ABCC1 p.Arg1046Asp
| Predicted by SNAP2: | A: D (85%), C: D (91%), D: D (95%), E: D (91%), F: D (95%), G: D (95%), H: D (91%), I: D (91%), K: D (80%), L: D (95%), M: D (85%), N: D (91%), P: D (95%), Q: D (85%), S: D (85%), T: D (85%), V: D (91%), W: D (95%), Y: D (95%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: N, L: D, M: D, N: D, P: D, Q: D, S: D, T: D, V: D, W: D, Y: D, |
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[hide] Mutational analysis of ionizable residues proximal... J Biol Chem. 2004 Sep 10;279(37):38871-80. Epub 2004 Jun 18. Situ D, Haimeur A, Conseil G, Sparks KE, Zhang D, Deeley RG, Cole SP
Mutational analysis of ionizable residues proximal to the cytoplasmic interface of membrane spanning domain 3 of the multidrug resistance protein, MRP1 (ABCC1): glutamate 1204 is important for both the expression and catalytic activity of the transporter.
J Biol Chem. 2004 Sep 10;279(37):38871-80. Epub 2004 Jun 18., 2004-09-10 [PMID:15208328]
Abstract [show]
The multidrug resistance protein MRP1 is an ATP-dependent transporter of organic anions and chemotherapeutic agents. A significant number of ionizable amino acids are found in or proximal to the 17 transmembrane (TM) helices of MRP1, and we have investigated 6 of these at the cytoplasmic interface of TM13-17 for their role in MRP1 expression and transport activity. Opposite charge substitutions of TM13 Arg(1046) and TM15 Arg(1131) did not alter MRP1 expression nor did they substantially affect activity. In contrast, opposite charge substitutions of TM16 Arg(1202) and Glu(1204) reduced protein expression by >80%; however, MRP1 expression was not affected when Arg(1202) and Glu(1204) were replaced with neutral or same-charge residues. In addition, organic anion transport levels of the R1202L, R1202G, and R1202K mutants were comparable with wild-type MRP1. In contrast, organic anion transport by E1204L was substantially reduced, whereas transport by E1204D was comparable with wild-type MRP1, with the notable exception of GSH. Opposite charge substitutions of TM16 Arg(1197) and TM17 Arg(1249) did not affect MRP1 expression but substantially reduced transport. Mutants containing like-charge substitutions of Arg(1197) or Arg(1249) were also transport-inactive and no longer bound leukotriene C(4). In contrast, substrate binding by the transport-compromised E1204L mutant remained intact. Furthermore, vanadate-induced trapping of azido-ADP by E1204L was dramatically increased, indicating that this mutation may cause a partial uncoupling of the catalytic and transport activities of MRP1. Thus, Glu(1204) serves a dual role in membrane expression of MRP1 and a step in its catalytic cycle subsequent to initial substrate binding.
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No. Sentence Comment
82 RESULTS Expression and Transport Properties of MRP1 Mutants Containing Opposite and Same Charge Substitutions of Arg1046 , Asp1084 , and Arg1131 in or Near TM13-15-In the first series of experiments, the importance of ionizable amino acids located in or near predicted TM13-15 for MRP1 protein expression and function were investigated by replacing Arg1046 and Arg1131 with Asp and Glu, respectively.
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ABCC1 p.Arg1046Asp 15208328:82:349
status: NEW83 Levels of expression of the R1046D and R1131E mutant proteins were determined by the immunoblotting of membrane vesicles prepared from the transfected HEK293T cells with mAb QCRL-1.
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ABCC1 p.Arg1046Asp 15208328:83:28
status: NEW87 To determine the functional consequences of replacing Arg1046 , Asp1084 , and Arg1131 with an oppositely charged amino acid, the ability of the R1046D, D1084R, and R1131E mutants to transport different organic anions was tested.
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ABCC1 p.Arg1046Asp 15208328:87:144
status: NEW88 Fig. 2, B and C, shows that uptake levels of E217betaG and LTC4, respectively, by the R1046D and R1131E mutants were moderately reduced (by 30-50%) compared with wild-type MRP1, whereas uptake by the D1084R mutant was substantially reduced by Ͼ90%.
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ABCC1 p.Arg1046Asp 15208328:88:86
status: NEW89 Similarly, levels of E1SO4 and MTX uptake by the R1046D and R1131E mutants were comparable with or moderately different from wild-type MRP1, whereas uptake of these organic anions by D1084R was substantially diminished (Յ25% of wild-type MRP1) (Fig. 2, D and E).
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ABCC1 p.Arg1046Asp 15208328:89:49
status: NEW93 A, MRP1 expression in membrane vesicles prepared from HEK293T cells transfected with empty vector [pcDNA3.1] and vector containing wild-type (WT-MRP1) and mutant (R1046D, D1084R, D1084E, and R1131E) cDNAs was determined by immunoblotting with mAb QCRL-1. Shown is a representative immunoblot of membrane vesicles (1 and 2 g of protein) from a single transfection.
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ABCC1 p.Arg1046Asp 15208328:93:163
status: NEW95 B-F, levels of 3 H-labeled organic anion uptake by the membrane vesicles shown in A were determined and corrected to take into account any differences in MRP1 protein expression (empty pcDNA3.1 vector control (open bars), wild-type MRP1 (black bars), and mutants R1046D, D1084R, D1084E, and R1131E (gray bars)).
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ABCC1 p.Arg1046Asp 15208328:95:263
status: NEW118 Immunoblots of membrane vesicle proteins prepared from cells expressing the Glu1204 mutants E1204L and E1204D were carried out as described in A. TABLE I Summary of organic anion transport activity of MRP1 mutants with substitutions of ionizable amino acids in and proximal to TM13 to TM17 of MSD3 Mutation % Wild-type MRP1 transport activitya E217betaG LTC4 E1SO4 MTX GSH TM13 R1046D 115 70 80 120 NDb TM14 D1084R Ͻ10 Ͻ10 15 25 Ͻ10 D1084E 80 20 65 90 20 TM15 R1131E 70 50 80 60 ND TM16 R1197E Ͻ10 Ͻ10 Ͻ15 Ͻ10 ND R1197K 20 Ͻ25 Ͻ20 Ͻ10 ND R1202G 115 115 75 70 ND R1202L 115 120 50 110 ND E1204L Ͻ10 50 10 110 Ͻ25 E1204D 100 115 100 115 Ͻ25 TM17 R1249D Ͻ10 Ͻ15 Ͻ10 Ͻ10 ND R1249K Ͻ10 10 Ͻ15 Ͻ10 ND a The values shown are means of duplicate or triplicate determinations and are derived from Fig. 2, 4, and 5 (see figure legends for details).
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ABCC1 p.Arg1046Asp 15208328:118:378
status: NEW