley RG
Photolabeling of human and murine multidrug resistance protein 1 with the high affinity inhibitor [125I]LY475776 and azidophenacyl-[35S]glutathione.
J Biol Chem. 2002 Sep 20;277(38):35225-31. Epub 2002 Jul 22., 2002-09-20 [PMID:12138119]

Abstract [show]
Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent transporter of structurally diverse organic anion conjugates. The protein also actively transports a number of non-conjugated chemotherapeutic drugs and certain anionic conjugates by a presently poorly understood GSH-dependent mechanism. LY475776is a newly developed (125)I-labeled azido tricyclic isoxazole that binds toMRP1 with high affinity and specificity in a GSH-dependent manner. The compound has also been shown to photolabel a site in the COOH-proximal region of MRP1's third membrane spanning domain (MSD). It is presently not known where GSH interacts with the protein. Here, we demonstrate that the photactivateable GSH derivative azidophenacyl-GSH can substitute functionally for GSH in supporting the photolabeling of MRP1 by LY475776 and the transport of another GSH-dependent substrate, estrone 3-sulfate. In contrast to LY475776, azidophenacyl-[(35)S] photolabels both halves of the protein. Photolabeling of the COOH-proximal site can be markedly stimulated by low concentrations of estrone 3-sulfate, suggestive of cooperativity between the binding of these two compounds. We show that photolabeling of the COOH-proximal site by LY475776 and the labeling of both NH(2)- and COOH- proximal sites by azidophenacyl-GSH requires the cytoplasmic linker (CL3) region connecting the first and second MSDs of the protein, but not the first MSD itself. Although required for binding, CL3 is not photolabeled by azidophenacyl-GSH. Finally, we identify non-conserved amino acids in the third MSD that contribute to the high affinity with which LY475776 binds to MRP1.

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164 In contrast, when the same preparations of HEK membranes were photolabeled with azidophenacyl-[35 S]GSH, the extent of labeling of T1242A MRP1 was higher than that of the wild type protein. Login to comment
230 Mutation of Thr-1242 to Ala, as found at the corresponding position in murine mrp1, decreases the ability to transport E217betaG and to confer drug resistance, but does not affect LTC4 transport. Login to comment

[hide] Drug binding domains of MRP1 (ABCC1) as revealed b... Curr Med Chem Anticancer Agents. 2004 Jan;4(1):19-30. Karwatsky JM, Georges E
Drug binding domains of MRP1 (ABCC1) as revealed by photoaffinity labeling.
Curr Med Chem Anticancer Agents. 2004 Jan;4(1):19-30., [PMID:14754409]

Abstract [show]
Drug resistance is a major impediment in the treatment of cancer patients receiving single or multiple drug treatment. Efforts to reverse drug resistance of tumor cells have not been successful. In recent years, considerable emphasis has been placed on understanding the underlying mechanisms that confer drug resistance. The expression of the multidrug resistance protein 1 (MRP1 or ABCC1) in cancer cells has been shown to confer resistance to diverse classes of anti-cancer drugs. MRP1 is a member of the ATP-binding cassette (ABC) family whose function, in tumor cells, is to reduce drug accumulation through energized drug efflux. To learn more about the functions of MRP1 in tumor drug resistance, knowledge of the protein binding characteristics and the location of its binding sites are essential. Photoaffinity labeling (PAL) has emerged as a leading technique that can rapidly shed light on a protein's drug binding characteristics and ultimately drug binding domains. Several MRP1-specific photoreactive probes have been developed. PAL of MRP1 was first demonstrated with the quinoline-based drug, IAAQ. Other studies showed that the high affinity endogenous substrate of MRP1, LTC(4), has intrinsic photoreactive properties and binds within both N- and C-terminal domains of MRP1. LTC(4) is conjugated to glutathione (GSH), a property common to several MRP1 substrates. In addition, several unconjugated drugs have been identified that interact with MRP1: [(3)H]VF-13,159, IAAQ, IACI and IAARh123. Mapping studies showed that IACI and IAARh123 bind two sites within transmembrane (TM) regions 10-11 and 16-17 of MRP1. Interestingly, the GSH-dependent PAL of [(125)I]azidoAG-A and [(125)I]LY475776 occurs within, or proximal to TM 16-17. The PAL with several analogs of GSH, IAAGSH and azidophenacyl-[(35)S]GSH found to interact specifically with MRP1 within TM 10-11 and TM 16-17 in addition to binding two cytoplasmic regions in MRP1, L0 and L1. This review focuses on the use of PAL for studying MRP1 interactions with various drugs and cell metabolites. Furthermore, knowledge of MRP1 drug binding domains, as identified by PAL with various photoreactive drug analogs, provides an important first step towards more detailed analyses of MRP1 binding domains.

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233 A mutation of Thr1242 to Ala in MRP1 decreases photoaffinity labeling with [125 I]LY475776 and supports the possibility of a drug binding site in TM 17 of MRP1. 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
146 Introduction of a second mutation based on mouse Mrp1 sequence into human MRP1/T1242A to generate a double mutant MRP1/ E1089Q/T1242A created a protein similar to mouse wild-type Mrp1 that restored resistance to vicristine and VP-16 but not to doxorubicin and epirubicin [85]. 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