ABCMdb

PMID: 11278867

Ito K, Olsen SL, Qiu W, Deeley RG, Cole SP
Mutation of a single conserved tryptophan in multidrug resistance protein 1 (MRP1/ABCC1) results in loss of drug resistance and selective loss of organic anion transport.
J Biol Chem. 2001 May 11;276(19):15616-24. Epub 2001 Feb 21., 2001-05-11 [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC1 p.Trp1246Cys ABCC1 p.Trp1246Cys In this study, we found that substitution of a highly conserved tryptophan at position 1246 with cysteine (W1246C-MRP1) in the putative last transmembrane segment (TM17) of MRP1 eliminated 17beta-estradiol 17-(beta-D-glucuronide) (E217betaG) transport by membrane vesicles prepared from transiently transfected human embryonic kidney cells while leaving the capacity for leukotriene C4- and verapamil-stimulated glutathione transport intact. Login to comment 2 ABCC1 p.Trp1246Cys In addition, in contrast to wild-type MRP1, leukotriene C4 transport by the W1246C-MRP1 protein was no longer inhibitable by E217betaG, indicating that the mutant protein had lost the ability to bind the glucuronide. Login to comment 3 ABCC1 p.Trp1246Ala ABCC1 p.Trp1246Phe A similar phenotype was observed when Trp1246 was replaced with Ala, Phe, and Tyr. Login to comment 5 ABCC1 p.Trp1246Cys In addition to the loss of E217betaG transport, HeLa cells stably transfected with W1246C-MRP1 cDNA were not resistant to the Vinca alkaloid vincristine and accumulated levels of [3 H]vincristine comparable to those in vector control-transfected cells. Login to comment 6 ABCC1 p.Trp1246Cys Cells expressing W1246C-MRP1 were also not resistant to cationic anthracyclines (doxorubicin, daunorubicin) or the electroneutral epipodophyllotoxin VP-16. Login to comment 47 ABCC1 p.Trp1246Cys ABCC1 p.Trp1246Ala ABCC1 p.Trp1246Phe ABCC1 p.Trp1246Tyr Mutagenesis was then performed according to the manufacturer`s instructions with the following sense mutagenic primers (substituted nucleotides are underlined): W1246C, 5Ј-CCACGTACT- TGAACTGCCTGGTTCGGATGTC-3Ј; W1246A, 5Ј-CCACGTACTTGAA- CGCGCTGGTTCGGATGTC-3Ј; W1246F, 5Ј-CCACGTACTTGAACTTC- CTGGTTCGGATGTC-3Ј; and W1246Y, 5Ј-CCACGTACTTGAACTATCT- GGTTCGGATGTC-3Ј. Login to comment 54 ABCC1 p.Trp1246Cys ABCC1 p.Trp1246Cys ABCC1 p.Trp1246Ala ABCC1 p.Trp1246Ala Mutant MRP1-GFP fusion proteins were generated by replacing the 1.3-kb BsmBI/EcoRI fragment in the pcDNA3.1(-)-MRP1K-GFP construct with the comparable fragment containing either the W1246C or W1246A mutation generated above and designated pcDNA-3.1(-)- W1246C-MRP1-GFP and pcDNA3.1(-)-W1246A-MRP1-GFP, respectively. Login to comment 103 ABCC1 p.Pro196Ala RESULTS Identification of Trp1246 as a Functionally Important Amino Acid in MRP1-During the course of a mutational analysis of proline residues in MRP1, we generated an MRP1 mutant in which proline at position 196 was replaced with alanine (P196A). Login to comment 105 ABCC1 p.Pro196Ala ABCC1 p.Pro196Ala In contrast, a second independently derived P196A mutant HeLa cell line displayed a phenotype similar to that of cells expressing wild-type MRP1.2 Using three sets of overlapping primers, the integrated MRP1-transfected cDNAs of the wild-type MRP1 and the first P196A mutant cell lines were amplified by polymerase chain reaction. Login to comment 106 ABCC1 p.Trp1246Cys ABCC1 p.Pro196Ala The DNA fragments obtained were of the size expected, and when sequenced, it was discovered that, in addition to the P196A mutation, the transfected copies of MRP1 cDNA in the first cell mutant line with an altered phenotype contained a G 3 C nucleotide mutation, resulting in substitution of cysteine for tryptophan at position 1246. Login to comment 108 ABCC1 p.Trp1246Cys A W1246C Substitution Causes Complete Loss of E217betaG Transport Activity in Membrane Vesicles from Transiently Transfected HEK293T Cells-As a first step to confirming the importance of Trp1246 , this amino acid was substituted with cysteine in pcDNA3.1(-)-MRP1K by site-directed mutagenesis, and the resulting construct was then transiently transfected into HEK293T cells. Login to comment 111 ABCC1 p.Trp1246Cys For the experiments shown in Fig. 2, the levels of W1246C-MRP1 in the transient transfectants were ϳ70% those of wild-type MRP1 (Fig. 2A). Login to comment 112 ABCC1 p.Trp1246Cys A time course of LTC4 uptake was performed, and the levels of uptake by the W1246C-MRP1 mutant relative to wild-type MRP1 were2 K. Ito, R. G. Deeley, and S. P. C. Cole, unpublished observations. FIG. 1. Login to comment 122 ABCC1 p.Trp1246Cys ABCC1 p.Trp1246Cys Subsequent kinetic analyses of LTC4 transport by the W1246C-MRP1 mutant indicated that its affinity for this substrate was reduced compared with wild-type MRP1 (Km ϭ 189 nM versus 79 nM), but the Vmax values for the two proteins were similar (40.6 versus 35.1 pmol/mg/min).3 These results indicate that substitution of Trp1246 with Cys reduces the affinity of MRP1 for this substrate, but that the transport capacities of the mutant and wild-type proteins are comparable. Login to comment 123 ABCC1 p.Trp1246Cys In contrast, uptake of E217betaG by the W1246C-MRP1 mutant was dramatically reduced and was comparable to that of the vector-transfected controls (Fig. 2C). Login to comment 125 ABCC1 p.Trp1246Cys Thus, the ability of E217betaG to inhibit LTC4 transport by the W1246C-MRP1 mutant was examined, and the results are shown in Fig. 2D. E217betaG (25 ␮M) inhibited LTC4 transport by wild-type MRP1 by 54%, as expected. Login to comment 126 ABCC1 p.Trp1246Cys In contrast, E217betaG had no effect on LTC4 transport by W1246C-MRP1, indicating that the loss of E217betaG transport by the mutant protein is associated with a loss of binding of this substrate. Login to comment 128 ABCC1 p.Trp1246Ala ABCC1 p.Trp1246Phe ABCC1 p.Trp1246Tyr These included substitution with a nonpolar non-aromatic amino acid (Ala; W1246A-MRP1) as well as conservative substitutions with polar (Tyr; W1246Y-MRP1) and nonpolar (Phe; W1246F-MRP1) aromatic amino acids. Login to comment 131 ABCC1 p.Trp1246Cys ABCC1 p.Trp1246Ala ABCC1 p.Trp1246Phe ABCC1 p.Trp1246Tyr The LTC4 transport levels of the W1246A-MRP1 mutant (Fig. 3B) and the W1246Y-MRP1 and W1246F-MRP1 mutants (Fig. 3C) were similar to those of wild-type MRP1 and the W1246C-MRP1 mutant. Login to comment 132 ABCC1 p.Trp1246Cys ABCC1 p.Trp1246Ala In contrast, like the W1246C mutant, the W1246A mutant did not transport E217betaG (Fig. 3D). Login to comment 133 ABCC1 p.Trp1246Phe ABCC1 p.Trp1246Tyr E217betaG transport by the W1246Y and W1246F mutants was also extremely low (ϳ10% of wild-type MRP1) (Fig. 3E). Login to comment 136 ABCC1 p.Trp1246Cys Transport activity of wild-type MRP1 and mutant W1246C-MRP1 in transiently transfected HEK293T cells. Login to comment 139 ABCC1 p.Trp1246Cys B, shown is the time course of LTC4 uptake in membrane vesicles prepared from HEK293T cells transiently transfected with wild-type MRP1 (q), mutant W1246C-MRP1 (f), and empty control (E) cDNA expression vectors. Membrane vesicles were incubated at 23 °C with 50 nM [3 H]LTC4 in transport buffer for the times indicated. Results shown are means Ϯ S.D. of triplicate determinations in a single experiment. Similar results were found in three additional independent experiments. Login to comment 140 ABCC1 p.Trp1246Cys C, shown is the time course of E217betaG uptake in membrane vesicles prepared from HEK293T cells transiently transfected with wild-type MRP1 (q), mutant W1246C-MRP1 (f), and control empty (E) cDNA expression vectors. Membrane vesicles were incubated at 37 °C with 400 nM [3 H]E217betaG in transport buffer for the times indicated. Results shown are means Ϯ S.D. of triplicate determinations in a single experiment. Similar results were found in three additional independent experiments. Login to comment 141 ABCC1 p.Trp1246Cys ABCC1 p.Trp1246Cys D, membrane vesicles prepared from wild-type MRP1and W1246C-MRP1-transfected cells (A) were incubated for 2 min with [3 H]LTC4 at 23 °C in the absence (white bars) and presence (black bars) of 25 ␮M E217betaG. Login to comment 144 ABCC1 p.Trp1246Cys ABCC1 p.Trp1246Phe ABCC1 p.Trp1246Tyr A, immunoblots of membrane vesicles prepared from HEK293T cells transiently transfected with pcDNA3.1(-)-MRP1K (wild-type MRP1 (WT-MRP1)), pcDNA3.1(-)-W1236A-MRP1, pcDNA3.1(-)-W1246C-MRP1, pcDNA3.1(-)-W1246F-MRP1, pcDNA3.1(-)-W1246Y-MRP1, and pcDNA3.1(-) alone as a control. Login to comment 146 ABCC1 p.Trp1246Cys ABCC1 p.Trp1246Ala ABCC1 p.Trp1246Phe ABCC1 p.Trp1246Tyr B and C, time course of [3 H]LTC4 uptake by inside-out membrane vesicles prepared from HEK293T cells expressing MRP1 mutants W1246A (Ⅺ) and W1246C (f) (B) and W1246F (Œ) and W1246Y () (C). Login to comment 149 ABCC1 p.Trp1246Cys ABCC1 p.Trp1246Ala ABCC1 p.Trp1246Phe ABCC1 p.Trp1246Tyr D and E, time course of [3 H]E217betaG uptake by inside-out membrane vesicles prepared from MRP1 mutants W1246A (Ⅺ) and W1246C (f) (D) and W1246F (Œ) and W1246Y () (E). Login to comment 156 ABCC1 p.Trp1246Cys ABCC1 p.Trp1246Ala Trp1246 Mutant MRP1 Molecules Are Correctly Routed to the Plasma Membrane-To ensure that the loss of transport activity in the Trp1246 MRP1 mutants was not caused by impaired trafficking of the mutant molecules to the plasma membrane, GFP-tagged constructs of wild-type MRP1 and mutants W1246C and W1246A were generated and transiently transfected into HEK293T cells. Login to comment 159 ABCC1 p.Trp1246Cys To examine whether Trp1246 plays a role in conferring drug resistance, stably transfected cell lines were established by transfection of HeLa cells with pcDNA3.1(-)-MRP1K and pcDNA3.1(-)-W1246C-MRP1. Login to comment 163 ABCC1 p.Trp1246Cys In contrast, the IC50 of the W1246C-MRP1 mutant for vincristine was similar to that of the vector control-transfected cell line. Login to comment 164 ABCC1 p.Trp1246Cys When accumulation of [3 H]vincristine was measured, steady-state concentrations of the drug in the W1246C-MRP1-transfected cells were comparable to those in vector control-transfected cells (33.83 Ϯ 1.94 versus 34.25 Ϯ 2.24 pmol/106 cells/h) (Fig. 6B). Login to comment 166 ABCC1 p.Trp1246Cys The drug resistance phenotype of the W1246C-MRP1-transfected cells was further characterized by determining the sensitivity of these cells to the cationic anthracyclines doxorubicin and daunorubicin as well as the electroneutral epipodophyllotoxin VP-16 (etoposide). Login to comment 167 ABCC1 p.Trp1246Cys As shown in Fig. 7, the IC50 values of the W1246C-MRP1 mutant cells for all three drugs were similar to those of the vector control-transfected cells. Login to comment 168 ABCC1 p.Trp1246Cys Thus, in addition to eliminating the ability to bind and transport the conjugated estrogen E217betaG, the W1246C substitution in MRP1 results in loss of resistance to all classes of natural product drugs tested. Login to comment 169 ABCC1 p.Trp1246Cys Resistance to Potassium Antimony Tartrate Is Retained, but Resistance to Sodium Arsenite Is Reduced, in W1246C-MRP1-expressing Cells-We have previously shown that in addition to conferring resistance to anticancer drugs, both human and murine MRP1 can confer low level resistance to arsenical and antimonial oxyanions (4, 28). Login to comment 170 ABCC1 p.Trp1246Cys When tested for sensitivity to potassium antimony tartrate, the IC50 of the W1246C mutant HeLa cells was comparable to that of cells expressing wild-type MRP1 (40 versus 30 ␮g/ml) (Fig. 8A). Login to comment 172 ABCC1 p.Trp1246Cys In contrast, W1246C mutant cells displayed reduced resistance to sodium arsenite (Fig. 8B). Login to comment 173 ABCC1 p.Trp1246Cys Thus, the IC50 of W1246C mutant HeLa cells for sodium arsenite was 1 ␮g/ml 4 W. Qiu, A. Haimeur, R. G. Deeley, and S. P. C. Cole, unpublished observations. FIG. 4. Login to comment 181 ABCC1 p.Trp1246Cys ABCC1 p.Trp1246Ala HEK293T cells were transfected with pcDNA3.1(-)-MRP1K-GFP (A, D, and G), pcDNA3.1(-)-W1246C-MRP1-GFP (B, E, and H), and pcDNA3.1(-)-W1246A-MRP1-GFP (C, F, and I); and 48 h later, cells were processed for confocal fluorescence microscopy as described under "Experimental Procedures." Login to comment 191 ABCC1 p.Pro196Ala In the course of a mutational analysis of this region of MRP1, we derived a mutant in which Pro196 was replaced with Ala by site-directed mutagenesis (34). Login to comment 195 ABCC1 p.Trp1246Cys A, vincristine sensitivity of HeLa cell lines stably transfected with the pcDNA3.1(-) vector (⅜), pcDNA3.1(-)-MRP1K (ⅷ), and pcDNA3.1(-)-W1246C-MRP1 (f) was determined using a tetrazolium salt-based chemosensitivity assay as described under "Experimental Procedures." Login to comment 197 ABCC1 p.Trp1246Cys B, [3 H]vincristine (VCR) accumulation in HeLa cell lines stably transfected with the pcDNA3.1(-) vector (white bar), pcDNA3.1(-)-MRP1K (wild-type MRP1 (WT-MRP1); black bar), and pcDNA3.1(-)-W1246C-MRP1 (gray bar). Login to comment 202 ABCC1 p.Trp1246Cys HeLa cell lines stably transfected with the pcDNA3.1(-) vector (⅜), pcDNA3.1(-)-MRP1K (ⅷ), and pcDNA3.1(-)-W1246C-MRP1 (f) were exposed to doxorubicin (A), daunorubicin (B), and VP-16 (C) for 72 h at 37 °C, and then cell viability was measured as described under "Experimental Procedures." Login to comment 205 ABCC1 p.Trp1246Cys ABCC1 p.Pro196Ala ABCC1 p.Pro196Ala In contrast, a second independently derived transfected cell line also expressing an MRP1 protein with the same P196A mutation displayed a phenotype similar to that of cells expressing wild-type MRP1.2 It was subsequently discovered that the transfected MRP1 in the first P196A mutant cell line had acquired a second non-engineered mutation causing the substitution of Cys for Trp at position 1246. Login to comment 206 ABCC1 p.Trp1246Cys ABCC1 p.Trp1246Cys ABCC1 p.Pro196Ala Consequently, we generated a single W1246C mutant of MRP1 so that its phenotype could be compared with that of the double P196A/W1246C mutant. Login to comment 207 ABCC1 p.Trp1246Cys When transiently transfected into HEK293T cells, W1246C-MRP1 could be expressed at levels comparable to those of wild-type MRP1 and also transported LTC4 with a similar efficiency. Login to comment 209 ABCC1 p.Trp1246Cys ABCC1 p.Trp1246Cys ABCC1 p.Trp1246Cys ABCC1 p.Pro196Ala However, as observed for the double P196A/ W1246C mutant, transport of E217betaG by W1246C-MRP1 membrane vesicles was not detectably different from that obtained with vesicles from vector control-transfected cells, and although LTC4 transport appeared unchanged in the W1246C mutants, it could no longer be inhibited by E217betaG. Login to comment 210 ABCC1 p.Trp1246Cys ABCC1 p.Pro196Ala Taken together, these results allowed us to conclude that the altered phenotype of the P196A/W1246C-transfected cells was attributable to the single tryptophan substitution at position 1246 in MSD3 and did not require the additional proline substitution at position 196 in the cytoplasmic loop between MSD1 and MSD2. Login to comment 211 ABCC1 p.Trp1246Cys ABCC1 p.Trp1246Ala Additional mutants in which Trp1246 was substituted with Ala, Phe, and Tyr displayed transport characteristics similar to those of W1246C-MRP1, indicating that replacement of the tryptophan residue rather than the introduced cysteine was responsible for the phenotype. Login to comment 212 ABCC1 p.Trp1246Cys To examine the drug resistance phenotype of the W1246C mutation, stably transfected HeLa cells were generated. Login to comment 213 ABCC1 p.Trp1246Cys In addition to the selective loss of E217betaG transport, we found that W1246C-MRP1-expressing cells were no longer resistant to natural product chemotherapeutic agents, including the electroneutral VP-16 and the cationic vincristine and anthracyclines. Login to comment 214 ABCC1 p.Trp1246Cys Consistent with this loss of drug resistance, vincristine accumulation in intact cells expressing W1246C-MRP1 was comparable to that in vector control-transfected cells, which was ϳ2-fold higher than vincristine accumulation in HeLa cells expressing wild-type MRP1. Login to comment 215 ABCC1 p.Trp1246Cys On the other hand, the W1246C-MRP1-expressing cells were still resistant to antimony tartrate and partially resistant to sodium arsenite. Login to comment 230 ABCC1 p.Trp1246Cys HeLa cell lines stably transfected with the pcDNA3.1(-) vector (⅜), pcDNA3.1(-)-MRP1K (q), and pcDNA3.1(-)- W1246C-MRP1 (f) were exposed to potassium antimony tartrate (A) or sodium arsenite (B) for 72 h at 37 °C, and then cell viability was measured as described under "Experimental Procedures." Login to comment 234 ABCC1 p.Trp1246Cys Thus, the absence of E217betaG transport activity and drug resistance in the W1246C-MRP1 mutant can be explained by the fact that although cysteine can participate in hydrogen bonding, it is considerably smaller than tryptophan and lacks aromaticity. Login to comment 241 ABCC1 p.Trp1246Ala Our recent finding that wild-type MRP1, but not W1246A-MRP1, transports the O-glucuronide of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol indicates that Trp1246 is important for the transport of other glucuronide conjugates as well.5 It has been proposed (and there is considerable evidence to support the notion) that substrates for P-glycoprotein are taken up from the inner leaflet of the plasma membrane (39). Login to comment