ABCMdb

PMID: 17662239

Telbisz A, Muller M, Ozvegy-Laczka C, Homolya L, Szente L, Varadi A, Sarkadi B
Membrane cholesterol selectively modulates the activity of the human ABCG2 multidrug transporter.
Biochim Biophys Acta. 2007 Nov;1768(11):2698-713. Epub 2007 Jul 10., [PubMed]

Sentences

No. Mutations Sentence Comment

8 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Interestingly, modulation of membrane cholesterol did not significantly affect the function of the R482G or R482T substrate mutant ABCG2 variants, or that of the MDR1 transporter. Login to comment 27 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly The R482G and R482T mutant variants of ABCG2, found only in drug-selected tumor cells, show a significantly altered drug resistance pattern, as compared to the wild-type protein. Login to comment 29 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly In contrast, the R482G and R482T variants practically do not transport methotrexate or drug conjugates [14-17]. Login to comment 30 ABCG2 p.Gln141Lys ABCG2 p.Val12Met There are several polymorphic variants of ABCG2 present in large percentage in the human population (e.g. V12M, Q141K), and the possible alterations in the transport capacity and substrate handling of these variants have been examined in numerous experimental systems [18-24]. Login to comment 47 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Interestingly, membrane cholesterol modulation under the same conditions had only a negligible effect on the activity of ABCG2-R482G and ABCG2-R482T mutant variants, or that of the MDR1 multidrug transporter. Login to comment 86 ABCG2 p.Arg482Gly Confocal microscopy HEK cells stably transfected with ABCG2 (wt) or ABCG2-R482G were seeded onto eight-well Nunc Lab-Tek II Chambered Coverglass (Nalge Nunc International, Rochester, NY) at 3×104 per well cell density, and grown for 48 h in D-MEM containing 10% FCS. Login to comment 104 ABCG2 p.Arg482Gly HEK cells, expressing the human wild-type ABCG2, the ABCG2-R482G variant, or the MDR1 protein, were pretreated in HPMI medium by 4 mM CD or 4 mM C-CD for 30 min at 37 °C, then washed twice to eliminate cyclodextrin. Login to comment 122 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly The Hoechst 33342 dye is a good substrate of both the wild-type and the R482G or R482T mutant variants of ABCG2 [1,3], as well as of the MDR1 transporter [4]. Login to comment 125 ABCG2 p.Arg482Gly The expression of ABCG2, its R482G mutant form, as well as MDR1 strongly decrease Hoechst dye accumulation. Login to comment 131 ABCG2 p.Arg482Gly As documented in Fig. 1A, cholesterol depletion of HEK cells by 4 mM CD for 30 min at 37 °C practically eliminated Hoechst dye extrusion by the wild-type ABCG2 (the activity factor decreased from 0.54 to 0.05), while this treatment had no effect on dye extrusion in ABCG2-R482G expressing, or MDR1 expressing HEK cells. Login to comment 144 ABCG2 p.Arg482Gly In experiments not documented here in detail, we have repeated these cellular transport experiments by measuring the uptake of mitoxantrone (MX) and Pheophorbide A (PheA), both transported compounds for both ABCG2 and its R482G variant, by using flow cytometry. Login to comment 153 ABCG2 p.Arg482Gly Again, dye extrusion activity by the ABCG2-R482G variant was practically unaffected by CD or C-CD treatment. Login to comment 154 ABCG2 p.Arg482Gly These experiments confirm that Hoechst 33342 dye extrusion in HEK cells, expressing the wild-type ABCG2, is strongly modulated by membrane cholesterol, while Hoechst transport by the ABCG2-R482G variant is not affected by this membrane lipid. Login to comment 161 ABCG2 p.Arg482Gly Moreover, the ATP-dependent transport by ABCG2-R482G or MDR1 was also not significantly affected under these conditions. Login to comment 163 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Experiments with isolated Sf9 cell membranes In order to explore the molecular details of the cholesterol effects observed in intact cells, ABCG2 and its R482G, R482T mutant variants were expressed in Sf9 cells. Login to comment 186 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly The R482G or R482T variants of ABCG2 have significantly different substrate handling properties than the wild-type protein. Login to comment 188 ABCG2 p.Arg482Gly As shown in Fig. 3B, MTX transport by the ABCG2-R482G variant was very low both in the control Fig. 2. Login to comment 192 ABCG2 p.Arg482Gly Hoechst dye accumulation was measured in cells expressing the human wild-type ABCG2 or the ABCG2-R482G variant at 37 °C, and cellular dye content was estimated based on the fluorescence in selected regions of interests. Login to comment 204 ABCG2 p.Arg482Thr Similarly, the MTX transport rate by the ABCG2-R482T variant was below 50 pmol/mg membrane protein/min, irrespective of the membrane cholesterol content. Login to comment 205 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly We found a similar lack of significant ESG and E3S transport by the R482G and R482T variants, irrespective of the cholesterol content of the membrane vesicles (not shown). Login to comment 214 ABCG2 p.Arg482Gly The apparent Km of MTX uptake was about 0.5 mM in both cases, but the proper determination of the Km and Vmax values in these experiments was hindered by the low solubility of MTX at higher than 3 mM concentrations. Fig. 4A also documents that the R482G variant of ABCG2 had a very low MTX transport activity, irrespective of the MTX concentrations examined. Login to comment 217 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Again, neither the R482G, nor the R482T variants showed any MTX transport activity, irrespective of the ATP concentration or the cholesterol content of the Sf9 cell membrane vesicles. Login to comment 228 ABCG2 p.Arg482Gly MTX uptake was measured at 100 μM MTX concentration for 5 min at 37 °C in membrane vesicles containing the human wild-type ABCG2 (WT), or R482G-ABCG2 (G) transporter. Login to comment 233 ABCG2 p.Arg482Gly MTX uptake was measured at 100 μM MTX concentration for 5 min at 37 °C in membrane vesicles containing the human wild-type ABCG2 (WT), or R482G-ABCG2 (G) transporter. Login to comment 244 ABCG2 p.Arg482Gly The experiments shown in Fig. 4 demonstrate that increased membrane cholesterol did not convert the ABCG2-R482G mutant into an efficient MTX or ESG transporter. Login to comment 245 ABCG2 p.Arg482Gly However, in order to further explore the effect of cholesterol on the substrate specificity of the mutant and wild-type ABCG2, we have also examined Rhodamine 123 (R123) uptake by the Sf9 membrane vesicles. R123 is a transported substrate of the ABCG2-R482G variant, while this compound is practically not transported by the wild-type protein. Login to comment 246 ABCG2 p.Arg482Gly As shown in Fig. 5, MgATP-dependent, rapid vesicular R123 uptake was well measurable by flow cytometry in Sf9 membrane vesicles, expressing the human R482G-ABCG2 protein. Login to comment 250 ABCG2 p.Arg482Gly These data indicate that cholesterol loading of the vesicles (in these experiments to 55-62 μg cholesterol/mg membrane protein) did not evoke R123 uptake in the vesicles containing the wild-type ABCG2, while slightly increased (as an average by 25-30%) both the initial rate and the maximum level of the R123 uptake in the R482G-ABCG2 vesicles. Login to comment 254 ABCG2 p.Arg482Gly MTX and ESG uptake was measured for 5 min at 37 °C at 5 mM ATP in membrane vesicles containing the human wild-type ABCG2 (WT), or R482G-ABCG2 (G) transporter. Login to comment 259 ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly ▪-MTX uptake in the control vesicles (8 μg cholesterol/mg membrane protein), containing the human wild-type ABCG2 (WT) transporter, •-MTX uptake in cholesterol-loaded vesicles (56 μg cholesterol/mg membrane protein), containing the human wild-type ABCG2 (WT) transporter, ▴-MTX uptake in the control vesicles (8 μg cholesterol/mg membrane protein), containing the human R482G ABCG2 (G) transporter, ▵-MTX uptake in cholesterol-loaded vesicles (62 μg cholesterol/ mg membrane protein), containing the human R482G ABCG2 (G) transporter. Login to comment 271 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly In contrast, many substrates caused a strong activation for the ABCG2-ATPase of the R482G or R482T variants [9,14]. Login to comment 273 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Fig. 6A shows the vanadate-sensitive ATPase activity of the wild-type ABCG2 as well as the R482G and the R482T variants, both in the absence and presence of two potential transported substrates. Login to comment 274 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly In these studies we selected prazosin, and the EKI-785 tyrosine kinase inhibitor (EKI), as these compounds were shown to be substrates both for the wild-type, as well as the R482G or R482T variants of ABCG2 [14,35]. Login to comment 279 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly However, cholesterol loading greatly increased the drug-stimulated ATPase activity of the wild-type ABCG2 in the presence of both substrates, while it had no such effect in the case of the R482G or R482T mutant variants. Login to comment 289 ABCG2 p.Arg482Gly Effect of cholesterol loading on ATP-dependent Rhodamine 123 (R123) uptake in Sf9 membrane vesicles. R123 uptake was measured by flow cytometry (see Materials and methods) at 1 μM R123 concentration, by taking 30-s time points for 5 min at 22 °C in membrane vesicles containing the human wild-type ABCG2 (WT), or R482G-ABCG2 (G) transporter. Login to comment 292 ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly ▪- R123 uptake in the control vesicles (8 μg cholesterol/mg membrane protein), containing the human wild-type ABCG2 (WT) transporter, •-R123 uptake in cholesterol-loaded vesicles (56 μg cholesterol/mg membrane protein), containing the human wild-type ABCG2 (WT) transporter, ▴-R123 uptake in the control vesicles (8 μg cholesterol/mg membrane protein), containing the human R482G ABCG2 (G) transporter, ▾-R123 uptake in cholesterol-loaded vesicles (62 μg cholesterol/mg membrane protein), containing the human R482G ABCG2 (G) transporter. Login to comment 293 ABCG2 p.Arg482Gly ×-R123 uptake in cholesterol-loaded vesicles (62 μg cholesterol/mg membrane protein), containing the human R482G ABCG2 (G) transporter, in the presence of 1 μM Ko143. Login to comment 296 ABCG2 p.Arg482Gly Fig. 6C also depicts the effect of membrane cholesterol on the prazosin and EKI stimulation of the ATPase activity of the ABCG2-R482G variant. Login to comment 306 ABCG2 p.Arg482Gly In contrast, nucleotide trapping in the ABCG2-R482G variant was significantly increased by transported substrates. Login to comment 322 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly Panel A Effect of cholesterol loading on the vanadate-sensitive ATPase activity in isolated Sf9 membrane preparations. ATPase activity in the vesicles was measured for 20 min at 37 °C in membranes containing the human wild-type ABCG2 (WT), the R482G-ABCG2 (R482G), or the R482T-ABCG2 (R482T) transporter. Login to comment 327 ABCG2 p.Arg482Gly Control membranes contained 8 μg cholesterol/mg membrane protein, while cholesterol-loaded membranes contained 56 μg cholesterol/mg membrane protein in the case of the wt ABCG2, 62 μg cholesterol/mg membrane protein in the case of the ABCG2-R482G, and 65 μg cholesterol/mg membrane protein in the case of the ABCG2-R482 T variant. Login to comment 335 ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly ATPase activity was measured for 20 min at 37 °C in membranes containing the human wild-type ABCG2 (WT), or the R482G-ABCG2 (R482G) transporter. Login to comment 339 ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly •-Per cent stimulation of the Ko143-sensitive ATPase activity by 20 μM Prazosin in membranes containing the human wild-type ABCG2 (WT) transporter, ▪- Per cent stimulation of the Ko143-sensitive ATPase activity by 1 μM EKI in membranes containing the human wild-type ABCG2 (WT) transporter, ○-Per cent stimulation of the Ko143-sensitive ATPase activity by 20 μM Prazosin in membranes containing the human R482G ABCG2 (G) transporter, □-Per cent stimulation of the Ko143-sensitive ATPase activity by 1 μM EKI in membranes containing the human R482G ABCG2 (G) transporter. Login to comment 343 ABCG2 p.Arg482Gly In the case of the ABCG2-R482G mutant variant, prazosin and EKI stimulation of nucleotide trapping was already present in the control membranes [17], and in this case we did not find any significant difference by cholesterol enrichment of the Sf9 cell membranes in the present study (data not shown). Login to comment 349 ABCG2 p.Arg482Gly This effect was fully reversible, and selective for the wild-type ABCG2, while the function of the highly active mutant variant ABCG2-R482G, found in drug-selected tumor cells, was not influenced by a similar cholesterol depletion. Login to comment 361 ABCG2 p.Arg482Gly Similarly to that seen in intact cells, this effect of membrane cholesterol was absent in the case of the R482G mutant variant of the transporter. Login to comment 366 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Interestingly, the ATPase activity of the R482G or R482T mutant variants of ABCG2 could be significantly enhanced by the respective substrate drugs both in the Sf9 and the mammalian cell membrane preparations [10,14]. Login to comment 374 ABCG2 p.Arg482Gly In contrast, transported substrates significantly increased nucleotide trapping by the ABCG2-R482G variant [17]. Login to comment 383 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Based on Sf9 membrane ATPase measurements, earlier we proposed that the R482G and R482T variants of ABCG2 may have "gain-of-function" properties [14]. Login to comment