ABCMdb

PMID: 12042670

Conrad S, Kauffmann HM, Ito K, Leslie EM, Deeley RG, Schrenk D, Cole SP
A naturally occurring mutation in MRP1 results in a selective decrease in organic anion transport and in increased doxorubicin resistance.
Pharmacogenetics. 2002 Jun;12(4):321-30., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC1 p.Arg433Ser In this study, we characterized a low-frequency (<1%) naturally occurring mutation in MRP1 expected to cause the substitution of a conserved arginine with serine at position 433 in a predicted cytoplasmic loop of the protein. Login to comment 5 ABCC1 p.Arg433Ser In contrast, 17â- oestradiol-17â-(D-glucuronide) transport by the Arg433 Ser mutant MRP1 was similar to that by wild-type MRP1. Login to comment 7 ABCC1 p.Arg433Ser In contrast to the reduced LTC4 and oestrone sulphate transport, stably transfected HeLa cells expressing Arg433 Ser mutant MRP1 were 2.1-fold more resistant to doxorubicin than cells expressing wild-type MRP1, while resistance to VP-16 and vincristine was unchanged. Login to comment 31 ABCC1 p.Arg433Ser In addition, we have recreated the Arg433 Ser substitution in a MRP1 mammalian expression vector and analysed its transport and drug resistance properties after transfection into human cells. Login to comment 38 ABCC1 p.Arg433Ser Site-directed mutagenesis The mutation Arg433 Ser was generated using the following sense primer 59P-GTGGACGCTCAGTC GTTCATGGACTTGGCC-39 (substituted nucleotides are underlined) with the USE Mutagenesis Kit (Amersham Pharmacia Biotech, PQ, Canada), according to the manufacturer`s instructions. Login to comment 79 ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser Results A heterozygous Arg433 Ser substitution in MRP1 does not result in any overt clinical symptoms A mutation in exon 10 of the human MRP1 gene expected to result in substitution of Arg433 to Ser was found in a single individual among 91 healthy Caucasian volunteers. Login to comment 81 ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser An Arg433 Ser substitution causes a reduction in MRP1-mediated LTC4 and oestrone sulphate transport activity, but leaves E217âG transport activity intact To determine whether MRP1-mediated transport of [3 H]LTC4, [3 H]oestrone sulphate and [3 H]E217âG was affected by a Arg433 Ser substitution, this mutation was generated in a mammalian MRP1 expression vector and transiently transfected into HEKSV293T cells. Login to comment 82 ABCC1 p.Arg433Ser Stably transfected clonal HeLa cell lines expressing the Arg433 Ser MRP1 mutant were also generated in order to confirm the results obtained with the transient transfectants and to be able to test drug resistance in longer term assays. Login to comment 85 ABCC1 p.Arg433Ser In contrast, levels of [3 H]E217âG uptake by the Arg433 Ser mutant MRP1 and wild-type MRP1 were similar and proportional to their relative protein expression levels (Fig. 2). Login to comment 87 ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser Arg433 Ser mutant MRP1 is correctly routed to the plasma membrane To determine whether the decrease in LTC4 and oestrone sulphate transport caused by the Arg433 Ser substitution might be associated with a change in the trafficking of MRP1 to the plasma membrane, the subcellular localization of the mutant and wild-type MRP1 was determined by indirect immunofluorescent confocal microscopy. Login to comment 88 ABCC1 p.Arg433Ser As shown in Fig. 4, both the wild-type and mutant proteins expressed in HeLa cells showed a pattern of strong plasma membrane staining, indicating that MRP1 trafficking in the transfected cells was unaffected by the Arg433 Ser substitution. Login to comment 89 ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser Kinetic analysis of ATP-dependent [3 H]LTC4 and [3 H]oestrone sulphate transport by Arg433 Ser mutant MRP1 Since the initial time courses of LTC4 and oestrone sulphate uptake indicated a two-fold reduction in transport activity of the Arg433 Ser mutant MRP1, the effect of this mutation on the transport kinetics of these substrates was examined. Login to comment 91 ABCC1 p.Arg433Ser A non-linear regression analysis of the data obtained showed small but not significant differences in the apparent Km values of the wild-type and Arg433 Ser mutant proteins for LTC4 and oestrone sulphate. Login to comment 92 ABCC1 p.Arg433Ser In contrast, the apparent Vmax values of the Arg433 Ser mutant for both substrates were approximately two-fold lower than for wild-type MRP1 when normalized for relative levels of protein expression. Login to comment 93 ABCC1 p.Arg433Ser Drug resistance profile of cells expressing Arg433 Ser MRP1 In addition to its ability to transport conjugated organic anions, MRP1 can also confer resistance to a variety of anticancer drugs by reducing intracellular drug accumulation [21,22]. Login to comment 94 ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser To determine whether the Arg433 Ser substitution affected the drug resistance properties of MRP1, the drug resistance profile of Arg433 Ser MRP1 Fig. 1 Expression and ATP-dependent [3 H]LTC4 transport activity of wild-type and mutant Arg433 .Ser MRP1 in membrane vesicles prepared from transfected cells. Login to comment 95 ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser (a) MRP1 expression levels in membrane vesicles of human embryonic kidney cells transfected with pcDNA3.1(-) as control, pcDNA3.1(-)wt-MRP1K (wtMRP1) and pcDNA3.1(-) MRP1K-Arg433 Ser (R433S) cDNA expression vectors were determined by dot blot analysis with MAb QCRL-1. Login to comment 97 ABCC1 p.Arg433Ser (b) Time course of [3 H]LTC4 uptake in membrane vesicles shown in (a) prepared from HEKSV293T cells transfected with wild-type MRP1 (wtMRP1, j), mutant (R433S, m) and control (pcDNA3.1(-), h) cDNA expression vectors. Results shown are means (Æ SD) of triplicate determinations in a single experiment. Similar results were obtained in two additional independent experiments with vesicles from transfected HEKSV293T cells and in two additional experiments with vesicles from transfected HeLa cells. Login to comment 99 ABCC1 p.Arg433Ser The Arg433 Ser mutation increased resistance to the cationic anthracycline agent, doxorubicin by approximately two-fold (P , 0.01) relative to wild-type MRP1 (Fig. 6a). Login to comment 100 ABCC1 p.Arg433Ser In contrast, the sensitivity of the Arg433 Ser mutant to the Vinca alkaloid, vincristine, and the electroneutral epipodophyllotoxin, VP-16 (Fig. 6b,c) was comparable to that conferred by wild-type MRP1. Login to comment 108 ABCC1 p.Arg433Ser Thus, it seems likely that this transport protein contains more than one Fig. 2 Expression and ATP-dependent [3 H]E217âG transport activity of wild-type and mutant Arg433 Ser MRP1 in membrane vesicles prepared from transfected cells. Login to comment 110 ABCC1 p.Arg433Ser (b) Time course of [3 H]E217âG uptake in membrane vesicles shown in (a) (wtMRP1, j; mutant R433S, m; control pcDNA3.1(-), h). Login to comment 114 ABCC1 p.Arg433Ser (b) Time course of [3 H]oestrone sulphate uptake in the presence of 1 mmol GSH in the membrane vesicles shown in (a) prepared from cells transfected with wild-type MRP1 (wtMRP1, j), mutant (R433S, m) and empty vector control (pcDNA3.1(-), h) cDNA expression vectors. Results shown are means (Æ SD) of triplicate determinations in a single experiment. Similar results were obtained in three additional independent experiments with vesicles from transfected HeLa cells and in two experiments with vesicles from transfected HEKSV293T cells. Login to comment 118 ABCC1 p.Gly671Val ABCC1 p.Arg433Ser Of all the mutations detected, two are predicted to cause amino acid substitutions in the protein, Gly671 Val and Arg433 Ser. Login to comment 119 ABCC1 p.Gly671Val The Gly671 Val polymorphism is located near the Walker A motif of the first nucleotide binding domain of MRP1. Login to comment 121 ABCC1 p.Arg433Ser According to most topological models of MRP1, the second mutation, an Arg433 Ser substitution, is located close to the COOH-proximal end of the cytoplasmic loop connecting the predicted transmembrane (TM)7 to TM8 in the second membrane spanning domain (MSD2) of MRP1 [41-43]. Login to comment 122 ABCC1 p.Gly671Val In the present study, we found that this mutation, in contrast to Gly671 Val, results in significant substrate-specific alterations of MRP1 function. Login to comment 123 ABCC1 p.Arg433Ser When the properties of a recombinant Arg433 Ser MRP1 mutant expressed in transfected human cells were examined, a two-fold reduction in LTC4 and GSH-stimulated oestrone sulphate transport activity was observed even though the expression levels and membrane localization of the mutant MRP1 and wild-type MRP1 were comparable. Login to comment 125 ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser Thus, the Vmax values for LTC4 and GSH-stimulated oestrone sulphate transport by the Arg433 Ser mutant MRP1 were approximately two-fold lower than Fig. 4 Confocal microscopy of transfected HeLa cells expressing wild-type and Arg433 Ser mutant MRP1 proteins. Login to comment 130 ABCC1 p.Arg433Ser (b) Transfected HeLa cells expressing Arg433 Ser mutant MRP1. Login to comment 132 ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser (a) ATP-dependent uptake of [3 H]LTC4 by the membrane vesicles shown in Fig. 1(a) was measured at various LTC4 concentrations (0.01-1 ìM) for 45 s at 23 8C [wild-type MRP1 (wtMRP1, j; and Arg433 Ser mutant MRP1 (R433S, m)]. Login to comment 135 ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser (b) ATP-dependent uptake of [3 H]oestrone sulphate by the membrane vesicles shown in Fig. 3(a) [wild-type MRP1 (wtMRP1, j) and Arg433 Ser mutant MRP1 (R433S, m)]. Login to comment 141 ABCC1 p.Arg433Ser In contrast, the levels of E217âG uptake by the Arg433 Ser mutant MRP1 were unchanged. Login to comment 154 ABCC1 p.Glu1089Gln Moreover, substitution of the acidic Glu residue at position 1089 with Gln can selectively alter the ability of MRP1 to confer resistance to anthracycline antibiotics, but has no effect on its ability to transport E217âG or LTC4 [39]. Login to comment 155 ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser HeLa cells expressing the Arg433 Ser mutant protein were observed to be two-fold more resistant to the anthracycline doxorubicin in addition to displaying a two-fold reduction in LTC4 and GSH-stimulated oestrone sulphate transport activity. Enhanced resistance was specific for this anthracycline as the resistance of cells expressing the Arg433 Ser mutant to the Vinca alkaloid, vincristine, and to the epipodophyllotoxin, VP-16, remained comparable to cells expressing wild-type MRP1. Login to comment 160 ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser Furthermore, TM segments corresponding to those identified in P-glycoprotein as being important for substrate binding and/or transport have also been Table 1 Kinetic parameters of [3 H]LTC4 and GSH-stimulated [3 H]oestrone sulphate uptake by membrane vesicles from cells transfected with expression vectors encoding wild-type and Arg433 Ser mutant MRP1 Km (ìM) Vmax (pmol/mg/min) Normalized Vmax (pmol/mg/min) Substrate wtMRP1 R433S wtMRP1 R433S wtMRP1 R433S LTC4 0.18 Æ 0.04 0.14 Æ 0.06 121 Æ 8 71 Æ 10 121 Æ 8 59 Æ 8 Oestrone sulphate 0.89 Æ 0.07 1.10 Æ 0.34 72 Æ 2 41 Æ 4 72 Æ 2 34 Æ 3 Apparent Km and Vmax values for LTC4 and GSH-stimulated oestrone sulphate uptake were determined as described in Materials and Methods and the legend to Fig. 5. Login to comment 169 ABCC1 p.Arg433Ser Thus, our data, demonstrating that the doxorubicin resistance of cells expressing the Arg433 Ser mutation is increased while vincristine and VP-16 resistance are unaffected, indicate that amino acid changes in this cytoplasmic loop of MRP1, similar to the comparable loop in P-glycoprotein, can selectively affect the drug resistance profile conferred by this transporter. Login to comment 171 ABCC1 p.Arg433Ser Cells stably transfected with wild-type MRP1 (wtMRP1, j), mutant (R433S, m) and control (pcDNA3.1(-), h) cDNA expression vectors were exposed to (a) doxorubicin, (b) vincristine and (c) etoposide at the concentrations indicated for 72 h at 37 8C. Login to comment 174 ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser 100 75 50 25 0.1 1 10 100 Vincristine (nM) %Controlabsorbance 100 75 50 25 1 10 100 Etoposide (µM) %Controlabsorbance Table 2 Relative drug resistance of HeLa cells expressing wild-type and Arg433 Ser mutant MRP1 Relative resistancea Drug wtMRP1 Arg433 Ser MRP1 Ratio Doxorubicin 2.5 Æ 0.6 (n ¼ 5) 5.3 Æ 1.0 (n ¼ 8)b 2.1 Vincristine 7.0 Æ 4.1 (n ¼ 5) 10.3 Æ 5.0 (n ¼ 10) 1.5 VP-16 3.4 Æ 1.4 (n ¼ 6) 3.8 Æ 1.6 (n ¼ 6) 1.1 a The resistance of stably transfected HeLa cells was determined using a tetrazolium-based microtitre plate assay. The relative resistance factors were obtained by dividing the IC50 values for wild-type or Arg433 Ser mutant MRP1 transfected cells by the IC50 values for empty vector control transfected cells and are normalized for differences in protein expression levels as appropriate. Login to comment 177 ABCC1 p.Arg433Ser Thus, the Arg433 Ser substitution results in an additional potential phosphorylation site which, if utilized, might in some direct or indirect way influence the activity of the protein. Login to comment 179 ABCC1 p.Arg433Ser The MRP1 mutation predicted to cause the Arg433 Ser substitution described here was present in a cohort of 91 healthy Caucasian volunteers with an estimated allelic frequency of ,1%. Login to comment 181 ABCC1 p.Arg433Ser However, the mutation was heterozygous in the case found and the alterations in Arg433 Ser MRP1 transport activity observed in the current study suggest that homozygous carriers should be examined before ruling out possible physiological consequences associated with this mutation in vivo. Login to comment 182 ABCC1 p.Arg433Ser Thus, the two-fold decrease in LTC4 and GSH-stimulated oestrone sulphate transport by the Arg433 Ser mutant MRP1 in vitro raises the possibility that individuals bearing this polymorphism might be subject to a variety of biological effects, such as an impaired response to an inflammatory stimulus [30-32]. Login to comment