ABCMdb

PMID: 15083066

Lee YM, Cui Y, Konig J, Risch A, Jager B, Drings P, Bartsch H, Keppler D, Nies AT
Identification and functional characterization of the natural variant MRP3-Arg1297His of human multidrug resistance protein 3 (MRP3/ABCC3).
Pharmacogenetics. 2004 Apr;14(4):213-23., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC3 p.Arg1297His Identification and functional characterization of the natural variant MRP3-Arg1297 His of human multidrug resistance protein 3 (MRP3/ABCC3) Young-Min A. Leea , Yunhai Cuia , Jo¨rg Ko¨niga , Angela Rischb , Birgit Ja¨gerb , Peter Dringsc , Helmut Bartschb , Dietrich Kepplera and Anne T. Niesa The human multidrug resistance protein 3 (MRP3, symbol ABCC3) is an ATP-binding cassette transporter that mediates the efflux of organic anions, including lipophilic substances conjugated with glucuronate, sulphate or glutathione, across the basolateral membrane of polarized cells (e.g. hepatocytes) into blood. Login to comment 4 ABCC3 p.Arg1297His The 3890G>A mutation, resulting in MRP3-Arg1297 His, was introduced into the ABCC3 cDNA which was stably transfected into MDCKII cells. Login to comment 5 ABCC3 p.Arg1297His For the functional characterization of MRP3-Arg1297 His in comparison with MRP3, ATP-dependent transport was analysed in isolated membrane vesicles. Login to comment 6 ABCC3 p.Leu548Gln ABCC3 p.Arg1297His Two non-synonymous MRP3 variants were identified with an allele frequency of 0.003 for 1643T>A (MRP3-Leu548 Gln) and 0.08 for 3890G>A (MRP3-Arg1297 His). Login to comment 7 ABCC3 p.Arg1297His Because of the high frequency of the 3890G>A mutation, and because of the close proximity of Arg1297 to the second nucleotide-binding domain, we pursued the functional characterization of the MRP3-Arg1297 His polymorphic variant. Login to comment 8 ABCC3 p.Arg1297His MRP3-Arg1297 His was correctly localized to the basolateral membrane of polarized MDCKII cells. Login to comment 9 ABCC3 p.Arg1297His We identified monoglucuronosyl bilirubin, bisglucuronosyl bilirubin and leukotriene C4 as substrates for both MRP3 and MRP3-Arg1297 His. Login to comment 10 ABCC3 p.Arg1297His Dehydroepiandrosterone-3-sulphate and 17â-glucuronosyl oestradiol were transported with similar kinetics by MRP3 and MRP3-Arg1297 His. Login to comment 44 ABCC3 p.Leu548Gln ABCC3 p.Arg1297His In addition to characterizing human MRP3 as a transporter for bilirubin glucuronosides, we identified two SNPs in the human ABCC3 gene that resulted in non-synonymous amino acid changes (i.e. MRP3-Leu548 Gln and MRP3-Arg1297 His). Login to comment 45 ABCC3 p.Arg1297His We functionally characterized the polymorphic variant MRP3-Arg1297 - His because of its high frequency in the Caucasian population and because amino acid Arg1297 is located close to the second nucleotide-binding domain (NBD). Login to comment 75 ABCC3 p.Arg1297His Site-directed mutagenesis The nucleotide exchange 3890G.A, leading to the amino acid exchange Arg1297 His, was introduced into the ABCC3 cDNA (GenBank/EBI data bank accession no. Login to comment 87 ABCC3 p.Arg1297His The fragment was then subcloned into the mammalian expression vector MRP3/pcDNA3.1(+) [8] between the restriction sites SanDI and AgeI, resulting in MRP3-Arg1297 His/pc DNA3.1. Login to comment 90 ABCC3 p.Arg1297His For stable transfection, parental MDCKII cells were grown in 10-cm diameter dishes until reaching confluency and transfected with the MRP3/pcDNA3.1-vector, MRP3-Arg1297 His/ pcDNA3.1-vector, or pcDNA3.1-vector (¼ control) using the polybrene method [36]. Login to comment 95 ABCC3 p.Arg1297His MRP3 and MRP3-Arg1297 His were detected with the FDS antiserum diluted 1 : 500 in Tris-buffered saline/Tween20 (20 mM Tris, 145 mM NaCl, 2.7 mM KCl, 0.05% Tween 20, pH 7.6) containing 5% milk powder. Login to comment 142 ABCC3 p.Arg1297His Because of the high frequency of the 3890G.A mutation in the Caucasian population, we analysed the functional consequence of the MRP3-Arg1297 His polymorphism. Login to comment 143 ABCC3 p.Arg1297His Immunoblot and immunofluorescence analysis of MRP3 and MRP3-Arg1297 His stably expressed in MDCK cells In line with previous studies [6,8], recombinant MRP3 appeared as a fully glycosylated form of 190 kDa and of a less glycosylated form of approximately 170 kDa in immunoblot analysis (Fig. 2a), the ratio of the 190 to the 170 kDa form being 1.8 Æ 0.3 (n ¼ 3) for this membrane vesicle preparation. Login to comment 145 ABCC3 p.Arg1297His The MRP3-Arg1297 His protein was also present in a 190 and a 170 kDa form, their ratio being 1.4 Æ 0.1 (n ¼ 3) in this preparation. Login to comment 146 ABCC3 p.Arg1297His Two other MRP3-Arg1297 His membrane vesicle preparations had ratios of 1.3 and 1.4. Login to comment 147 ABCC3 p.Arg1297His In the given blot (Fig. 2a), the level of the synthesized 190 kDa form of MRP3-Arg1297 His was 1.04 of that of MRP3 and 1.15 when both glycosylated forms were taken into account. Login to comment 149 ABCC3 p.Arg1297His ABCC3 p.Arg1297His Using confocal laser scanning microscopy (Fig. 2b-e), MRP3 and MRP3-Arg1297 His were localized to the basolateral membrane of MDCKII cells, indicating the synthesis of a full-length MRP3-Arg1297 His and its correct basolateral routing. Login to comment 151 ABCC3 p.Arg1297His ABCC3 p.Arg1297His ATP-dependent transport of 17â-glucuronosyl [3 H]oestradiol, [3 H]LTC4 and [3 H]DHEAS by MRP3 and MRP3-Arg1297 His For the functional characterization of MRP3-Arg1297 His, transport assays with [3 H]E217âG, [3 H]LTC4 and [3 H]DHEAS were performed. Login to comment 152 ABCC3 p.Arg1297His ATP-dependent transport of all three substances was detected for MRP3 and MRP3-Arg1297 His. Login to comment 153 ABCC3 p.Arg1297His Transport by MRP3-Arg1297 His showed similar transport kinetics as MRP3 (Fig. 3). Login to comment 156 ABCC3 p.Leu548Gln ABCC3 p.Arg1297His ABCC3 p.Arg1297His Unauthorized reproduction of this article is prohibited. Table 4 Allelic variations in human MRP3 Exon Nucleotide variation Amino acid variation Allele frequency Type of mutation 13 1643T.A Leu548 Gln T ¼ 0.997 Non-synonymous A ¼ 0.003 22 3039C.T Gly1013 Gly C ¼ 0.94 Synonymous T ¼ 0.06 27 3890G.A Arg1297 His G ¼ 0.92 Non-synonymous A ¼ 0.08 27 3942C.T His1314 His C ¼ 0.76 Synonymous T ¼ 0.24 29 4266C.T Gly1422 Gly C ¼ 0.99 Synonymous T ¼ 0.01 31 4509A.G Glu1503 Glu A ¼ 0.80 Synonymous G ¼ 0.20 (c) (e) (d) MRP3MRP3-Arg1297 His(b) 180 kDa M RP3-Arg1297 H is C o M RP3 (a) Fig. 2 Analysis of the synthesis of MRP3 and MRP3-Arg1297 His in polarized MDCKII cells. Login to comment 157 ABCC3 p.Arg1297His (a) Immunoblot analysis using the FDS antiserum directed against the C-terminus of human MRP3 [8] indicates synthesis of full-length and fully glycosylated MRP3 and MRP3-Arg1297 His in transfected MDCKII cells. Login to comment 160 ABCC3 p.Arg1297His (b-e) Confocal laser scanning micrographs after reaction with the FDS antiserum showed basolateral localization of MRP3-Arg1297 His (b,c) and MRP3 (d,e) in polarized MDCKII cells. Login to comment 162 ABCC3 p.Arg1297His Scale bar ¼ 20 ìm. values) of vesicles from MRP3-, MRP3-Arg1297 His-transfected and control cells for E217âG were 24.2 Æ 5.8 ìM (71.5 Æ 6.3 pmol/mg protein per min), 16.0 Æ 10.9 ìM (72.3 Æ 19.5 pmol/mg protein per min) and 13.4 Æ 7.5 ìM (25.0 Æ 5.2 pmol/mg protein per min), respectively. Login to comment 164 ABCC3 p.Arg1297His Km values (and Vmax values) for vesicles from MRP3- and MRP3-Arg1297 His-transfected cells for DHEAS were 46.3 Æ 7.3 (281 Æ 21 pmol/mg protein per min) and 34.6 Æ 5.7 ìM (269 Æ 19 pmol/mg protein per min), respectively. Login to comment 165 ABCC3 p.Arg1297His ABCC3 p.Arg1297His ATP-dependent transport of [3 H]-monoglucuronosyl bilirubin and [3 H]-bisglucuronosyl bilirubin by MRP3 and MRP3-Arg1297 His Membrane vesicles from MRP3- or MRP3-Arg1297 His-transfected MDCKII cells showed significant ATP-dependent transport (P , 0.001 compared to controls) of [3 H]MGB (12 nM) with a transport rate of 0.12 pmol/ Copyright (c) Lippincott Williams & Wilkins. Login to comment 167 ABCC3 p.Arg1297His ABCC3 p.Arg1297His ABCC3 p.Arg1297His ABCC3 p.Arg1297His Time (min) MRP3 MRP3-Arg1297 His Control 9 6 3 0 ATP-dependent[3 H]LTC4transport (pmol/mgprotein) (b) 0 2 4 6 8 10 MRP3 MRP3-Arg1297 His Control 120 80 40 0 ATP-dependent[3 H]DHEAStransport (pmol/mgprotein) (c) 0 2 4 6 8 10 MRP3 MRP3-Arg1297 His Control 290 150 100 50 0 ATP-dependent[3H]E217βGtransport (pmol/mgprotein) (a) 0 2 4 6 8 10 Fig. 3 ATP-dependent transport of (a) 17â-glucuronosyl oestradiol (E217âG), (b) leukotriene C4 (LTC4) and (c) dehydroepiandrosterone-3-sulphate (DHEAS) into membrane vesicles from MRP3-, MRP3-Arg1297 His-transfected and control MDCKII cells. Login to comment 171 ABCC3 p.Arg1297His ABCC3 p.Arg1297His ABCC3 p.Arg1297His MRP3 Km 24µM E217βG 60 40 20 0 [3H]E217βGtransport (pmol/mgproteinpermin) (a) 0 20 40 60 80 E217βG (µM) MRP3-Arg1297His Km 16µM E217βG 60 40 20 0 (b) 0 10 20 40 50 E217βG (µM) 30 MRP3-Arg1297His Km 35µM DHEAS250 150 50 0 (d) 0 30 60 90 120 DHEAS (µM) 200 100 MRP3 Km 46µM DHEAS250 200 100 0 (c) 0 30 60 90 120 DHEAS (µM) 150 50 [3H]DHEAStransport (pmol/mgproteinpermin) Fig. 4 Kinetic analysis of MRP3- and MRP3-Arg1297 His-mediated transport of (a,b) glucuronosyl oestradiol (E217âG) or (c,d) dehydroepiandrosterone-3-sulphate (DHEAS). Login to comment 172 ABCC3 p.Arg1297His Rates of ATP-dependent transport of [3 H]E217âG or [3 H]DHEAS were determined in membrane vesicles from MRP3- or MRP3-Arg1297 His-transfected MDCKII cells at the indicated substrate concentrations after 5 min. Login to comment 177 ABCC3 p.Arg1297His For each substrate, the transport kinetics of MRP3 and MRP3-Arg1297 His were similar. Login to comment 178 ABCC3 p.Arg1297His Only the 10-min value of the [3 H]BGB transport was significantly higher for MRP3-Arg1297 His than for MRP3 (Fig. 5f). Login to comment 181 ABCC3 p.Arg1297His ABCC3 p.Arg1297His ABCC3 p.Arg1297His ABCC3 p.Arg1297His ABCC3 p.Arg1297His ATP 5'-AMP MRP3 MGB (a) 2 1 0 [3 H]MGBtransport(pmol/mgprotein) 0 5 10 ATP 5'-AMP MRP3-Arg1297His MGB (b) 2 1 0 [3 H]MGBtransport(pmol/mgprotein) 0 5 10 MRP3 MRP3-Arg1297 His (c) 2 1 0 ATP-dependent[3H]MGBtransport (pmol/mgprotein) 0 5 10 Control Time (min) MRP3 MRP3-Arg1297 His (f) 2 1 0 ATP-dependent[3 H]BGBtransport (pmol/mgprotein) 0 5 10 Control Time (min) * ATP 5'-AMP MRP3-Arg1297 His BGB (e) 2 1 0 [3 H]BGBtransport(pmol/mgprotein) 0 5 10 ATP 5'-AMP MRP3 BGB (d) 2 1 0[3 H]BGBtransport(pmol/mgprotein) 0 5 10 Fig. 5 Transport of (a-c) [3 H]monoglucuronosyl bilirubin (MGB) and (d-f) [3 H]bisglucuronosyl bilirubin (BGB) into membrane vesicles from MRP3- and MRP3-Arg1297 His-transfected MDCKII cells. Login to comment 193 ABCC3 p.Leu548Gln ABCC3 p.Arg1297His In addition to several synonymous mutations that have been described in a Japanese population [29], we identified two non-synonymous mutations in MRP3 (i.e. MRP3-Leu548 Gln and MRP3-Arg1297 His) (Fig. 1, Table 4). Login to comment 194 ABCC3 p.Arg1297His We pursued the functional characterization of MRP3-Arg1297 His because Arg1297 is located close to the Walker A motif of the second NBD (Fig. 1). Login to comment 196 ABCC3 p.Arg1297His Recombinant MRP3 and MRP3-Arg1297 His were present in 190- and 170-kDa forms (Fig. 2a), both of which are most likely differentially glycosylated forms [6,8]. Login to comment 197 ABCC3 p.Arg1297His The level of the less glycosylated form appeared to be higher for MRP3-Arg1297 His than for MRP3. However, the proportion of both forms may vary as observed for the different MRP3 membrane vesicle preparations. Login to comment 200 ABCC3 p.Arg1297His Therefore, the MRP3-Arg1297 His protein is probably also localized in the basolateral membrane of human hepatocytes. Login to comment 201 ABCC3 p.Arg1297His However, this remains to be proven because human liver samples were not available from patients with the MRP3-Arg1297 His polymorphic variant. Login to comment 202 ABCC3 p.Arg1297His ABCC3 p.Arg1297His Because the Arg1297 His polymorphism had no effect on maturation and basolateral localization of MRP3 (Fig. 2), we analysed whether MRP3-Arg1297 His differs in its transport properties from MRP3. Login to comment 204 ABCC4 p.Leu1084Lys For example, induced mutations in rat Mrp2 resulted in acquired transport activity of Mrp2 for taurocholate, whereas mutation of the corresponding position in Mrp3 (Mrp3- Leu1084 Lys) resulted in loss of transport activity of Mrp3 for taurocholate and glucuronoside conjugates [41]. Login to comment 209 ABCC1 p.Arg433Ser However, a naturally occurring variant (MRP1-Arg433 Ser) has been described, which was correctly routed to the plasma membrane but showed a selective decrease in organic anion transport [45]. Login to comment 210 ABCC1 p.Gly671Val By contrast, another natural polymorphic variant, MRP1-Gly671 Val, which is located near the first NBD of MRP1, had similar transport characteristics as MRP1 [46]. Login to comment 211 ABCC3 p.Arg1297His In the case of MRP3-Arg1297 His, we also observed similar transport characteristics as for MRP3 (Fig. 3) with the established MRP3 substrates LTC4 and E217âG [1-4]. Login to comment 212 ABCC3 p.Arg1297His The Km values for E217âG of MRP3 and MRP3-Arg1297 His (Fig. 4) were in the range of those reported by Zeng et al. [2] (26 ìM) and Akita et al. [4] (43 ìM). Login to comment 213 ABCC3 p.Arg1297His We additionally examined the bilirubin glucuronosides MGB and BGB, and the steroid DHEAS as MRP3 substrates (Figs 3-5), and showed that they were transported with similar kinetic characteristics by MRP3-Arg1297 His as well as by MRP3. Login to comment 214 ABCC3 p.Arg1297His In the case of significant transport differences between MRP3-Arg1297 His and MRP3, studies would be of interest to elucidate whether interindividual variations of MRP3 affect the response of an individual to chemotherapy. Login to comment 227 ABCC3 p.Arg1297His In conclusion, we identified two non-synonymous SNPs and functionally characterized for the first time a natural variant of MRP3, MRP3-Arg1297 His. Login to comment 229 ABCC3 p.Arg1297His Thus, based on our membrane vesicle transport assays, individuals with the MRP3-Arg1297 His variant are not expected to be affected in their ability to export MRP3 substrates into blood. Login to comment