ABCG2 p.Leu558Ala
| Predicted by SNAP2: | A: D (75%), C: D (71%), D: D (85%), E: D (85%), F: N (53%), G: D (85%), H: D (71%), I: N (57%), K: D (80%), M: D (71%), N: D (80%), P: D (80%), Q: D (75%), R: D (75%), S: D (59%), T: D (75%), V: N (57%), W: D (85%), Y: D (75%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: N, G: D, H: D, I: N, K: D, M: N, N: D, P: D, Q: D, R: D, S: D, T: D, V: N, W: D, Y: N, |
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[hide] A functional steroid-binding element in an ATP-bin... Mol Pharmacol. 2008 Jan;73(1):12-7. Velamakanni S, Janvilisri T, Shahi S, van Veen HW
A functional steroid-binding element in an ATP-binding cassette multidrug transporter.
Mol Pharmacol. 2008 Jan;73(1):12-7., [PMID:18094074]
Abstract [show]
The human breast cancer resistance protein is an ATP-binding cassette (ABC) multidrug transporter that affects the bioavailability of chemotherapeutic drugs and can confer drug resistance on cancer cells. It is the second member of the ABCG subfamily, other members of which are associated with human steroid disorders such as hypercholesterolemia, sitosterolemia, and atherosclerosis. The molecular bases of protein-steroid interactions in ABC transporters are unknown. Here, we identify a steroid-binding element in the membrane domain of ABCG2 with a similarity to steroid hormone/nuclear receptors. The element facilitates steroid hormone binding and mediates modulation of ABCG2 activity. The identification of this element might provide an opportunity for the development of new therapeutic ligands for ABCG2.
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No. Sentence Comment
28 Mutations in the ABCG2R482G gene were introduced using the forward primer 5b18;-TTT TTT CAC GTC TGT TGG TCA ATC TCA C-3b18; and the reverse primer 5b18;-ATT GAC CAA CAG ACG TGA AAA AAT CAT C-3b18; for G553R, the forward primer 5b18;-GAT GAT TTT TAT GGG TCT GTT GGT CAA TCT CAC-3b18; and reverse primer 5b18;-CCA ACA GAC CCA TAA AAA TCA TCA TAA ACA C-3b18; for S552M, and the forward primer 5b18;-GGT CTG GGG GTC AAT GGC ACA ACC ATT GCA TCT TGG-3b18; and reverse primer 5b18;- ATG GTT GTG CCA TTG ACC CCC AGA CCT GAA AAA ATC-3b18; for L555A L558A.
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ABCG2 p.Leu558Ala 18094074:28:576
status: NEW[hide] Regulation of the function of the human ABCG2 mult... Drug Metab Dispos. 2014 Apr;42(4):575-85. doi: 10.1124/dmd.113.055731. Epub 2014 Jan 2. Telbisz A, Hegedus C, Varadi A, Sarkadi B, Ozvegy-Laczka C
Regulation of the function of the human ABCG2 multidrug transporter by cholesterol and bile acids: effects of mutations in potential substrate and steroid binding sites.
Drug Metab Dispos. 2014 Apr;42(4):575-85. doi: 10.1124/dmd.113.055731. Epub 2014 Jan 2., [PMID:24384916]
Abstract [show]
ABCG2 (ATP-binding cassette, subfamily G, member 2) is a plasma membrane glycoprotein that actively extrudes xenobiotics and endobiotics from the cells and causes multidrug resistance in cancer. In the liver, ABCG2 is expressed in the canalicular membrane of hepatocytes and excretes its substrates into the bile. ABCG2 is known to require high membrane cholesterol content for maximal activity, and by examining purified ABCG2 reconstituted in proteoliposomes we have recently shown that cholesterol is an essential activator, while bile acids significantly modify the activity of this protein. In the present work, by using isolated insect cell membrane preparations expressing human ABCG2 and its mutant variants, we have analyzed whether certain regions in this protein are involved in sterol recognition. We found that replacing ABCG2-R482 with large amino acids does not affect cholesterol dependence, but changes to small amino acids cause altered cholesterol sensitivity. When leucines in the potential steroid-binding element (SBE, aa 555-558) of ABCG2 were replaced by alanines, cholesterol dependence of ABCG2 activity was strongly reduced, although the L558A mutant variant when purified and reconstituted still required cholesterol for full activity. Regarding the effect of bile acids in isolated membranes, we found that these compounds decreased ABCG2-ATPase in the absence of drug substrates, which did not significantly affect substrate-stimulated ATPase activity. These ABCG2 mutant variants also altered bile acid sensitivity, although cholic acid and glycocholate were not transported by the protein. We suggest that the aforementioned two regions in ABCG2 are important for sterol sensing and may represent potential targets for pharmacologic modulation of ABCG2 function.
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No. Sentence Comment
5 When leucines in the potential steroid-binding element (SBE, aa 555-558) of ABCG2 were replaced by alanines, cholesterol dependence of ABCG2 activity was strongly reduced, although the L558A mutant variant when purified and reconstituted still required cholesterol for full activity.
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ABCG2 p.Leu558Ala 24384916:5:185
status: NEW38 In our present study, we provide a detailed mutational analysis of the cholesterol-sensing capability of different ABCG2 R482 mutants as well as mutants carrying the L555A, L558A, or L555A/L558A point mutations.
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ABCG2 p.Leu558Ala 24384916:38:173
status: NEWX
ABCG2 p.Leu558Ala 24384916:38:189
status: NEW49 The steroid-binding element mutants were created by site-directed polymerase chain reaction (PCR) mutagenesis using the following complementary primer pairs: L555A: 59 T TCA GGT CTC GCG GTC AAT CT and 59AG ATT GAC CGC GAG ACC TGA A; L558A: 59 GT CTG TTG GTG AAT GCC ACA ACC ATT and 59 AAT GGT TGT GGC ATT CAC CAA CAG AC; L555/558A: 59 GT CTC GCG GTG AAT GCC ACA ACC ATT and 59 AAT GGT TGT GGC ATT CAC CGC GAG AC.
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ABCG2 p.Leu558Ala 24384916:49:233
status: NEW55 The His6-tagged L558A and L555/ 558A mutants were created by cloning the PstI-SacI site from pAcUW21-L/ ABCG2-L558A or L555/558A into the pAcUW21-L/His6-ABCG2.
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ABCG2 p.Leu558Ala 24384916:55:16
status: NEWX
ABCG2 p.Leu558Ala 24384916:55:110
status: NEW119 We have generated the Leu to Ala mutations L555A, L558A, and L555A/L558A in this motif.
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ABCG2 p.Leu558Ala 24384916:119:50
status: NEWX
ABCG2 p.Leu558Ala 24384916:119:67
status: NEW122 As shown in Fig. 2B, the L555A, L558A, and L555A/L558A mutants exhibited a well-measurable vanadate-sensitive ATPase activity.
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ABCG2 p.Leu558Ala 24384916:122:32
status: NEWX
ABCG2 p.Leu558Ala 24384916:122:49
status: NEW123 However, given the similar expression levels of the wild-type and the mutant proteins, we found that L555A and L555A/L558A had only about one-third the basal ATPase activity as compared with wtABCG2.
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ABCG2 p.Leu558Ala 24384916:123:117
status: NEW124 Moreover, in the case of the L555A and L555A/L558A mutants, ATPase turnover in the presence of quercetin was also well below of that measured for the wild-type protein or the L558A mutant.
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ABCG2 p.Leu558Ala 24384916:124:45
status: NEWX
ABCG2 p.Leu558Ala 24384916:124:175
status: NEW133 In these experiments, we examined the effect of cholesterol on the [3 H]methotrexate ([3 H]MTX) and [3 H]estradiol-glucuronide ([3 H]ESG) transport activity of ABCG2 L555A, L558A, and L555A/L558A mutant variants expressed in Sf9 insect cells.
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ABCG2 p.Leu558Ala 24384916:133:173
status: NEWX
ABCG2 p.Leu558Ala 24384916:133:190
status: NEW136 We found that despite their comparable expression level to wtABCG2, the L555A and L555A/L558A mutants did not show any detectable vesicular transport activity for MTX in either control or cholesterol-rich membranes.
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ABCG2 p.Leu558Ala 24384916:136:88
status: NEW137 Even in the case of the L558A mutant, which showed high ATPase activity, we could detect only very low MTX transport activity, similar to that observed in the R482 mutants (Supplemental Figs.
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ABCG2 p.Leu558Ala 24384916:137:24
status: NEW139 When we analyzed [3 H]MTX transport by the L558A mutant in membranes loaded with cholesterol, we found only a nonsignificant increase in this transport activity (Supplemental Fig. 3A).
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ABCG2 p.Leu558Ala 24384916:139:43
status: NEW147 To analyze the cholesterol sensing of the purified SBE mutants, we have generated N-terminally His6-tagged versions of the L558A and L555/558A variants.
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ABCG2 p.Leu558Ala 24384916:147:123
status: NEW148 The His6-L558A and His6-L555A/ L558A ABCG2 mutants were successfully expressed in Sf9 cells, and we also found that tagging did not alter their functionality (data not shown).
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ABCG2 p.Leu558Ala 24384916:148:9
status: NEWX
ABCG2 p.Leu558Ala 24384916:148:31
status: NEW149 The membrane isolation as well as the purification and reconstitution of the L558A variant were successful.
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ABCG2 p.Leu558Ala 24384916:149:77
status: NEW159 the expression level of the His6-L555A/L558A variant in the Sf9 cells was comparable to that of the other variants, the purification yielded a much lower amount of this mutant (data not shown).
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ABCG2 p.Leu558Ala 24384916:159:39
status: NEW160 We analyzed the ATPase activity of the purified L558A variant, reconstituted in E. coli lipids in the absence and in the presence of cholesterol.
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ABCG2 p.Leu558Ala 24384916:160:48
status: NEW204 (A) ATPase activity of purified wtABCG2, ABCG2-L558A, and L555/558A in proteoliposomes.
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ABCG2 p.Leu558Ala 24384916:204:47
status: NEW240 Figure 6C shows that CA does not influence ATP hydrolysis of the L555A/L558A mutant-that is, both basal and substrate-stimulated activities remained unaltered.
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ABCG2 p.Leu558Ala 24384916:240:71
status: NEW241 GC and TC also did not influence the activity of the L555A/L558A mutant (data not shown).
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ABCG2 p.Leu558Ala 24384916:241:59
status: NEW243 In the case of the L558A mutant, a similar effect of bile acids was observed as in the case of the R482G variant: CA and TC inhibited both basal and substrate-stimulated ATPase activity (Supplemental Fig. 5), but the relative substrate activation was practically unchanged (Fig. 6D).
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ABCG2 p.Leu558Ala 24384916:243:19
status: NEW244 GC had no effect on either baseline or substrate-stimulated activity of the L558A mutant (Supplemental Fig. 5).
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ABCG2 p.Leu558Ala 24384916:244:76
status: NEW258 In their study, Velamakanni et al. (2008) found that the ABCG2-L555A/L558A mutant does not have an altered cholesterol sensing, but progesterone and estradiol binding as well as transport were abolished.
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ABCG2 p.Leu558Ala 24384916:258:69
status: NEW259 In our present work, we expressed and analyzed in detail the SBE (or LxxL motif) mutants L555A, L558A, and L555/558A of human ABCG2.
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ABCG2 p.Leu558Ala 24384916:259:96
status: NEW262 When examining the effect of cholesterol on their function, we found that although a slight increase in the baseline ATP hydrolysis of the L555A and L558A mutants occurred in cholesterol-enriched membranes (fold activation was 1.260.1 and 1.560.1, respectively), their relative substrate stimulation (ratio of ATP hydrolysis in the presence and absence of substrates) did not change (Fig. 2B and Supplemental Fig. 2).
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ABCG2 p.Leu558Ala 24384916:262:149
status: NEW264 Moreover, the L555A/L558A mutant was absolutely insensitive to cholesterol loading in the ATPase-activity measurements (Fig. 2B).
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ABCG2 p.Leu558Ala 24384916:264:20
status: NEW265 This apparent cholesterol independence of the L555A/L558A Fig. 5.
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ABCG2 p.Leu558Ala 24384916:265:52
status: NEW271 mutant contradicted the results described by Velamakanni et al. (2008), which may be due to the fact that they investigated a triple mutant of ABCG2, which had R482G besides the L555A/L558A mutation, whereas we performed our experiments using the wild-type ABCG2 (482R) as a background.
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ABCG2 p.Leu558Ala 24384916:271:184
status: NEW273 To solve this contradiction, we also generated the triple mutant R482G/L555A/L558A of ABCG2 and expressed this protein in insect cells.
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ABCG2 p.Leu558Ala 24384916:273:77
status: NEW275 Because even very low levels of membrane sterols may affect ABCG2 function, we have purified and reconstituted the L558A and L555A/L558A mutants in cholesterol-free liposomes.
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ABCG2 p.Leu558Ala 24384916:275:115
status: NEWX
ABCG2 p.Leu558Ala 24384916:275:131
status: NEW276 Surprisingly, we found that the L558A mutant also needs cholesterol for its full activity.
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ABCG2 p.Leu558Ala 24384916:276:32
status: NEW