ABCC1 p.Asp336Arg
| Predicted by SNAP2: | A: D (80%), C: D (80%), E: D (85%), F: D (85%), G: D (85%), H: D (91%), I: D (85%), K: D (95%), L: D (91%), M: D (85%), N: D (80%), P: D (91%), Q: D (91%), R: D (95%), S: D (75%), T: D (80%), V: D (85%), W: D (91%), Y: D (91%), |
| Predicted by PROVEAN: | A: D, C: 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, W: D, Y: D, |
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[hide] Charged amino acids in the sixth transmembrane hel... J Biol Chem. 2002 Nov 1;277(44):41326-33. Epub 2002 Aug 18. Haimeur A, Deeley RG, Cole SP
Charged amino acids in the sixth transmembrane helix of multidrug resistance protein 1 (MRP1/ABCC1) are critical determinants of transport activity.
J Biol Chem. 2002 Nov 1;277(44):41326-33. Epub 2002 Aug 18., 2002-11-01 [PMID:12186871]
Abstract [show]
The multidrug resistance protein, MRP1 (ABCC1), is an ATP-binding cassette transporter that confers resistance to chemotherapeutic agents. MRP1 also mediates transport of organic anions such as leukotriene C(4) (LTC(4)), 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG), estrone 3-sulfate, methotrexate (MTX), and GSH. We replaced three charged amino acids, Lys(332), His(335), and Asp(336), predicted to be in the sixth transmembrane (TM6) helix of MRP1 with neutral and oppositely charged amino acids and determined the effect on substrate specificity and transport activity. All mutants were expressed in transfected human embryonic kidney cells at levels comparable with wild-type MRP1, and confocal microscopy showed that they were correctly routed to the plasma membrane. Vesicular transport studies revealed that the MRP1-Lys(332) mutants had lost the ability to transport LTC(4), and GSH transport was reduced; whereas E(2)17betaG, estrone 3-sulfate, and MTX transport were unaffected. E(2)17betaG transport was not inhibited by LTC(4) and could not be photolabeled with [(3)H]LTC(4), indicating that the MRP1-Lys(332) mutants no longer bound this substrate. Substitutions of MRP1-His(335) also selectively diminished LTC(4) transport and photolabeling but to a lesser extent. Kinetic analyses showed that V(max) (LTC(4)) of these mutants was decreased but K(m) was unchanged. In contrast to the selective loss of LTC(4) transport in the Lys(332) and His(335) mutants, the MRP1-Asp(336) mutants no longer transported LTC(4), E(2)17betaG, estrone 3-sulfate, or GSH, and transport of MTX was reduced by >50%. Lys(332), His(335), and Asp(336) of TM6 are predicted to be in the outer leaflet of the membrane and are all capable of forming intrahelical and interhelical ion pairs and hydrogen bonds. The importance of Lys(332) and His(335) in determining substrate specificity and of Asp(336) in overall transport activity suggests that such interactions are critical for the binding and transport of LTC(4) and other substrates of MRP1.
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No. Sentence Comment
47 The sequences of the individual sense strands, with the altered codons underlined and the corresponding changes in amino acids indicated in parentheses were as follows: (K332D) 5Ј-CTC ATG AGC TTC TTC TTC GAC GCC ATC CAC GAC CTG-3Ј; (K332L) 5Ј-CTC ATG AGC TTC TTC TTC CTG GCC ATC CAC GAC CTG-3Ј; (H335E) 5Ј-GC TTC TTC TTC AAG GCC ATC GAG GAC CTG ATG ATG-3Ј; (H335L) 5Ј-GC TTC TTC TTC AAG GCC ATC TTG GAC CTG ATG ATG-3Ј; (H335Q) 5Ј-GC TTC TTC TTC AAG GCC ATC CAG GAC CTG ATG ATG-3Ј; (D336R) 5Ј-C AAG GCC ATC CAC CGG CTG ATG ATG TTT TCG-3Ј; (D336L) 5Ј-C AAG GCC ATC CAC CTG CTG ATG ATG TTT TCG-3Ј.
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ABCC1 p.Asp336Arg 12186871:47:542
status: NEW104 Representative confocal micrographs of cells expressing GFP-tagged wild-type MRP1 and mutants K332D, H335E, and D336R are shown in Fig. 2.
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ABCC1 p.Asp336Arg 12186871:104:112
status: NEW106 In the case of the MRP1-Lys332 mutants K332D and K332L (Fig. 3B) and the MRP1-Asp336 mutants D336L and D336R (Fig. 3D), LTC4 uptake was reduced to levels that were indistinguishable from those observed with vesicles prepared from empty vector-transfected control cells.
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ABCC1 p.Asp336Arg 12186871:106:103
status: NEW125 In contrast, substitution of Asp336 with either Leu or Arg led to a complete loss of E217betaG uptake.
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ABCC1 p.Asp336Arg 12186871:125:29
status: NEW126 Thus, as shown in Fig. 5C, MRP1-Asp336 mutants D336L and D336R exhibited the same E217betaG uptake activity as the empty vector control.
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ABCC1 p.Asp336Arg 12186871:126:57
status: NEW142 D, wild-type MRP1 (f), MRP1 mutants D336L (Œ) and D336R (‚), and control empty pcDNA3.1(-) vector (E).
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ABCC1 p.Asp336Arg 12186871:142:56
status: NEW152 However, this band is not detectable in [3 H]LTC4 photolabeled proteins from cells expressing comparable levels of the MRP1 mutants K332D and K332L or mutants D336L and D336R, indicating that these mutations abrogate photolabeling and hence binding of this compound.
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ABCC1 p.Asp336Arg 12186871:152:169
status: NEW165 C, time courses of [3 H]E217betaG uptake by membrane vesicles prepared from HEK293T cells transfected with cDNAs encoding wild-type MRP1 (f), TM6 mutants D336L (Œ), and D336R (‚), and the empty pcDNA3.1(-) vector control (E).
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ABCC1 p.Asp336Arg 12186871:165:175
status: NEW177 Substitution of Asp336 with Leu (D336L) reduced GSH uptake to ϳ25% of wild-type MRP1 levels, whereas substitution of this negatively charged residue with a positively charged residue (D336R) further reduced uptake of this tripeptide to just above basal levels observed with vesicles from the empty vector control (Fig. 7C).
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ABCC1 p.Asp336Arg 12186871:177:190
status: NEW185 In contrast, MTX uptake was reduced by ϳ50% when Asp336 was substituted with Leu (D336L) and by ϳ65% when substituted with Arg (D336R) (not shown).
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ABCC1 p.Asp336Arg 12186871:185:140
status: NEW204 Membrane vesicles were preincubated with acivicin and then incubated with [3 H]GSH in the presence of 30 M apigenin in transport buffer for 20 min at 37 °C. A, K332D and K332L; B, H335E, H335L, and H335Q; C, D336L and D336R.
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ABCC1 p.Asp336Arg 12186871:204:231
status: NEW208 F, WT-MRP1 (f); D336L (Œ); D336R (‚); vector control (E).
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ABCC1 p.Asp336Arg 12186871:208:33
status: NEW217 Substitutions of MRP1-Asp336 had a more dramatic effect on MRP1 transport activity than substitutions of either Lys332 or His335 in that the D336L and D336R mutants did not transport four of the five organic anion substrates tested, and transport of the fifth, MTX, was reduced by at least 50%.
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ABCC1 p.Asp336Arg 12186871:217:151
status: NEW[hide] Mutations of charged amino acids in or near the tr... Mol Pharmacol. 2004 Jun;65(6):1375-85. Haimeur A, Conseil G, Deeley RG, Cole SP
Mutations of charged amino acids in or near the transmembrane helices of the second membrane spanning domain differentially affect the substrate specificity and transport activity of the multidrug resistance protein MRP1 (ABCC1).
Mol Pharmacol. 2004 Jun;65(6):1375-85., [PMID:15155831]
Abstract [show]
Multidrug resistance protein 1 (MRP1) belongs to the ATP-binding cassette superfamily of transport proteins. In addition to drugs, MRP1 mediates the active transport of many conjugated and unconjugated organic anions. MRP1 consists of two membrane-spanning domains (MSD2 and MSD3) each followed by a nucleotide binding domain plus a third NH2-terminal MSD1. MSD2 contains transmembrane (TM) helices 6 through 11, and previously, we identified two charged residues in TM6 as having important but markedly different roles in MRP1 transport activity and substrate specificity by characterizing mutants containing nonconservative substitutions of Lys332 and Asp336. We have now extended these studies and found that the same-charge TM6 mutant K332R, like the nonconservatively substituted Lys332 mutants, exhibits a selective decrease in leukotriene C4 (LTC4) transport, associated with substantial changes in both Km and Vmax and LTC4 binding. The overall organic anion transport activity of the same-charge mutant of Asp336 (D336E) also remained very low, as observed for D336R. In addition, nonconservative substitutions of TM6-associated Lys319 and Lys347 resulted in a selective decrease in GSH transport. Of eight other charged residues in or proximal to TM7 to TM11 that were investigated, nonconservative substitutions of three of them [Lys396 (TM7), Asp436 (TM8), and Arg593 (TM11)] caused a substantial and global reduction in transport activity. However, unlike TM6 Asp336, wild-type transport activity could be reestablished in these MRP1 mutants by conservative substitutions. We conclude that MSD2-charged residues in or proximal to TM6, TM7, TM8, and TM11 play critical but differential roles in MRP1 transport activity and substrate specificity.
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No. Sentence Comment
5 The overall organic anion transport activity of the same-charge mutant of Asp336 (D336E) also remained very low, as observed for D336R.
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ABCC1 p.Asp336Arg 15155831:5:129
status: NEW123 In our earlier study, we showed that substitution of the negatively charged TM6 Asp336 residue with Arg or Leu essentially eliminated the transport of both conjugated and unconjugated MRP1 organic anion substrates (Haimeur et al., 2002).
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ABCC1 p.Asp336Arg 15155831:123:80
status: NEW