ABCMdb

PMID: 12374800

Ozvegy C, Varadi A, Sarkadi B
Characterization of drug transport, ATP hydrolysis, and nucleotide trapping by the human ABCG2 multidrug transporter. Modulation of substrate specificity by a point mutation.
J Biol Chem. 2002 Dec 13;277(50):47980-90. Epub 2002 Oct 8., 2002-12-13 [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly An altered drug resistance profile and substrate recognition were suggested for wild-type ABCG2 and its mutant variants (R482G and R482T); the mutations were found in drug-selected tumor cells. Login to comment 2 ABCG2 p.Lys86Met In order to characterize the different human ABCG2 transporters without possible endogenous dimerization partners, we expressed these proteins and a catalytic center mutant (K86M) in Sf9 insect cells. Login to comment 4 ABCG2 p.Lys86Met We found that the K86M mutant had no transport or ATP hydrolytic activity, although its ATP binding was retained. Login to comment 5 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly The wild-type ABCG2 and its variants, R482G and R482T, showed characteristically different drug and dye transport activities; mitoxantrone and Hoechst 33342 were transported by all transporters, whereas rhodamine 123 was only pumped by the R482G and R482T mutants. Login to comment 7 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly A relatively high basal ABCG2-ATPase, inhibited by fumitremorgin C, was observed in all active proteins, but specific drug stimulation could only be observed in the case of R482G and R482T mutants. Login to comment 9 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Nucleotide trapping was stimulated by the transported compounds in the R482G and R482T variants but not in the wild-type ABCG2. Login to comment 23 ABCG2 p.Arg482Ser ABCG2 p.Arg482Met Interestingly, in some drug-selected mouse cell lines, mutation of the equipositional amino acid in mouse ABCG2 occurred, causing altered drug resistance profiles for the mutant variants R482S and R482M (21). Login to comment 38 ABCG2 p.Arg482Gly Previously, we demonstrated that the ABCG2-R482G multidrug transporter could also be expressed in insect cells, allowing the investigation of its membrane ATPase activity (18). Login to comment 43 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly We generated and expressed the human wild-type ABCG2 and its variants R482G and R482T in Sf9 cells, and we characterized their transport and ATP hydrolytic activity. Login to comment 44 ABCG2 p.Arg482Gly ABCG2 p.Lys86Met As a control, we mutated a crucial amino acid in the catalytic center of ABCG2-R482G (Lys-86 was changed to Met) in hope of producing a non-functional transporter. Login to comment 59 ABCG2 p.Arg482Gly Generation of Transfer Vector Possessing Different Human ABCG2 cDNAs-pAcUW21-L/ABCG2 (R482G) was constructed as described (18). Login to comment 60 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Lys86Met Wild-type ABCG2 (Arg-482) and its variants (R482T and K86M) were created using ABCG2-R482G cDNA as a template (4) by overlap extension PCR (30). Login to comment 61 ABCG2 p.Arg482Thr ABCG2 p.Lys86Met Two internal complementary primer pairs were used with each containing the specific mutation as follows: the Arg- primer pairs were 5Ј-TTATTACCAATGCGCATGTTACC-3Ј and 5Ј-GG- TAACATGCGCATTGGTAATAA-3Ј; the R482T primer pairs were 5Ј-T- TATTACCTATGACGATGTTACC-3Ј and 5Ј-GGTAACATCGTCATAG- GTAATAA-3Ј; and the K86M primer pairs were 5Ј-TGGAGGCATGTC- TTCGTTATT-3Ј and 5Ј-TAATAACGAAGACATGCCTCCA-3Ј, respectively. Login to comment 62 ABCG2 p.Lys86Met The two outer primer pairs were 5Ј-CTTGGGATACTTGAATC- AGC-3Ј and 5Ј-GGTCATGAGAAGTGTTGCTA-3Ј for the wild-type and the amino acid 482 variants and 5Ј-GTATATTAATTAAAATACTATA- CTG-3Ј and 5Ј-GGCTCATCCAAGAACAAGAT-3Ј for the K86M mutant. Login to comment 64 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Lys86Met The PCR products containing the Arg-482 or R482T coding sequence were digested with PstI and MscI enzymes and ligated between the corresponding sites of the pAcUW21-L/ABCG2 vector. The PCR product coding for the K86M variant was digested with NotI and SpeI enzymes and ligated to the NotI and SpeI sites of the pAcUW21-L/ABCG2 (R482G) vector. The mutations were confirmed by sequencing the PstI-MscI or the NotI-SpeI fragments of the constructs, respectively. Login to comment 108 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Lys86Met In the present study we utilized this expression system to produce the wild-type human ABCG2 protein and its variants, R482G and R482T, as well as a catalytic center mutant, K86M. Login to comment 109 ABCG2 p.Arg482Thr ABCG2 p.Lys86Met Site-directed mutagenesis was performed on a human ABCG2 cDNA, which possesses Gly at position 482 (4), to create the wild-type (Arg-482) and the R482T and K86M variants of the ABCG2 half-transporter. Login to comment 125 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly ABCG2 p.Lys86Met We found equal expression levels for the wild-type, R482G, R482T, and K86M/ R482G mutant variants (data not shown). Login to comment 132 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly ABCG2 p.Lys86Met Lane 1, R482T/Sf9; lane 2, wild-type ABCG2/Sf9; lane 3, R482G/Sf9; lane 4, K86M-R482G/Sf9; lane 5, wild-type ABCG2/ HL60; lane 6, wild-type ABCG2/HL60 grown in the presence of 2.5 ␮g/ml tunicamycin; and lane 7, beta-galactosidase/Sf9. Login to comment 138 ABCG2 p.Lys86Met In Sf9 cells, expressing the K86M mutant, MX uptake is significantly higher and reaches the level of MX accumulation found in the FTC-inhibited cells. Login to comment 140 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Lys86Met These experiments indicate that wtABCG2 and the R482G or R482T variants actively extrude MX in the intact Sf9 cells, whereas the K86M mutant is inactive in this respect. Login to comment 141 ABCG2 p.Lys86Met Prazosin, a known substrate of ABCG2 (16, 37), also increased the level of MX accumulation in insect cells expressing the active ABCG2 proteins, whereas it had no effect on the cells expressing the K86M mutant (see Fig. 2A). Login to comment 144 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly As documented, at increasing MX concentrations, the wild-type (R482) ABCG2 was found to be somewhat less effective in protecting Sf9 cells against MX accumulation than the other two amino acid 482 variants; the accumulation of MX was about 15 Ϯ 4% more in wtABCG2- expressing cells than in cells containing the R482G or R482T variants. Login to comment 145 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly In addition, the MX transport capacities of the amino acid 482 variants R482G and R482T were found to be similar in these experiments. Login to comment 146 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Next we performed similar transport studies for the fluorescent dye rhodamine 123, which was indicated to be a transported substrate of the ABCG2 variants R482G and R482T (20). Login to comment 147 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Lys86Met As shown in Fig. 3, we found that insect cells expressing the wild-type ABCG2 or the K86M mutant accumulated significantly more rhodamine 123 than cells expressing the R482G or R482T variants. Login to comment 148 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly In contrast to that found for the wild-type ABCG2, in the R482G or R482T variants the addition of FTC greatly increased rhodamine 123 accumulation indicating an ABCG2-dependent extrusion of this compound. Login to comment 149 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly These data support the fact that MX is a substrate for wtABCG2 and its amino acid 482 variants, although MX transport may be less efficient by the wild-type protein than by the R482G or R482T variants. Login to comment 150 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly In contrast, the wild-type ABCG2 is practically inactive for rhodamine 123 transport, whereas the R482G and R482T mutants actively extrude this fluorescent dye. Login to comment 158 ABCG2 p.Arg482Gly ABCG2 p.Lys86Met Fig. 4A shows a typical Hst dye uptake experiment using intact Sf9 cells harvested post-40 h of recombinant baculovirus infection, expressing either ABCG2-R482G or its K86M mutant variant. Login to comment 161 ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly ABCG2 p.Lys86Met As documented, Hst dye uptake is significantly slower in the Sf9 cells that express the ABCG2-R482G protein (Fig. 4A, line A) than in cells expressing the K86M/R482G mutant (line B). Login to comment 163 ABCG2 p.Arg482Gly The rate of Hst dye uptake in cells expressing ABCG2-R482G (Fig. 4A, line A) greatly increases upon the addition of the specific inhibitor FTC (or Ko 143, not shown). Login to comment 164 ABCG2 p.Arg482Gly ABCG2 p.Lys86Met However, there is no change in the rate of Hst accumulation in cells expressing the K86M/R482G mutant (Fig. 4A, line B) or beta-galactosidase (not shown). Login to comment 167 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Fig. 4, B and C, shows that the rate of Hst influx was low and was greatly increased by the inhibitor FTC in the wild-type ABCG2 (Fig. 4B) and also in the R482G and R482T variants (Fig. 4, B and C). Login to comment 168 ABCG2 p.Lys86Met The K86M mutant was found to be inactive at all Hst dye concentrations examined (Fig. 4C). Login to comment 172 ABCG2 p.Arg482Gly We have shown earlier (18) that ABCG2-R482G expressed in Sf9 cells had a high level of vanadate-sensitive membrane ATPase activity, and this activity could be significantly increased by substrates and decreased by the inhibitors of this protein. Login to comment 173 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Lys86Met In the present study we have compared the ATPase activity of the wild-type human ABCG2 and its variants (R482G, R482T, and K86M) in the presence and absence of a variety of potential ABCG2 substrates or inhibitors. Login to comment 174 ABCG2 p.Arg482Gly ABCG2 p.Lys86Met As shown in Fig. 5A, the basal, vanadate-sensitive ATPase activity was significantly higher in membranes containing any of the wtABCG2 or its amino acid 482 variants than in those containing ABCG2-K86M/R482G or beta-galactosidase (not shown here). Login to comment 175 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Lys86Met Despite the similar ABCG2 expression levels, this basal ATPase activity was ϳ1.5-fold higher in case of the R482G variant (71 Ϯ 10.8 nmol of Pi/mg of membrane protein/ min) than in case of the wild-type ABCG2 or the R482T variant (45 Ϯ 8.85 and 46 Ϯ 9.04 nmol of Pi/mg of membrane protein/ min, respectively) and negligible in the ABCG2-K86M mutant (5 Ϯ 0.5 nmol of Pi/mg of membrane protein/min). Login to comment 177 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly In wtABCG2 and in R482G and R482T proteins FTC (or Ko 143) powerfully inhibited the vanadate-sensitive ATPase activity. Login to comment 179 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly In case of the R482G and R482T mutants, vanadate-sensitive ATPase activity could be greatly stimulated by several potential ABCG2 substrates (e.g. prazosin, mitoxantrone, adriamycin, rhodamine 123, benzamil, and camptothecin), corresponding to the transport activity of these proteins. Login to comment 182 ABCG2 p.Lys86Met It is worthwhile to note that Hst had no effect on the ATPase activity of Sf9 membranes containing ABCG2-K86M or beta-galactosidase (data not shown). Login to comment 183 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly We found that the effect of different ABCG2 substrates on the ATPase activity of variants R482G and R482T was almost similar. Login to comment 186 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Rhodamine 123, a specific substrate of the amino acid 482 mutants, gave a maximum of 30% stimulation, and the Kact values were 4.5 and 4 ␮M, respectively, for the R482G and R482T mutants. Login to comment 187 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly The maximum extent of stimulation of the R482G and R482T-ATPases by prazosin was 100 and 70%, and the Kact values were 7 and 3 ␮M. Login to comment 188 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Mitoxantrone showed a maximum of 50% stimulation of the ATPase activity of the R482G and R482T variants, with Kact values of 1 and 0.8 ␮M, respectively. Login to comment 189 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly FTC was found to be an effective inhibitor for the vanadate-sensitive ABCG2-ATPase activity both for the wild-type enzyme (76% inhibition at 10 ␮M) and the R482G variant (74% inhibition at 10 ␮M); however, its inhibitory effect was smaller and required higher FTC concentrations in the case of the R482T variant (37% inhibition at 10 ␮M) (see Fig. 5). Login to comment 190 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly The concentration of FTC causing half-maximal inhibition of the ABCG2-ATPases was 0.4 ␮M for the wild-type enzyme, 0.5 ␮M for the R482G variant, and 0.7 ␮M for the R482T mutant. Login to comment 192 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Lys86Met A, MX accumulation in Sf9 cells expressing beta-galactosidase (beta-gal), wild-type ABCG2 (Arg-482), and the R482G, R482T, or K86M mutants. Login to comment 202 ABCG2 p.Arg482Gly We found no significant effect of any of these conditions on the ATPase activity of the wild-type ABCG2 or the R482G variant; neither their basal activity nor the effects of drugs were changed. Login to comment 207 ABCG2 p.Arg482Gly In the presence of 3.1 mM Mg-8-azido-ATP, the vanadate-sensitive ATPase activity of ABCG2-R482G was 77% compared with the activity measured in the presence of a similar concentration of MgATP (data not shown). Login to comment 214 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Lys86Met We found that 8-azido-ATP binding was similar in the wild-type and in the R482G, R482T, and K86M mutant variants under these conditions (Fig. 6B). Login to comment 218 ABCG2 p.Arg482Gly As shown in Fig. 7B, in the presence of 1 mM vanadate and 5 ␮M Co-8-azido-[␣-32 P]ATP, ABCG2 (R482G) showed a high level of nucleotide trapping (lane 3). Login to comment 221 ABCG2 p.Arg482Gly ABCG2 p.Lys86Met As documented in Fig. 7B, the functionally inactive ABCG2-K86M/R482G did not show any nucleotide trapping activity under these conditions (lane 5). Login to comment 224 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly ABCG2 p.Lys86Met Rhodamine 123 accumulation in Sf9 cells expressing the wild-type (Arg-482), R482G, R482T, or K86M/R482G variants of ABCG2. Login to comment 228 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly As shown in Fig. 8, we found significant differences in the adenine nucleotide trapping of the wild-type, R482G, and R482T ABCG2 transporters. Login to comment 229 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly When we analyzed these data with a quantitative PhosphorImager, in the absence of added drugs the wild-type ABCG2 (Fig. 8, lane 4) showed a more pronounced nucleotide trapping activity, 2.6 Ϯ 0.03- and 2.05 Ϯ 0.5-fold of the R482G and R482T variants (lanes 7 and 10), respectively. Login to comment 230 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly The addition of prazosin, an activator of the ABCG2-ATPase of the R482G and R482T variants, significantly stimulated nucleotide trapping in the same variants (see Fig. 8, lanes 6 and 9, the stimulations were 2.0 Ϯ 0.22- and 1.7 Ϯ 0.04-fold, respectively). Login to comment 237 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Lys86Met DISCUSSION In this paper we describe the expression and detailed functional analysis of the wild-type human ABCG2 multidrug transporter and its mutant variants R482G, R482T, and K86M. Login to comment 241 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly According to the established sequence in the human genome data base (GenBankTM accession number AF103796), the wild-type ABCG2 contains arginine at this position, whereas the other two variants R482G and R482T most probably were generated during drug selection in tumor cell lines (20, 22). Login to comment 245 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly The aim of the present study was to provide a quantitative characterization both for the transport and the ATP hydrolytic activity of the wtABCG2 and its variants R482G and R482T. Login to comment 247 ABCG2 p.Lys86Met As documented under "Results," we obtained a uniformly high level expression of the wtABCG2 and its amino acid 482 variants, and as a negative control also expressed a Walker A Lys mutant of this protein (K86M). Login to comment 248 ABCG2 p.Arg482Gly ABCG2 p.Lys86Met We introduced the K86M mutation into the R482G variant of ABCG2, because we expected that the mutation of the key Lys will abolish the function of ABCG2 regardless the amino acid found in position 482. Login to comment 254 ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly ABCG2 p.Lys86Met A shows the increase in fluorescence due to the uptake of 2.5 ␮M Hst in ABCG2-R482G- (line A) or ABCG2-K86M/R482G (line B) -expressing Sf9 cells. Login to comment 255 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly ABCG2 p.Lys86Met B and C, the rate of Hst influx (⌬ fluorescence/⌬ time) into Sf9 cells expressing the wtABCG2 or the R482T mutant (B) and R482G or K86M/R482G (C) was determined at different Hst concentrations with or without the ABCG2 inhibitor FTC. Login to comment 258 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly ABCG2 p.Lys86Met ATPase activity measured in membranes of Sf9 cells expressing the wild-type, R482G, R482T, or K86M/R482G variants of human ABCG2. Login to comment 263 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ATPase activity determined in membranes of wtABCG2- (B), R482G- (C), or R482T (D)-expressing Sf9 cells. Login to comment 267 ABCG2 p.Lys86Met The K86M mutant of ABCG2/R482 was already investigated for Hoechst 33342 transport in mammalian cells (35), and it was found to be inactive. Login to comment 268 ABCG2 p.Arg482Gly On the other hand the R482G variant could be easily investigated in all four assays as described under "Results." Login to comment 273 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Comparing the transport activity of the wtABCG2 and its mutant variants, we found that the wild-type and the two amino acid 482 variants actively exported mitoxantrone, whereas rhodamine 123 extrusion could only be observed in cells expressing the R482G and R482T proteins. Login to comment 279 ABCG2 p.Arg482Gly We have already described a high level, vanadate-sensitive ATPase activity, stimulated by several transported compounds, for the human ABCG2-R482G variant in isolated Sf9 cell membranes (18). Login to comment 280 ABCG2 p.Arg482Thr In the present experiments a relatively high basal ATPase activity was found in case of the wild-type ABCG2 and the R482T variant, too. Login to comment 281 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly However, stimulation of the ABCG2-ATPase activity was observed only in Sf9 membranes containing the R482G or R482T variants and not the wild-type ABCG2. Login to comment 283 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly ABCG2 p.Lys86Met [␣-32 P]8-azido-ATP binding of the wild-type and R482G, R482T, or K86M/R482G mutant ABCG2 proteins. Login to comment 290 ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly ABCG2 p.Lys86Met [␣-32 P]8-azido-ATP trapping of the R482G and K86M/ R482G mutant ABCG2 proteins. Login to comment 291 ABCG2 p.Arg482Gly ABCG2 p.Lys86Met ABCG2 p.Lys86Met Sf9 membranes containing ABCG2R482G (see A, lane 2, and B, lanes 1-3), ABCG2-K86M (B, lane 5), or beta-galactosidase (beta-gal) (see A, lane 1 and B, lane 4) were incubated for 5 min at 37 °C with 5 ␮M 8-azido-[␣-32 P]ATP, 1 mM sodium orthovanadate (except for lane 1) and 2 mM Mg2ϩ (A) or 2 mM Co2ϩ (B) as described under "Experimental Procedures." Login to comment 296 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Comparison of the effect of different compounds on the [␣-32 P]8-azido-ATP trapping of the wild-type, and R482G or R482T variants of the ABCG2 protein. Login to comment 297 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Sf9 membranes (150 ␮g) containing wtABCG2 (lanes 2-4), ABCG2-R482G (lanes 5-7), ABCG2-R482T (lanes 8-10), or beta-galactosidase (lane 1) were incubated for 2 min at 37 °C with 2 ␮M 8-azido-[␣-32 P]ATP, 1 mM sodium orthovanadate, and 2 mM Co2ϩ as described under "Experimental Procedures." Login to comment 309 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly In case of the R482G and R482T variants, this endogenous stimulation (caused by endogenous activators, producing what we observe as a high base-line ATPase) is only partial, and further stimulation by the transported compounds is observed. Login to comment 321 ABCG2 p.Lys86Met We have documented that the 8-azido-ATP labeling of the wtABCG2 and its amino acid 482 variants and that of the K86M mutant was similar, which is to say that they seem to have similar ATP binding capacities. Login to comment 323 ABCG2 p.Lys86Met Clearly, the K86M mutant was unable to form the transition state intermediate, in agreement with the inactivity of this mutant in the ATPase and transport measurements. Login to comment 325 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly We found that the nucleotide trapping characteristics of the R482G or R482T variants were different from the wild-type ABCG2. Login to comment 326 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Whereas this transition state formation in variants R482G and R482T could be significantly stimulated by various ABCG2 substrates (prazosin and mitoxantrone), the transition state formation of the wild-type ABCG2 could not be stimulated but rather was inhibited by these compounds. Login to comment