ABCC4 p.Arg375Ala
| Predicted by SNAP2: | A: D (59%), C: D (59%), D: D (75%), E: D (66%), F: D (59%), G: N (61%), H: N (61%), I: D (59%), K: N (93%), L: D (53%), M: N (53%), N: N (53%), P: D (80%), Q: N (66%), S: N (53%), T: N (78%), V: D (63%), W: D (75%), Y: D (63%), |
| Predicted by PROVEAN: | A: N, C: D, D: D, E: N, F: D, G: D, H: N, I: D, K: N, L: D, M: N, N: N, P: D, Q: N, S: N, T: N, V: D, W: D, Y: D, |
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[hide] Functional role of arginine 375 in transmembrane h... Mol Pharmacol. 2008 Oct;74(4):964-71. Epub 2008 Jul 8. El-Sheikh AA, van den Heuvel JJ, Krieger E, Russel FG, Koenderink JB
Functional role of arginine 375 in transmembrane helix 6 of multidrug resistance protein 4 (MRP4/ABCC4).
Mol Pharmacol. 2008 Oct;74(4):964-71. Epub 2008 Jul 8., [PMID:18612080]
Abstract [show]
Multidrug resistance protein (MRP) 4 transports a variety of endogenous and xenobiotic organic anions. MRP4 is widely expressed in the body and specifically localized to the renal apical proximal tubule cell membrane, where it mediates the excretion of these compounds into urine. To characterize the MRP4 substrate-binding site, the amino acids Phe368, Phe369, Glu374, Arg375, and Glu378 of transmembrane helix 6, and Arg998 of helix 12, localized in the intracellular half of the central pore, were mutated into the corresponding amino acids of MRP1 and MRP2. Membrane vesicles isolated from human embryonic kidney 293 cells overexpressing these mutants showed significantly reduced methotrexate (MTX) and cGMP transport activity compared with vesicles that expressed wild-type MRP4. The only exception was substitution of Arg375 with serine, which had no effect on cGMP transport but significantly decreased the affinity of MTX. Substitution of the same amino acid with a positively charged lysine returned the MTX affinity to that of the wild type. Furthermore, MTX inhibition of MRP4-mediated cGMP transport was noncompetitive, and the inhibition constant was increased by introduction of the R375S mutation. A homology model of MRP4 showed that Arg375 and Arg998 face right into the central aqueous pore of MRP4. We conclude that positively charged amino acids in transmembrane helices 6 and 12 contribute to the MRP4 substrate-binding pocket.
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No. Sentence Comment
215 Deletion of the hydroxyl group of MRP1 residue 605 (S605A), corresponding to R375A in MRP4, decreased resistance to vincristine, etoposide (VP-16), and doxorubicin (Zhang et al., 2004).
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ABCC4 p.Arg375Ala 18612080:215:77
status: NEW61 Twelve mutants of the human MRP4 were generated: FF/L- (F368L and F369-), ERE/ SSQ (E374S, R375S, and E378Q), FFERE/L-SSQ (F368L, F369-, E374S, R375S, and E378Q), F368L, F369-, E374S, R375S, R375A, R375K, R375E, E378Q, and R998A.
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ABCC4 p.Arg375Ala 18612080:61:191
status: NEW126 Mean values Ϯ S.E. of three enzyme preparations are shown. Fig. 6. Western blot analysis (A), 0.5 M [3 H]MTX (B), and 1 M [3 H]cGMP (C) transport activity of wild-type, R375A, R375K, R375E, and R998A MRP4 transporter proteins.
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ABCC4 p.Arg375Ala 18612080:126:191
status: NEW127 A, top, Western blot of membrane vesicles isolated from HEK293 cells overexpressing MRP4 or MRP4 mutants R375A, R375K, R375E, and R998A, as well as negative control detected by polyclonal anti-human MRP4 (representative of three).
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ABCC4 p.Arg375Ala 18612080:127:105
status: NEW157 Concentration-dependent uptake of [3 H]MTX and [3 H]cGMP in membrane vesicles expressing human MRP4 mutants. Control (Œ), wild-type (f), R375A (F), R375K (E), R375E (छ), and R998A (x) MRP4 membrane vesicles were incubated with [3 H]MTX (top) or [3 H]cGMP (bottom) concentrations indicated in the figure.
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ABCC4 p.Arg375Ala 18612080:157:143
status: NEW165 Figure 6A shows an equal level of protein expression of wild type as well as R375A, R375K, R375E, and R988A mutants.
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ABCC4 p.Arg375Ala 18612080:165:77
status: NEW170 The transport activities of R375A, R375E, and R988A for MTX and cGMP did not differ from that of the negative control.
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ABCC4 p.Arg375Ala 18612080:170:28
status: NEW188 Nevertheless, several MRP4 mutants (FF/L-, ERE/SSQ, FFERE/L-SSQ, F368L, F369-, E374S, R375A, R375E, E378Q, and R998A) showed significantly diminished transport activity of either MTX or cGMP compared with that of wild-type MRP4.
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ABCC4 p.Arg375Ala 18612080:188:86
status: NEW221 When this charge is removed (R375S), the MTX affinity decreases or transport activity is absent (R375A and R375E).
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ABCC4 p.Arg375Ala 18612080:221:97
status: NEW223 As soon as this hydroxyl group is removed (R375A) or replaced with a (negatively charged) acidic group (R375E), transport of cGMP is absent.
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ABCC4 p.Arg375Ala 18612080:223:43
status: NEW