ABCC4 p.Phe368Leu
| Predicted by SNAP2: | A: N (53%), C: N (72%), D: D (75%), E: D (71%), G: D (63%), H: D (53%), I: N (57%), K: D (66%), L: N (53%), M: N (66%), N: D (63%), P: D (80%), Q: D (53%), R: D (71%), S: D (63%), T: D (59%), V: N (53%), W: D (59%), Y: N (72%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: 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: N, |
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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
61 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.Phe368Leu 18612080:61:56
status: NEWX
ABCC4 p.Phe368Leu 18612080:61:123
status: NEWX
ABCC4 p.Phe368Leu 18612080:61:163
status: NEW91 Each experi- Fig. 3. Western blot analysis (A), 0.5 M [3 H]MTX (B), and 1 M [3 H]cGMP (C) transport activity of wild-type, F368L, F369-, E374S, R375S, and E378Q MRP4 transporter proteins.
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ABCC4 p.Phe368Leu 18612080:91:139
status: NEW92 A, top, Western blot of membrane vesicles isolated from HEK293 cells overexpressing MRP4 or MRP4 mutants F368L, F369-, E374S, R375S, and E378Q, as well as negative control detected by polyclonal anti-human MRP4 (representative of three).
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ABCC4 p.Phe368Leu 18612080:92:105
status: NEW95 Concentration-dependent uptake of [3 H]MTX and [3 H]cGMP in membrane vesicles expressing human MRP4 mutants. Control (Œ), wild-type (f), F368L (F), F369- (Ⅺ), E374S (᭛), R375S (E), and E378Q (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.Phe368Leu 18612080:95:143
status: NEW142 To investigate the substitution of the amino acids of the previous mutants in more detail, we constructed the single mutants F368L, F369-, E374S, R375S, and E378Q.
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ABCC4 p.Phe368Leu 18612080:142:125
status: NEW144 The transport activity of both [3 H]MTX and [3 H]cGMP (0.5 and 1 M, respectively) was significantly reduced in mutants F368L, F369-, E374S, and E378Q compared with wild-type MRP4 (Fig. 3).
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ABCC4 p.Phe368Leu 18612080:144:127
status: NEW148 Mutants F368L, F369-, E374S, and E378Q showed transport activity levels that were comparable with that of the negative control at all concentrations tested for both MTX and cGMP.
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ABCC4 p.Phe368Leu 18612080:148:8
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.Phe368Leu 18612080:188:65
status: NEW