ABCG2 p.Tyr413Ser
| Predicted by SNAP2: | A: D (75%), C: D (66%), D: D (85%), E: D (85%), F: N (87%), G: D (85%), H: D (80%), I: D (66%), K: D (85%), L: D (59%), M: D (75%), N: D (85%), P: D (91%), Q: D (80%), R: D (85%), S: D (80%), T: D (80%), V: D (75%), W: D (53%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: N, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, |
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[hide] Mutations of the central tyrosines of putative cho... Biochim Biophys Acta. 2015 Feb;1848(2):477-87. doi: 10.1016/j.bbamem.2014.11.006. Epub 2014 Nov 14. Gal Z, Hegedus C, Szakacs G, Varadi A, Sarkadi B, Ozvegy-Laczka C
Mutations of the central tyrosines of putative cholesterol recognition amino acid consensus (CRAC) sequences modify folding, activity, and sterol-sensing of the human ABCG2 multidrug transporter.
Biochim Biophys Acta. 2015 Feb;1848(2):477-87. doi: 10.1016/j.bbamem.2014.11.006. Epub 2014 Nov 14., [PMID:25445676]
Abstract [show]
Human ABCG2 is a plasma membrane glycoprotein causing multidrug resistance in cancer. Membrane cholesterol and bile acids are efficient regulators of ABCG2 function, while the molecular nature of the sterol-sensing sites has not been elucidated. The cholesterol recognition amino acid consensus (CRAC, L/V-(X)(1-5)-Y-(X)(1-5)-R/K) sequence is one of the conserved motifs involved in cholesterol binding in several proteins. We have identified five potential CRAC motifs in the transmembrane domain of the human ABCG2 protein. In order to define their roles in sterol-sensing, the central tyrosines of these CRACs (Y413, 459, 469, 570 and 645) were mutated to S or F and the mutants were expressed both in insect and mammalian cells. We found that mutation in Y459 prevented protein expression; the Y469S and Y645S mutants lost their activity; while the Y570S, Y469F, and Y645F mutants retained function as well as cholesterol and bile acid sensitivity. We found that in the case of the Y413S mutant, drug transport was efficient, while modulation of the ATPase activity by cholesterol and bile acids was significantly altered. We suggest that the Y413 residue within a putative CRAC motif has a role in sterol-sensing and the ATPase/drug transport coupling in the ABCG2 multidrug transporter.
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No. Sentence Comment
6 We found that in the case of the Y413S mutant, drug transport was efficient, while modulation of the ATPase activity by cholesterol and bile acids was significantly altered.
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ABCG2 p.Tyr413Ser 25445676:6:33
status: NEW112 We found that mutation of Tyr to Ser at position 469 or 645 resulted in the loss of ATP hydrolysis (even if the activity is corrected for the lower expression level of the Y469S mutant); while mutations in the other two positions apparently did not alter ABCG2 functionality, as both the Y413S and Y570S mutants showed a high level of ATPase activity, which could be inhibited by a general ATPase inhibitor vanadate or the specific ABCG2 inhibitor Ko143 (Fig. 2B).
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ABCG2 p.Tyr413Ser 25445676:112:288
status: NEW123 Conversely, we found that in the presence of cholesterol, the ATPase activity of ABCG2-Y413S was distinct from that of the wild-type transporter.
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ABCG2 p.Tyr413Ser 25445676:123:87
status: NEW124 In the case of Y413S, cholesterol resulted in a major (50 +/- 8%; p b 0.01) enhancement of the basal ATP hydrolysis, which could be only slightly stimulated by the addition of prazosin or quercetin (Figs. 2B and C, and 4B).
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ABCG2 p.Tyr413Ser 25445676:124:15
status: NEW125 Next, we investigated whether other compounds which are known substrates of wtABCG2 also differently modify the Y413S-ATPase in the presence of cholesterol (Fig. 3).
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ABCG2 p.Tyr413Ser 25445676:125:112
status: NEW127 In contrast, the Y413S mutant had a significantly altered ATPase modulation pattern.
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ABCG2 p.Tyr413Ser 25445676:127:17
status: NEW129 For a detailed analysis of the effects of cholesterol loading, the basal and drug-stimulated ATPase activities of the wild-type and Y413S ABCG2 mutants were compared at increasing membrane cholesterol concentrations.
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ABCG2 p.Tyr413Ser 25445676:129:132
status: NEW130 As shown in Fig. 4A, the baseline ATPase of Y413S is activated by lower cholesterol concentrations than that of the wild-type protein, with a lower apparent half-maximum cholesterol activation concentration (KA value of 0.37 +/- 0.27 mM) as compared to the apparent affinity of the wild-type protein for cholesterol (KA for cholesterol of 0.70 +/- 0.09 mM).
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ABCG2 p.Tyr413Ser 25445676:130:44
status: NEW131 In contrast, in the case of the substrate-stimulated ATPase activities, wtABCG2 showed higher sensitivity to cholesterol loading (Fig. 4B): in the presence of quercetin the calculated KA value for cholesterol activation of the wild-type protein was 0.22 +/- 0.20 mM, while, due to the low level of cholesterol activation a reliable KA for the Y413S protein could not be determined.
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ABCG2 p.Tyr413Ser 25445676:131:343
status: NEW133 Altered bile acid sensitivity of the ABCG2-Y413S mutant The experiments described above showed that both the baseline and the drug-stimulated ATPase activity of the Y413S mutant differ significantly from that of the wild-type protein when excess cholesterol is present.
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ABCG2 p.Tyr413Ser 25445676:133:43
status: NEWX
ABCG2 p.Tyr413Ser 25445676:133:165
status: NEW144 bile acids on the ATPase activity of the Y413S mutant.
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ABCG2 p.Tyr413Ser 25445676:144:41
status: NEW146 Therefore the net result of bile acid effects was only a slight increase in the relative substrate stimulation of the Y413S mutant (Fig. 5B-D).
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ABCG2 p.Tyr413Ser 25445676:146:118
status: NEW159 As documented in Fig. 6, all the examined ABCG2 mutants, including Y413S, exhibited a methotrexate transport activity that, similarly to the wild-type transporter, was significantly (3- to 4-fold) enhanced by cholesterol loading of the membranes.
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ABCG2 p.Tyr413Ser 25445676:159:67
status: NEW161 Expression and transport activity of ABCG2 CRAC mutants in HEK 293 cells - effect of cholesterol depletion According to the presented results the baseline ATPase activity of the ABCG2-Y413S mutant showed a sterol sensitivity that significantly differed from that of the wild-type protein, while in the vesicular transport measurement the effect of cholesterol on this mutant did not differ from that measured for wtABCG2.
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ABCG2 p.Tyr413Ser 25445676:161:184
status: NEW163 Effect of known wtABCG2 substrates on the ATPase activity of wtABCG2 and the Y413S mutant.
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ABCG2 p.Tyr413Ser 25445676:163:77
status: NEW168 Effect of cholesterol on the wtABCG2 and Y413S-ATPase.
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ABCG2 p.Tyr413Ser 25445676:168:41
status: NEW174 generated mammalian HEK 293 cells stably expressing the Y413S, Y469F, Y570S and Y645F mutants.
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ABCG2 p.Tyr413Ser 25445676:174:56
status: NEW181 As shown in Fig. 7, all ABCG2 mutants, including Y413S, were able to actively transport Hoechst 33342, and cholesterol depletion significantly decreased the Hoechst 33342 transport activity of all the CRAC mutants similarly to wild-type ABCG2.
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ABCG2 p.Tyr413Ser 25445676:181:49
status: NEW183 Putative CRAC mutants of ABCG2, including Y413S, effectively transport substrate drugs and protect HEK 293 cells against cytotoxic substrates The differential effect of the transported substrates on the wild-type and the Y413S ABCG2 ATPase activity may reflect different transport properties.
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ABCG2 p.Tyr413Ser 25445676:183:42
status: NEWX
ABCG2 p.Tyr413Ser 25445676:183:221
status: NEW185 In order to further explore the relationship of altered drug-stimulated ATPase and transport activities, the transport of various fluorescent ABCG2 substrates was compared in HEK 293 cells overexpressing wtABCG2 and the Y413S mutant, respectively.
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ABCG2 p.Tyr413Ser 25445676:185:220
status: NEW187 Effect of bile acids on the wtABCG2 and Y413S-ATPase.
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ABCG2 p.Tyr413Ser 25445676:187:40
status: NEW199 Interestingly, in spite of its different behavior in the ATPase assay, the Y413S mutant also did not differ significantly from the wild-type protein, with respect to the transport of the above mentioned substrates.
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ABCG2 p.Tyr413Ser 25445676:199:75
status: NEW200 Among the cytotoxic drugs, the Y413S mutant ABCG2 protein (similarly to the wild-type) was able to transport doxorubicin (Fig. 8A) and provided protection against doxorubicin, SN-38, flavopiridol and topotecan, strongly indicating an active transport of these compounds by this mutant (Fig. 8B and Supplementary Fig. S3).
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ABCG2 p.Tyr413Ser 25445676:200:31
status: NEW239 However, the Y413S mutant exhibited a distinct ATPase activity.
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ABCG2 p.Tyr413Ser 25445676:239:13
status: NEW250 BODIPY-prazosin Pheophorbide A Hoechst 33342 mitoxantrone wtABCG2 + + + + Y413S + + + + Y469F + + + + Y570S + + + + Y645F + + + + Fig. 8.
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ABCG2 p.Tyr413Ser 25445676:250:74
status: NEW257 HEK 293 parental cells or stably expressing wtABCG2 or its Y413S mutant were incubated with 50 nM SN-38, 50 nM topotecan (TOP), 60 nM doxorubicin (DOX) or 500 nM flavopiridol (FLP) for 72 hours.
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ABCG2 p.Tyr413Ser 25445676:257:59
status: NEW262 Moreover, potential substrates that are known to activate the wild-type ABCG2-ATPase, rather inhibited the ATPase activity of the Y413S mutant (Fig. 3).
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ABCG2 p.Tyr413Ser 25445676:262:130
status: NEW263 In addition to this altered cholesterol-sensing, the ATPase activity of the Y413S mutant also showed an altered bile acid sensitivity, as this mutant was less sensitive to bile acids than the wild-type protein (Fig. 5).
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ABCG2 p.Tyr413Ser 25445676:263:76
status: NEW267 In the case of the Y413S mutant, cholesterol may promote "uncoupling" of the ATP hydrolysis in the ABCG2 protein.
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ABCG2 p.Tyr413Ser 25445676:267:19
status: NEW268 Alternatively, if we consider the possibility that cholesterol is transported by ABCG2, the results could be explained by a higher affinity to cholesterol by the ABCG2-Y413S mutant, also observed in the case of the R482G mutant [15].
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ABCG2 p.Tyr413Ser 25445676:268:168
status: NEW270 If ABCG2 can transport cholesterol, our data can be interpreted to suggest that the Y413S mutant is a more efficient cholesterol transporter.
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ABCG2 p.Tyr413Ser 25445676:270:84
status: NEW273 Since ABCG2-mediated cholesterol transport has not been experimentally confirmed, we speculate that the "cholesterol-stimulated" ATPase activity of the ABCG2-Y413S mutant is a result of its differential sensitivity to the modulatory effect of cholesterol.
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ABCG2 p.Tyr413Ser 25445676:273:158
status: NEW274 In contrast to these findings related to the ABCG2-ATPase activity, we did not find any significant alteration in the cholesterol sensitivity of the ABCG2-Y413S mediated transport either in insect cell vesicular transport, or mammalian intact cell transport assays.
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ABCG2 p.Tyr413Ser 25445676:274:155
status: NEW275 Active substrate transport by the Y413S mutant was clearly activated by cholesterol in both systems, and by all the compounds examined (Figs. 6 and 7).
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ABCG2 p.Tyr413Ser 25445676:275:34
status: NEW276 Moreover, the Y413S mutant protected the cells against toxic drugs similarly to the wild-type ABCG2.
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ABCG2 p.Tyr413Ser 25445676:276:14
status: NEW277 All these experimental data suggest that although the ABCG2-Y413S mutant has an altered cholesterol interaction, it is rather manifested in a partial uncoupling of the ATPase activity of the transporter in the absence of transported substrates.
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ABCG2 p.Tyr413Ser 25445676:277:60
status: NEW