ABCG2 p.Leu555Ala
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PMID: 24384916
[PubMed]
Telbisz A et al: "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."
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Sentence
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38
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.Leu555Ala 24384916:38:166
status: NEWX
ABCG2 p.Leu555Ala 24384916:38:183
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.Leu555Ala 24384916:49:158
status: NEW119 We have generated the Leu to Ala mutations L555A, L558A, and L555A/L558A in this motif.
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ABCG2 p.Leu555Ala 24384916:119:43
status: NEWX
ABCG2 p.Leu555Ala 24384916:119:61
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.Leu555Ala 24384916:122:25
status: NEWX
ABCG2 p.Leu555Ala 24384916:122:43
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.Leu555Ala 24384916:123:101
status: NEWX
ABCG2 p.Leu555Ala 24384916:123:111
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.Leu555Ala 24384916:124:29
status: NEWX
ABCG2 p.Leu555Ala 24384916:124:39
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.Leu555Ala 24384916:133:166
status: NEWX
ABCG2 p.Leu555Ala 24384916:133:184
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.Leu555Ala 24384916:136:72
status: NEWX
ABCG2 p.Leu555Ala 24384916:136:82
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.Leu555Ala 24384916:148:24
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.Leu555Ala 24384916:159:33
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.Leu555Ala 24384916:240:65
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.Leu555Ala 24384916:241:53
status: NEW242 Similarly, the activity of the L555A was not altered by the presence of bile acids.
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ABCG2 p.Leu555Ala 24384916:242:31
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.Leu555Ala 24384916:258:63
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.Leu555Ala 24384916:259:89
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.Leu555Ala 24384916:262:139
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.Leu555Ala 24384916:264:14
status: NEW265 This apparent cholesterol independence of the L555A/L558A Fig. 5.
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ABCG2 p.Leu555Ala 24384916:265:46
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.Leu555Ala 24384916:271:178
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.Leu555Ala 24384916:273:71
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.Leu555Ala 24384916:275:125
status: NEW