ABCMdb

PMID: 25445676

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., [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCG2 p.Tyr645Phe ABCG2 p.Tyr469Phe ABCG2 p.Tyr645Ser ABCG2 p.Tyr570Ser ABCG2 p.Tyr469Ser 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. Login to comment 6 ABCG2 p.Tyr413Ser 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. Login to comment 45 ABCG2 p.Lys86Met The generation of the vector construct with the ABCG2-K86M mutant was described earlier [26]. Login to comment 54 ABCG2 p.Arg482Gly In cholesterol depletion experiments we used the HEK 293 cell line stably expressing the R482G mutant established earlier [15]. Login to comment 112 ABCG2 p.Tyr570Ser ABCG2 p.Tyr469Ser ABCG2 p.Tyr469Ser ABCG2 p.Tyr413Ser 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). Login to comment 113 ABCG2 p.Tyr645Ser ABCG2 p.Tyr469Ser In order to test if the inactivity of the Y469S and Y645S mutants was due to a specific loss of Tyr at this position, we also mutated these tyrosines to phenylalanines. Login to comment 114 ABCG2 p.Tyr645Phe ABCG2 p.Tyr469Phe We found that the Y469F and Y645F mutants were active (Fig. 2B), indicating the importance of the phenyl ring, but not of a hydroxyl group at these positions for protein function. Login to comment 120 ABCG2 p.Tyr645Phe ABCG2 p.Tyr469Phe ABCG2 p.Tyr570Ser We found that the basal ATPase activity of the Y469F, Y570S and Y645F mutants showed a moderate (approximately 20%, p b 0.05) increase upon cholesterol addition, while the substrate stimulated ATP hydrolysis of the same mutants was significantly (50-100% increase, p b 0.01) accelerated by cholesterol loading (Fig. 2C). Login to comment 123 ABCG2 p.Tyr413Ser Conversely, we found that in the presence of cholesterol, the ATPase activity of ABCG2-Y413S was distinct from that of the wild-type transporter. Login to comment 124 ABCG2 p.Tyr413Ser 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). Login to comment 125 ABCG2 p.Tyr413Ser 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). Login to comment 127 ABCG2 p.Tyr413Ser In contrast, the Y413S mutant had a significantly altered ATPase modulation pattern. Login to comment 129 ABCG2 p.Tyr413Ser 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. Login to comment 130 ABCG2 p.Tyr413Ser 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). Login to comment 131 ABCG2 p.Tyr413Ser 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. Login to comment 133 ABCG2 p.Tyr413Ser ABCG2 p.Tyr413Ser 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. Login to comment 144 ABCG2 p.Tyr413Ser bile acids on the ATPase activity of the Y413S mutant. Login to comment 146 ABCG2 p.Tyr413Ser 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). Login to comment 159 ABCG2 p.Tyr413Ser 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. Login to comment 161 ABCG2 p.Tyr413Ser 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. Login to comment 163 ABCG2 p.Tyr413Ser Effect of known wtABCG2 substrates on the ATPase activity of wtABCG2 and the Y413S mutant. Login to comment 168 ABCG2 p.Tyr413Ser Effect of cholesterol on the wtABCG2 and Y413S-ATPase. Login to comment 174 ABCG2 p.Tyr645Phe ABCG2 p.Tyr469Phe ABCG2 p.Tyr413Ser generated mammalian HEK 293 cells stably expressing the Y413S, Y469F, Y570S and Y645F mutants. Login to comment 181 ABCG2 p.Tyr413Ser 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. Login to comment 183 ABCG2 p.Tyr413Ser ABCG2 p.Tyr413Ser 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. Login to comment 185 ABCG2 p.Tyr413Ser 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. Login to comment 187 ABCG2 p.Tyr413Ser Effect of bile acids on the wtABCG2 and Y413S-ATPase. Login to comment 199 ABCG2 p.Tyr413Ser 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. Login to comment 200 ABCG2 p.Tyr413Ser 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). Login to comment 221 ABCG2 p.Tyr459Ser Tyr to Ser mutations at positions 459, 469 and 645 resulted in the loss of protein function, and the Tyr 459 to Ser mutation resulted in a complete loss of ABCG2 expression (Fig. 2). Login to comment 223 ABCG2 p.Tyr459Ser The fact that the Y459S mutant could not be expressed properly in insect cells suggests that the tyrosine at this amino acid position is crucial for the proper folding and processing of the protein and therefore might be an important determinant of ABCG2 structure. Login to comment 224 ABCG2 p.Tyr459Cys Recently, the Y459C heterozygous mutation was reported to occur in patients with renal cancer [38]. Login to comment 226 ABCG2 p.Tyr645Ser ABCG2 p.Tyr469Ser The Y469S and Y645S mutants could be expressed in comparable amounts to the wild-type protein, however, they were found to be non-functional (Fig. 2). Login to comment 231 ABCG2 p.Tyr645Ser ABCG2 p.Tyr469Ser Our experiments, in which we tested the conformation of the ABCG2 mutants by labeling them with the conformation sensitive anti-ABCG2 5D3 antibody, revealed that the Y469S and Y645S mutants have decreased 5D3 binding capacity (Supplementary Fig. S4). Login to comment 232 ABCG2 p.Tyr645Ser ABCG2 p.Tyr469Ser Therefore the loss of the activity of the Y469S and Y645S mutants is most probably due to their improper conformation and not by their altered cholesterol-sensing. Login to comment 233 ABCG2 p.Tyr645Phe Previously, the Y645F mutation has been found to be functional, although has shown slightly decreased Hoechst 33342 transport capacity [39]. Login to comment 239 ABCG2 p.Tyr413Ser However, the Y413S mutant exhibited a distinct ATPase activity. Login to comment 250 ABCG2 p.Tyr645Phe ABCG2 p.Tyr469Phe ABCG2 p.Tyr570Ser ABCG2 p.Tyr413Ser BODIPY-prazosin Pheophorbide A Hoechst 33342 mitoxantrone wtABCG2 + + + + Y413S + + + + Y469F + + + + Y570S + + + + Y645F + + + + Fig. 8. Login to comment 257 ABCG2 p.Tyr413Ser 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. Login to comment 262 ABCG2 p.Tyr413Ser Moreover, potential substrates that are known to activate the wild-type ABCG2-ATPase, rather inhibited the ATPase activity of the Y413S mutant (Fig. 3). Login to comment 263 ABCG2 p.Tyr413Ser 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). Login to comment 267 ABCG2 p.Tyr413Ser In the case of the Y413S mutant, cholesterol may promote "uncoupling" of the ATP hydrolysis in the ABCG2 protein. Login to comment 268 ABCG2 p.Arg482Gly ABCG2 p.Tyr413Ser 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]. Login to comment 270 ABCG2 p.Tyr413Ser If ABCG2 can transport cholesterol, our data can be interpreted to suggest that the Y413S mutant is a more efficient cholesterol transporter. Login to comment 273 ABCG2 p.Tyr413Ser 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. Login to comment 274 ABCG2 p.Tyr413Ser 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. Login to comment 275 ABCG2 p.Tyr413Ser 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). Login to comment 276 ABCG2 p.Tyr413Ser Moreover, the Y413S mutant protected the cells against toxic drugs similarly to the wild-type ABCG2. Login to comment 277 ABCG2 p.Tyr413Ser 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. Login to comment