ABCMdb

PMID: 14645676

Nakanishi T, Doyle LA, Hassel B, Wei Y, Bauer KS, Wu S, Pumplin DW, Fang HB, Ross DD
Functional characterization of human breast cancer resistance protein (BCRP, ABCG2) expressed in the oocytes of Xenopus laevis.
Mol Pharmacol. 2003 Dec;64(6):1452-62., [PubMed]

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0 ABCG2 p.Arg482Thr Functional Characterization of Human Breast Cancer Resistance Protein (BCRP, ABCG2) Expressed in the Oocytes of Xenopus laevis TAKEO NAKANISHI, L. AUSTIN DOYLE, BRET HASSEL, YUETONG WEI, KENNETH S. BAUER, SUHLAN WU, DAVID W. PUMPLIN, HONG-BIN FANG, and DOUGLAS D. ROSS The Program in Experimental Therapeutics (T.N., L.A.D., B.H., Y.W., K.S.B., S.W.) and Division of Biostatistics (H.-B.F.), University of Maryland Greenebaum Cancer Center, the Division of Hematology and Oncology, Departments of Medicine (T.N., L.A.D., D.D.R.), Anatomy and Neurobiology (D.W.P.), Microbiology (B.H.), and Epidemiology and Preventative Medicine (H.-B.F.), University of Maryland School of Medicine, Baltimore Maryland; School of Pharmacy, University of Maryland, Baltimore, Baltimore, Maryland (K.S.B.); and the Baltimore Veterans Administration Medical Center, Baltimore Maryland (D.D.R.) Received May 16, 2003; accepted August 26, 2003 This article is available online at http://molpharm.aspetjournals.org ABSTRACT To evaluate the function and substrate specificity of human breast cancer resistance protein (BCRP, ABCG2) in the absence of cofactors or heterologous partner proteins, Xenopus laevis oocytes were injected with cRNA of wild-type or mutant (R482T) BCRP. Login to comment 2 ABCG2 p.Arg482Thr Accumulation and efflux assays revealed that oocytes expressing R482T transported daunorubicin (DNR), mitoxantrone (MX), rhodamine 123, and flavopiridol (FLV), whereas wild-type BCRP transported only MX and FLV, in agreement with observations in mammalian and other systems. Login to comment 4 ABCG2 p.Ser187Thr ABCG2 p.Ser187Ala Injection of oocytes with cRNA containing mutations of serine 187 in the ATP-binding cassette signature motif (S187T or S187A) resulted in strong expression of the mutant forms; however, these oocytes were devoid of transporter activity. Login to comment 5 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Ser187Thr ABCG2 p.Ser187Thr When oocytes were coinjected with R482T and R482T/S187T, DNR transport was inhibited in a manner dependent on the amount of R482T/S187T cRNA added, consistent with the idea that the active form of BCRP is a homodimer or homomultimer. Login to comment 28 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Mutant forms of BCRP with threonine or glycine in place of arginine at codon 482 (R482T or R482G) have been described in drug-selected cell lines (Honjo et al., 2001; Komatani et al., 2001), including the original isolate of BCRP from MCF-7/ AdrVp cells (Doyle et al., 1998), which express the R482T mutation. Login to comment 29 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Compared with the wild-type form, the R482T or R482G mutations are able to transport anthracyclines and Rho123 (Honjo et al., 2001; Ozvegy et al., 2002) but not methotrexate (Volk et al., 2002). Login to comment 57 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Ser187Thr ABCG2 p.Ser187Ala The PCR product was ligated into the pCR Blunt TOPO II TABLE 1 Summary of cDNA constructs made as templates for producing BCRP cRNA Construct Plasmid Features of the BCRP cRNA Produced RNA Polymerase Consensus Sequence Upstream from BCRP cDNA I pSP64poly(A) R482T-poly(A) SP6 II pCR Blunt TOPO II Kozak-R482T-poly(A) T7 III pSD64TR 5ЈUTR-Kozak-R482T-3ЈUTR-poly(A) other BCRP forms: 5ЈUTR-Kozak-R482-3ЈUTR-poly(A) 5ЈUTR-Kozak-R482T/S187T- 3ЈUTR-poly(A) 5ЈUTR-Kozak-R482T/ S187A-3ЈUTR-poly(A) SP6 III- pSD64TR 5ЈUTR-R482T-3ЈUTR-poly(A) SP6 5ЈUTR, 3ЈUTR, portions of the 5Ј- and 3Ј-UTR of the Xenopus laevis beta-globin gene; Kozak, modified sequences proximate to the start codon, as described under Materials and Methods; Poly(A), addition of a poly(A) tail, as described under Materials and Methods. Login to comment 60 ABCG2 p.Ser187Thr ABCG2 p.Ser187Ala Introduction of S187T and S187A Mutations into Human BCRP cDNA. Login to comment 64 ABCG2 p.Ser187Thr ABCG2 p.Ser187Ala The S187T primer pair was 5Ј-G TTT ATC CGT GGT GTG AC T GGA GGA GAA AG-3Ј and 5Ј-CT TTC TCC TCC AGT CAC ACC ACG GAT AAA C-3Ј, and the S187A primer pair was 5Ј-G TTT ATC CGT GGT GTG GC T GGA GGA GAA AG-3Ј and 5Ј-CT TTC TCC TCC AGC CAC ACC ACG GAT AAA C-3Ј. Login to comment 105 ABCG2 p.Arg482Thr Oocytes injected with water or BCRP cRNA (wild type, R482T) were fixed in 4% paraformaldehyde in PBS, then immersed overnight in 30% sucrose in PBS. Login to comment 116 ABCG2 p.Arg482Thr However, injection of oocytes with BCRP cRNA directly transcribed from BCRP (R482T) cDNA (Doyle et al., 1998) failed to produce BCRP protein, as detected by Western blot examination or by functional assays. Login to comment 122 ABCG2 p.Ser2Ala The Kozak modification of BCRP in construct III resulted in a change from serine to alanine at amino acid 2. Login to comment 128 ABCG2 p.Arg482Thr Oocytes were injected with cRNA coding for the wild-type (R482) or mutant R482T form of BCRP, then fixed and sectioned as described under Materials and Methods. Login to comment 129 ABCG2 p.Arg482Thr Exposure of these sections to the BXP-34 monoclonal antibody to BCRP resulted in immunoreactivity only in the plasma membranes of oocytes expressing wild-type (Fig. 2A) or the mutant R482T form of BCRP (Fig. 2B), as detected by immunofluorescence microscopic analysis. Login to comment 132 ABCG2 p.Arg482Thr Functional expression of BCRP (R482T) was examined by 10 ␮M DNR accumulation (A) or Western blot (B) in oocytes injected with 50 nl of water (control) or cRNA (1 ␮g/␮l) transcribed from construct I, II, or III. Login to comment 133 ABCG2 p.Arg482Thr For convenience, the R482T form of BCRP was used to enable monitoring of function by DNR accumulation. Login to comment 140 ABCG2 p.Arg482Thr Western blot examination of oocytes injected with wild-type or BCRP (R482T) cRNA at the time of the immunofluorescence experiments confirmed robust expression of BCRP, with a molecular mass of 70 kDa (Fig. 2D). Login to comment 141 ABCG2 p.Arg482Thr Accumulation and Efflux of MX or DNR in Oocytes Expressing BCRP (R482T). Login to comment 142 ABCG2 p.Arg482Thr To determine whether functional BCRP was expressed in X. laevis oocytes, we monitored the accumulation of DNR and [3 H]MX in control or BCRP(R482T) injected oocytes. Login to comment 144 ABCG2 p.Arg482Thr Oocytes expressing BCRP (R482T) showed a remarkable reduction in the accumulation of DNR or MX over the 120-min period. Login to comment 147 ABCG2 p.Arg482Thr To examine whether the diminished accumulation of DNR or MX in the BCRP (R482T)-expressing oocytes was caused by enhanced drug efflux, the efflux of these compounds in the presence or absence of 5 ␮M FTC was compared with that of control oocytes. Login to comment 148 ABCG2 p.Arg482Thr Before the efflux studies, the drug of interest was preloaded into the control or BCRP (R482T)-expressing oocytes by incubating with drug for 90 min. Login to comment 149 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr In the case of BCRP (R482T)-expressing oocytes, 5 ␮M FTC was added, which resulted in intracellular drug accumulation comparable with levels attained in the control (water injected) oocytes; after preloading and subtraction of background, the accumulation of DNR in BCRP (R482T)-injected oocytes was 3.79 Ϯ 0.15 pmol/oocyte, whereas that in control was 4.55 Ϯ 0.31 pmol/oocyte. Login to comment 150 ABCG2 p.Arg482Thr [3 H]MX accumulation in control and preloaded R482T-expressing oocytes was 0.87 Ϯ 0.16 and 0.96 Ϯ 0.91 pmol/oocyte, respectively. Login to comment 151 ABCG2 p.Arg482Thr Both DNR and MX were effluxed more rapidly from the BCRP (R482T)-expressing oocytes, compared with control (Fig. 3, D and E). Login to comment 152 ABCG2 p.Arg482Thr This rapid efflux was not observed in R482T-expressing oocytes in the presence of FTC. Login to comment 156 ABCG2 p.Arg482Thr Substrate Specificity of Wild-Type and Mutant (R482T) BCRP Expressed in X. laevis Oocytes. Login to comment 157 ABCG2 p.Arg482Thr Accumulation assays using DNR, MX, FLV, and Rho123 were performed to study the effect of the Arg-to-Thr mutation at codon 482 on the substrate specificity of BCRP in the X. laevis oocyte system. Login to comment 158 ABCG2 p.Arg482Thr Figure 4 shows the intracellular accumulation of the four compounds in oocytes injected with R482 or R482T BCRP in the absence or presence of FTC. Login to comment 159 ABCG2 p.Arg482Thr Accumulation of all four compounds in the BCRP (R482T)-expressing oocytes was markedly reduced. Login to comment 162 ABCG2 p.Arg482Thr A Mutation in the ABC Signature Motif Inactivates BCRP (R482T) Transport of DNR; BCRP Expressed in X. laevis Oocytes Functions as a Homodimer or Homomultimer. Login to comment 163 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Ser187Thr ABCG2 p.Ser187Ala To determine whether dimerization is required for BCRP activity, a mutant construct was created by substituting the highly conserved serine at residue 187 in the ABC signature motif with threonine (R482T/S187T) or alanine (R482T/S187A). Login to comment 165 ABCG2 p.Arg482Thr As shown in Fig. 5A, no difference was observed in expression levels of BCRP protein among oocytes injected with cRNA encoding BCRP (R482T) or these codon 187 mutant BCRP constructs. Login to comment 167 ABCG2 p.Arg482Thr Confocal immunofluorescence microscopic analysis was performed in oocytes injected with 50 nl of cRNA of BCRP (wild-type, R482) (A), BCRP (R482T) (B), or water as control (C), using the BXP-34 antibody. Login to comment 168 ABCG2 p.Arg482Thr A and B show the distribution of BCRP (R482 or R482T) at the oocyte surface (C). Login to comment 170 ABCG2 p.Arg482Thr Cell lysate (25 ␮g) from the oocytes injected with water (control), BCRP (R482), or BCRP (R482T) cRNA was loaded per lane of the gel. Login to comment 173 ABCG2 p.Arg482Thr Accumulation of 25 ␮M DNR (A), 10.8 ␮M MX (B) or varying concentrations of DNR (C, DNR: 10 to 250 ␮M) in control (E) or BCRP (R482T)-expressing oocytes (F). Login to comment 174 ABCG2 p.Arg482Thr Efflux of DNR (D) or MX (E) from control (E) or BCRP (R482T)-expressing oocytes (F) afterpreloading with DNR or MX in the presence of 5 ␮M FTC was monitored at room temperature, as described under Materials and Methods. Login to comment 175 ABCG2 p.Arg482Thr Œ, drug efflux in BCRP (R482T)-expressing oocytes in the presence of 5 ␮M FTC. Login to comment 178 ABCG2 p.Arg482Thr *, statistically significant difference (p Ͻ 0.05, student`s t test) between control and R482T-expressing oocytes. Login to comment 180 ABCG2 p.Arg482Thr a manner analogous to a dominant-negative mutation, if coinjected with the active BCRP (R482T) cRNA. Login to comment 181 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Ser187Thr ABCG2 p.Ser187Thr Indeed, when BCRP (R482T) was coinjected with varying amounts of the "dominant-negative" construct S187T/R482T, the transport of DNR was significantly inhibited in a manner dependent on the amount of the S187T mutant construct added (Fig. 5C), strongly suggesting that homodimerization or multimerization is essential for BCRP activity. Login to comment 182 ABCG2 p.Ser187Thr The degree of inhibition seemed to plateau after the addition of 48 ng of the "dominant-negative" construct; for technical reasons, we were unable to inject the oocytes with amounts of S187T mutant cRNA in excess of 72 ng. Login to comment 185 ABCG2 p.Arg482Thr First, DNR accumulation in BCRP (R482T)-expressing oocytes was studied (Fig. 6A). Login to comment 188 ABCG2 p.Arg482Thr In contrast, MX and Rho123 only partially inhibited DNR transport by BCRP (R482T), and 10-fold molar excess of TPT was not inhibitory at all. Login to comment 189 ABCG2 p.Arg482Thr When BCRP substrate inhibition of MX accumulation was studied (Fig. 6B) using a 23-fold molar excess of Rho123, TPT, DNR, or FLV as competitors, only FLV caused partial but statistically significant reversal of BCRP R482 or R482T transport of MX. Login to comment 190 ABCG2 p.Arg482Thr Interestingly, when the competition studies were done with respect to FLV accumulation (Fig. 6C), none of the competing BCRP substrates was able to inhibit FLV efflux by wild-type or R482T BCRP, despite their presence in a 10-fold or, in the case of DNR, a 100-fold molar excess relative to FLV. Login to comment 193 ABCG2 p.Arg482Thr Accumulation of BCRP substrates DNR (25 ␮M, A), MX (0.5 ␮M, B), FLV (25 ␮M, C), and Rho123 (5 ␮M, D) was measured in control (Ⅺ), BCRP (R482T)-expressing oocytes (f) or BCRP (R482, wild-type)-expressing oocytes (u). Login to comment 199 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Ser187Thr ABCG2 p.Ser187Ala Expression of dominant-negative construct was confirmed by Western blot (A) and by DNR accumulation (25 ␮M, B) in control or R482T-expressing oocytes (f), R482T/S187T- expressing oocytes (o), or R482T/S187A-expressing oocytes (gray o). Login to comment 201 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Ser187Thr DNR accumulation (25 ␮M, C) was determined in oocytes coinjected with 24 ng of R482T cRNA and different amounts (0 to 72 ng) of the R482T/S187T construct cRNA (f). Login to comment 202 ABCG2 p.Arg482Thr ABCG2 p.Ser187Thr As control, the accumulation of 25 ␮M DNR was also determined in control (Ⅺ) or oocytes injected with 24 ng of R482T/S187T cRNA only (o). Login to comment 204 ABCG2 p.Arg482Thr *, p Ͻ 0.05; **, p Ͻ 0.01; and ***, p Ͻ 0.001 represent statistically significant differences (Student`s t test) in coinjected oocytes compared with accumulation in oocytes injected with R482T cRNA only. Login to comment 207 ABCG2 p.Arg482Thr Our results further demonstrate that oocytes injected with mutant R482T or wild-type BCRP cRNA express BCRP in the oocyte plasma membrane, as evidenced by confocal immunofluorescence microscopic analysis. Login to comment 212 ABCG2 p.Arg482Thr Validation of the assay is based our observation that the function of BCRP expressed in the oocytes parallels that observed in mammalian systems (Doyle et al., 1998; Honjo et al., 2001; Robey et al., 2001; Minderman et al., 2002); the R482T mutant form of BCRP expressed in the oocytes efficiently transported DNR, Rho123, MX, and FLV, whereas the oocyte-expressed wild-type form transported only MX and FLV. Login to comment 213 ABCG2 p.Arg482Thr Furthermore, FTC, in concentrations known to inhibit BCRP function in mammalian systems (Rabindran et al., 2000) also caused complete inhibition of both mutant R482T and wild-type BCRP forms expressed in the oocytes. Login to comment 216 ABCG2 p.Arg482Thr In our studies, the diminished accumulation of DNR or MX displayed by BCRP R482T-expressing oocytes correlated with greater initial efflux rates of these drugs compared with control oocytes. Login to comment 224 ABCG2 p.Arg482Thr Ⅺ, accumulation of these drugs in control oocytes for each condition (100%); f, accumulation in BCRP (R482T)-expressing oocytes; u, accumulation in BCRP (R482, wild-type)-expressing oocytes. Login to comment 233 ABCG2 p.Arg482Thr ABCG2 p.Ser187Thr ABCG2 p.Ser187Ala Hence, we made two mutations of serine 187 in this signature motif of BCRP R482T, one to an amino acid that, like serine, has a polar side chain (threonine, S187T) and the other to a nonpolar side chain amino acid (alanine, S187A). Login to comment 234 ABCG2 p.Arg482Thr The R482T mutant form of BCRP was used because it allowed us to monitor BCRP function by DNR accumulation. Login to comment 239 ABCG2 p.Arg482Thr ABCG2 p.Ser187Thr ABCG2 p.Ser187Thr ABCG2 p.Ser187Thr When the S187T mutation was coexpressed with BCRP R482T in the oocytes, the S187T mutant protein acted as would a dominant-negative inhibitor of BCRP transport of DNR, with the degree of inhibition increasing in proportion to the amount of S187T cRNA injected. Login to comment 241 ABCG2 p.Ser187Thr ABCG2 p.Ser187Ala Although it is possible that the inhibition caused by the S187A/T mutants was the result of the mutant saturating a limiting and essential oocyte cellular component crucial for protein transport or maturation, it is more likely that the inhibition was analogous to a "dominant-negative" effect of the S187T mutant. Login to comment 242 ABCG2 p.Lys86Met ABCB1 p.Leu553Pro The latter is in agreement with previous observations of dominant-negative BCRP constructs possessing a mutation in the Walker A motif (K86M) (Ozvegy et al., 2002) or putative 5th transmembrane domain (L553P) (Kage et al., 2002), supporting the notion that the active form of BCRP is a homodimer. Login to comment 245 ABCG2 p.Ser187Thr This may have occurred because the affinity of A-to-A and I-to-I homodimeric partners may be greater than that of the S187T mutant-to-active BCRP heterodimeric partners. Login to comment 247 ABCG2 p.Ser187Thr Because of injection volume constraints and the viscosity of highly concentrated cRNA solutions, we were technically unable to inject the oocytes with amounts of the S187T mutant form in excess of 72 ng. Login to comment 249 ABCG2 p.Arg482Thr In the substrate interaction studies in BCRP expressing X. laevis oocytes presented here (Fig. 6 and Table 2), we found a significant inhibitory effect of FLV on BCRP-mediated MX transport, in good agreement with Robey et al. (2001); furthermore, FLV and MX significantly inhibited DNR accumulation in R482T-expressing oocytes. Login to comment 255 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr It is possible that BCRP also has multiple substrate interaction sites; however, because we did TABLE 2 Summary of substrate interaction studies Substrate Competitor Inhibitor Substrate (BCRP form) DNR MX FLV TPT Rho123 FTC DNR (R482T) Partial Yes No Ϯ Yes MX (W or R482T) No Partial No No Yes FLV (W or R482T) No No No N.D. Yes W, wild type; R482T, BCRP R482T; No, no inhibition by competitor; Yes, inhibition by competitor, P Ͻ 0.01; Partial, partial (approximately 50%) inhibition by competitor, P Ͻ 0.01; Ϯ, slight (approximately 20%) inhibition by competitor, P Ͻ 0.05; N.D., no data. Login to comment