ABCMdb

PMID: 16847575

Pozza A, Perez-Victoria JM, Sardo A, Ahmed-Belkacem A, Di Pietro A
Purification of breast cancer resistance protein ABCG2 and role of arginine-482.
Cell Mol Life Sci. 2006 Aug;63(16):1912-22., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCG2 p.Arg482Thr Interestingly, the R482T point mutation increased both maximal hydrolysis rate and affinity for MgATP, and lowered sensitivity to vanadate inhibition. Login to comment 5 ABCG2 p.Arg482Thr The R482T mutation only produced a limited change, if any, on the binding of drug substrates, indicating that methotrexate, on the one hand, and rhodamine 123 or doxorubicin, on the other hand, bound similarly to wild-type and mutant transporters whether or not they were subject to cellular transport. Login to comment 23 ABCG2 p.Arg482Thr To decide between these alternatives and establish structure-function relationships, the aim of this study was to overexpress and purify both wild-type (R482) and mutant (R482T) ABCG2 to determine parameters ofATPase activity and measure direct binding of ligands, i.e. nucleotides, substrate drugs and inhibitors. Login to comment 26 ABCG2 p.Arg482Thr The present results show that the R482T mutation represents a gain-of-function for ATPase activity by increasing both maximal rate of hydrolysis and affinity for MgATP. Login to comment 36 ABCG2 p.Arg482Thr The pcDNA3 plasmid containing R482T-ABCG2 cDNA, provided by Dr. D. D. Ross, was used for subcloning into the pTriex-4-Neo plasmid (Novagen, VWR, Fontenay-sous- Bois, France). Login to comment 37 ABCG2 p.Arg482Thr The R482T cDNA was mutated to obtain R482 cDNA by site-directed mutagenesis using a QuickChange Site-directed mutagenesis Kit (Stratagene, La Jolla). Login to comment 40 ABCG2 p.Arg482Thr Recombinant baculoviruses carrying either R482T- or R482-ABCG2 human cDNA were generated with a BacVector transfection kit (Novagen), according to manufacturer`s instructions. Login to comment 52 ABCG2 p.Arg482Thr Inverted membrane vesicles of High Five cells infected by baculovirus vector encoding the R482 or R482T transporter were treated with solubilization buffer {50 mM HEPES/NaOH, pH 8, 18 mM 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 0.5 M NaCl, 10 mM imidazole, 20% glycerol} at a final protein concentration of 2 mg/ml, for 30 min with gently shaking at 4 °C. Login to comment 72 ABCG2 p.Arg482Thr Lane 1: prestained molecular weight markers; lane 2: inverted membrane vesicles of High Five cells infected with baculovirus vector encoding R482 or R482T ABCG2; lane 3: supernatant after solubilization; lane 4: supernatant after centrifugation (15000 g, 30 min); lane 5: detergent-insoluble pellet after centrifugation; lane 6: supernatant after binding for 2 h; lane 7: Ni-NTA agarose gel after binding for 2 h; lane 8: washing; lane 9: Ni-NTA agarose gel after washing; lane 10: elution; lane 11: Ni-NTA agarose gel after elution; lane 12: after imidazole removal by gel filtration. Login to comment 74 ABCG2 p.Arg482Thr Buffer-corrected fluorescence emission spectra for purified wild-type R482 (■) or mutant R482T (▲) transporter at (0.045 mg protein/ml); the fluorescence emission spectra were recorded at 25 °C upon excitation at 295 nm. Login to comment 89 ABCG2 p.Arg482Thr The ATPase activity of purified R482 (■) or R482T (▲) ABCG2 was measured with 0.5 μg protein in the presence of 50 μg azolectin at 37 °C for 40 min. Login to comment 91 ABCG2 p.Arg482Thr The inhibition of ATPase activity of R482 (■) and R482T transporter (▲) in the presence of 5 mM MgATP was measured at increasing orthovanadate concentrations (0.001-10 mM). Login to comment 92 ABCG2 p.Arg482Thr (c, d) nucleotide binding as monitored by quenching of the intrinsic tryptophan fluorescence of purified R482 (■) and R482T (▲) transporter at 0.045 mg/ml: ATP (c) and ADP/Mg2+ (d). Login to comment 97 ABCG2 p.Arg482Thr A similar procedure was used to overexpress, solubilize and purify mutant R482T ABCG2: qualitatively similar results were obtained as in Fig. 1a and b, but with a twofold lower yield (data not shown). Login to comment 99 ABCG2 p.Arg482Thr It is worthwhile mentioning that the R482T point mutation significantly increased fluorescence intensity, by nearly 20%, giving rise to a more symmetrical emission spectrum. Login to comment 103 ABCG2 p.Arg482Thr Interestingly, the R482T point mutation both increased Vmax, up to 1111 ± 79 nmol ATP hydrolyzed/min/mg, and lowered the Km(MgATP) to 1.0 ± 0.1 mM. Login to comment 107 ABCG2 p.Arg482Thr Interestingly, the R482T mutation increased the binding affinity for ATP (in the absence of magnesium ions to avoid hydrolysis), with a shift in apparent KD from 81.3 ± 10.4 to 36.1 ± 7.4 μM, whereas it lowered the binding affinity for MgADP, the hydrolysis product, with a shift in KD from 82.0 ± 13.5 to 149 ± 22 μM (cf. Table 1). Login to comment 112 ABCG2 p.Arg482Thr Interestingly, the R482T point mutation, reported to abolish methotrexate transport by whole cells and inverted membrane vesicles, was found here to only slightly alter KD1 (7.30 ± 0.57 μM), while KD2 remained unchanged (87.0 ± 29.4 μM), whereas maximal quenching was higher for mutant as compared with wild-type ABCG2. Login to comment 113 ABCG2 p.Arg482Thr Conversely, although wild-type ABCG2 was known not to transport rhodamine 123, in contrast to R482T mutant [8], displaying here apparent KD1 and KD2 values of 0.52 ± 0.10 and 16.6 ± 6.6 μM, it in fact bound the fluo- Table 1. Login to comment 119 ABCG2 p.Arg482Thr Wild-type R482 Mutant R482T KD1 (μM) ΔF1 (%) KD2 (μM) ΔFmax (%) KD1 (μM) ΔF1 (%) KD2 (μM) ΔFmax (%) Nucleotides ATP 81.3 ± 10.4 5.42 ± 0.19 36.1 ± 7.4 2.99 ± 0.14 MgADP 82.0 ± 13.5 6.42 ± 0.23 149 ± 22 11.1 ± 0.4 Substrate drugs Methotrexate 4.48 ± 0.40 13.7 ± 3.4 84.2 ± 25.6 66.3 ± 3.4 7.30 ± 0.57 15.7 ± 4.7 87.0 ± 29.4 84.3 ± 4.7 Rhodamine 123 0.52 ± 0.10 5.66 ± 1.21 16.6 ± 6.6 24.4 ± 1.2 0.42 ± 0.09 6.11 ± 1.23 15.4 ± 6.6 23.9 ± 1.2 Doxorubicin 1.63 ± 0.45 12.4 ± 3.4 22.2 ± 11.1 47.6 ± 3.4 2.91 ± 0.60 7.32 ± 2.36 25.9 ± 9.0 40.7 ± 2.4 Mitoxantrone 0.65 ± 0.06 4.03 ± 0.42 20.3 ± 4.3 16.0 ± 0.4 0.80 ± 0.10 4.53 ± 0.10 17.8 ± 4.6 25.5 ± 0.1 Pheophorbide a 0.94 ± 0.12 10.0 ± 1.3 17.7 ± 4.2 40.0 ± 1.3 1.50 ± 0.23 6.77 ± 1.75 21.7 ± 6.3 38.2 ± 1.8 Hoechst33342 2.04 ± 0.67 12.8 ± 2.3 28.7 ± 19.6 32.2 ± 2.3 1.69 ± 0.40 14.0 ± 4.1 23.1 ± 9.6 66.0 ± 4.1 Inhibitors GF120918 2.71 ± 0.66 12.6 ± 5.1 25.7 ± 10.9 72.4 ± 5.1 6.23 ± 1.46 9.6 ± 5.2 30.9 ± 11.7 70.9 ± 5.2 6-prenylchrysin 0.80 ± 0.11 8.22 ± 2.39 26.8 ± 9.2 51.8 ± 2.4 0.57 ± 0.07 11.0 ± 2.7 23.4 ± 6.1 69.0 ± 2.7 Ko143 0.012 ± 0.0016 13.2 ± 7.9 0.14 ± 0.11 66.8 ± 7.9 0.012 ± 0.0005 8.63 ± 3.63 0.096 ± 0.02 91.4 ± 3.6 Novobiocin 2.56 ± 0.56 4.45 ± 2.07 23.8 ± 10.8 27.6 ± 2.1 1.49 ± 0.33 8.75 ± 2.87 21.1 ± 10.0 41.3 ± 2.9 rescent dye similarly (0.42 ± 0.09 and 15.4 ± 6.6 μM, respectively) (Fig. 3b, and Table 1). Login to comment 128 ABCG2 p.Arg482Thr Fluorescence emission was recorded, as in Fig. 2c, d for R482 (■) and R482T (▲) transporters, in the presence of increasing concentrations of either substrate drugs such as methotrexate (a) and rhodamine 123 (b), or known inhibitors such as GF120918 (c) and 6-prenylchrysin (d). Login to comment 145 ABCG2 p.Arg482Thr The R482T mutant ABCG2 could be also prepared by the same procedure despite a twofold lower yield that might indicate some loss in protein stability. Login to comment 148 ABCG2 p.Arg482Thr The functionality of both recombinant purified transporters was assessed by (i) the high ATPase activity, sensitive to vanadate and dependent on the R482T mutation, (ii) the direct binding of nucleotides, in the presence or absence of magnesium ions and their dependence on the mutation, (iii) the binding of most substrate drugs and inhibitors in the micromolar range, consistent with their known cellular effects, and (iv) the very high-affinity binding, in the nanomolar range, of Ko143 recognized as the most efficient ABCG2 inhibitor. Login to comment 149 ABCG2 p.Arg482Thr The R482T hot-spot mutation as a gain-of-function for ATP hydrolysis. Login to comment 152 ABCG2 p.Arg482Thr It is further shown here that the gain-of-function induced by the R482T mutation is also characterized by a twofold increase in affinity for MgATP, concomitant with a better binding of substrate ATP and a better release of product ADP. Login to comment 182 ABCG2 p.Arg482Thr In conclusion, the R482T mutation, which occurs in vitro on cell cultures submitted to high drug pressure, induces a gain-of-function for ATPase activity and for substrate-drug transport, but not drug binding. Login to comment 313 ABCG2 p.Arg482Thr 39 Alqawi, O., Bates, S. and Georges, E. (2004) Arginine482 to threonine mutation in the breast cancer resistance protein ABCG2 inhibits rhodamine 123 transport while increasing binding. Login to comment