ABCMdb

PMID: 18612080

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., [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCC4 p.Arg375Ser 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. Login to comment 7 ABCC4 p.Arg375Ser Furthermore, MTX inhibition of MRP4-mediated cGMP transport was noncompetitive, and the inhibition constant was increased by introduction of the R375S mutation. Login to comment 61 ABCC4 p.Arg375Ser ABCC4 p.Arg375Ser ABCC4 p.Arg375Ser ABCC4 p.Arg998Ala ABCC4 p.Glu378Gln ABCC4 p.Glu378Gln ABCC4 p.Glu378Gln ABCC4 p.Arg375Glu ABCC4 p.Arg375Ala ABCC4 p.Glu374Ser ABCC4 p.Glu374Ser ABCC4 p.Glu374Ser ABCC4 p.Phe368Leu ABCC4 p.Phe368Leu ABCC4 p.Phe368Leu ABCC4 p.Arg375Lys 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. Login to comment 91 ABCC4 p.Arg375Ser ABCC4 p.Glu378Gln ABCC4 p.Glu374Ser ABCC4 p.Phe368Leu 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. Login to comment 92 ABCC4 p.Arg375Ser ABCC4 p.Glu378Gln ABCC4 p.Glu374Ser ABCC4 p.Phe368Leu 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). Login to comment 95 ABCC4 p.Arg375Ser ABCC4 p.Glu378Gln ABCC4 p.Glu374Ser ABCC4 p.Phe368Leu 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. Login to comment 99 ABCC4 p.Arg375Ser To evaluate the inhibitory effects of MTX on [3 H]cGMP uptake in MRP4 and MRP4-R375S membrane vesicles, the previously mentioned transport assay was performed using 1, 10, and 100 ␮M cGMP, in the absence or presence of MTX concentrations ranging from 1 to 600 ␮M. Login to comment 122 ABCC4 p.Arg375Ser Dixon plot showing the MTX inhibition of cGMP transport by human MRP4 and mutant R375S. Login to comment 124 ABCC4 p.Arg375Ser Specific uptake of MRP4 (top) and mutant R375S (bottom) was determined after subtraction of the negative control. Login to comment 126 ABCC4 p.Arg998Ala ABCC4 p.Arg375Glu ABCC4 p.Arg375Ala ABCC4 p.Arg375Lys 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. Login to comment 127 ABCC4 p.Arg998Ala ABCC4 p.Arg375Glu ABCC4 p.Arg375Ala ABCC4 p.Arg375Lys 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). Login to comment 142 ABCC4 p.Arg375Ser ABCC4 p.Glu378Gln ABCC4 p.Glu374Ser ABCC4 p.Phe368Leu 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. Login to comment 144 ABCC4 p.Glu378Gln ABCC4 p.Glu374Ser ABCC4 p.Phe368Leu 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). Login to comment 145 ABCC4 p.Arg375Ser Interestingly, cGMP transport activity of mutant R375S was 98 Ϯ 2% of wild type, whereas its MTX transport activity was only 55 Ϯ 2%. Login to comment 148 ABCC4 p.Glu378Gln ABCC4 p.Glu374Ser ABCC4 p.Phe368Leu 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. Login to comment 149 ABCC4 p.Arg375Ser The maximum transport rate (Vmax) values for the wild-type and R375S mutant were 280 Ϯ 60 and 270 Ϯ 120 pmol/mg protein/min for MTX and 370 Ϯ 30 and 270 Ϯ 70 pmol/mg protein/min for cGMP, respectively. Login to comment 150 ABCC4 p.Arg375Ser The apparent affinity (Km) values of wild-type MRP4 and mutant R375S were 230 Ϯ 90 and 720 Ϯ 320 ␮M for MTX and 610 Ϯ 70 and 610 Ϯ 80 ␮M for cGMP, respectively. Login to comment 151 ABCC4 p.Arg375Ser The Km value for cGMP was not influenced by substitution of Arg375 with Ser, whereas it was increased 3-fold for MTX. Login to comment 152 ABCC4 p.Arg375Ser To test the interaction of MTX and cGMP in more detail, we analyzed the possible inhibitory effect of MTX on [3 H]cGMP uptake for wild-type and R375S mutant MRP4. Login to comment 153 ABCC4 p.Arg375Ser A Dixon plot of net cGMP transport by wild type and R375S in the absence or presence of increasing MTX concentrations was constructed and analyzed by linear regression (Fig. 5). Login to comment 154 ABCC4 p.Arg375Ser Remarkably, the intersection of the three lines representing MTX inhibition curves at different cGMP concentrations was at the x-axis for both wild type and R375S mutant, indicating a noncompetitive inhibitory effect. Login to comment 155 ABCC4 p.Arg375Ser The inhibition constant value (intersection with the x-axis), Ki, for wild-type MRP4 was 164 Ϯ 4 ␮M, compared with 470 Ϯ 70 ␮M for mutant R375S. Login to comment 157 ABCC4 p.Arg998Ala ABCC4 p.Arg375Glu ABCC4 p.Arg375Ala ABCC4 p.Arg375Lys 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. Login to comment 165 ABCC4 p.Arg375Glu ABCC4 p.Arg375Ala ABCC4 p.Arg375Lys Figure 6A shows an equal level of protein expression of wild type as well as R375A, R375K, R375E, and R988A mutants. Login to comment 170 ABCC4 p.Arg375Glu ABCC4 p.Arg375Ala The transport activities of R375A, R375E, and R988A for MTX and cGMP did not differ from that of the negative control. Login to comment 171 ABCC4 p.Arg375Ser The Vmax values for MTX were 250 Ϯ 20 and 190 Ϯ 10 pmol/mg protein/min and those for cGMP were 420 Ϯ 10 and 420 Ϯ 20 pmol/mg protein/min for wild type and R375S mutant, respectively. Login to comment 172 ABCC4 p.Arg375Lys The Km values of wild-type MRP4 and R375K were 230 Ϯ 90 and 250 Ϯ 20 ␮M for MTX and 610 Ϯ 70 and 640 Ϯ 60 ␮M for cGMP, respectively. Login to comment 173 ABCC4 p.Arg375Lys Thus, R375K retained substrate affinity comparable with the wild type for both MTX and cGMP. Login to comment 185 ABCC4 p.Arg375Ser The only exception was substitution of Arg375 with serine, which had no effect on cGMP transport, but significantly decreased the affinity for MTX. Substitution of the same amino acid with a positively charged lysine returned the MTX affinity to that of the wild type. Login to comment 188 ABCC4 p.Arg998Ala ABCC4 p.Glu378Gln ABCC4 p.Arg375Glu ABCC4 p.Arg375Ala ABCC4 p.Glu374Ser ABCC4 p.Phe368Leu 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. Login to comment 211 ABCC4 p.Glu374Ser Zhang et al. (2004) showed that replacement of MRP1 Ser604 (corresponding to E374S in MRP4) with alanine selectively increased 17beta-estradiol 17-(beta-D-glucuronide) transport. Login to comment 215 ABCC1 p.Ser605Ala ABCC4 p.Arg375Ala 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). Login to comment 217 ABCC4 p.Arg375Ser In the present study, substitution of MRP4 Arg375 with serine resulted in a lower affinity for MTX, but the affinity for cGMP did not change (Table 1). Login to comment 218 ABCC4 p.Arg375Ser Furthermore, the MTX inhibition constant for cGMP transport by mutant R375S was significantly lower than that of the wild type. Login to comment 221 ABCC4 p.Arg375Ser ABCC4 p.Arg375Glu ABCC4 p.Arg375Ala When this charge is removed (R375S), the MTX affinity decreases or transport activity is absent (R375A and R375E). Login to comment 222 ABCC4 p.Arg375Ser This positive charge is less important for cGMP transport, because in the presence of a hydroxyl group (R375S), the transport properties seem unchanged. Login to comment 223 ABCC4 p.Arg375Glu ABCC4 p.Arg375Ala As soon as this hydroxyl group is removed (R375A) or replaced with a (negatively charged) acidic group (R375E), transport of cGMP is absent. Login to comment 224 ABCC4 p.Arg375Ser Our observation that the R375S mutation has a larger effect on MTX transport could be explained by the fact that MTX contains two negative charges that need to be compensated, whereas cGMP only has one. Login to comment 230 ABCC1 p.Arg1249Ala ABCC4 p.Arg998Ala Moreover, alanine substitution of Arg1249 in MRP1 (also corresponding to R998A) impaired MRP1-mediated LTC4 transport and reduced vincristine resistance (Ren et al., 2002). Login to comment 233 ABCC4 p.Arg998Lys ABCC4 p.Arg998Glu Glutamic acid (R998E) and lysine (R998K) mutants would be needed to strengthen this conclusion. Login to comment 237 ABCC4 p.Arg375Ser Mutant R375S and wild-type MRP4 possessed similar affinities for cGMP, but the MTX affinity of this mutant was nearly 3-fold lower than that of the wild type. Login to comment 244 ABCC4 p.Arg375Ser ABCC4 p.Arg375Lys 3 and 4 MRP4 230 Ϯ 90 280 Ϯ 60 610 Ϯ 70 370 Ϯ 30 164 Ϯ 4 R375S 720 Ϯ 320 270 Ϯ 120 610 Ϯ 80 270 Ϯ 70 470 Ϯ 70 Data from Fig. 4 MRP4 230 Ϯ 90 250 Ϯ 20 610 Ϯ 70 420 Ϯ 10 R375K 250 Ϯ 20 190 Ϯ 10 640 Ϯ 60 420 Ϯ 20 substrate-binding site. Login to comment