ABCMdb

PMID: 24384916

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., [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCG2 p.Leu558Ala 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. Login to comment 26 ABCG2 p.Arg482Gly Still, in isolated ABCG2 preparations even the R482G mutant required low levels of cholesterol for full function (Telbisz et al., 2013). Login to comment 38 ABCG2 p.Leu558Ala ABCG2 p.Leu558Ala ABCG2 p.Leu555Ala ABCG2 p.Leu555Ala 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. Login to comment 48 ABCG2 p.Lys86Met Generation of the baculovirus transfer vector (pAcUW21-L) harboring the cDNA for wild-type (wt) ABCG2 or the R482 and K86M mutants was described previously (Ozvegy et al., 2002; Ozvegy-Laczka et al., 2005). Login to comment 49 ABCG2 p.Leu558Ala ABCG2 p.Leu555Ala 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. Login to comment 52 ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly The R482G/L555/ 558A triple mutant was created by replacing the DNA fragment between the PstI-NcoI sites of the pAcUW21-L/R482G with that of derived from the pAcUW21-L/L555/558A vector. Login to comment 55 ABCG2 p.Leu558Ala ABCG2 p.Leu558Ala 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. Login to comment 91 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly In contrast to the wild-type protein, in Sf9 cell membranes increasing the membrane cholesterol levels did not significantly influence the activity of the R482G and R482T mutants (Telbisz et al., 2007), although experiments on purified ABCG2 reconstituted in proteoliposomes revealed that cholesterol is also essential for the function of the R482G variant (Telbisz et al., 2013). Login to comment 92 ABCG2 p.Arg482Asp To examine how the characteristics of amino acid 482 influence the cholesterol-sensing capability of ABCG2, we have analyzed seven additional R482 mutants (R482D, I, M, N, S, Y, and K). Login to comment 95 ABCG2 p.Arg482Asp In cholesterol-loaded membranes, the basal ATPase activity of most of these variants increased (see Supplemental Table 1), but the increase in membrane cholesterol levels did not change the relative substrate stimulation of the R482D, G, N, S, and T variants (Fig. 1A). Login to comment 96 ABCG2 p.Arg482Ile In contrast, in the case of the R482I, K, M, and Y variants, similarly to the wild-type ABCG2, cholesterol enrichment significantly improved the ratio of substrate stimulation, as examined after the addition of prazosin (Fig. 1A). Login to comment 108 ABCG2 p.Arg482Lys ABCG2 p.Arg482Tyr Similar to earlier findings, there was a well-measurable Ko143-sensitive Hst dye transport both in the cells expressing wtABCG2 and in those expressing most R482 mutants, with only very low activity in the case of the R482K and R482Y variants (Fig. 1B). Login to comment 109 ABCG2 p.Arg482Ile When the cells were loaded with cholesterol, this Hst dye uptake was significantly improved in the wild-type and the R482I and M variants. Login to comment 110 ABCG2 p.Arg482Lys Moreover, significant Hst transport activity occurred in the case of the R482K and Y variants. Login to comment 111 ABCG2 p.Arg482Asp In contrast, we did not observe a significant effect of cholesterol on the Hst transport by the R482D, G, N, S, and T variants. Login to comment 115 ABCG2 p.Arg482Ile Cholesterol loading significantly increased R123 extrusion in cells expressing the R482I and M variants, while there was no measurable effect in the D, G, N, S, or T variants. Login to comment 116 ABCG2 p.Arg482Lys In the case of the wild-type protein and the R482K and Y variants, there was no detectable R123 extrusion in either the absence or presence of cholesterol. Login to comment 119 ABCG2 p.Leu558Ala ABCG2 p.Leu558Ala ABCG2 p.Leu555Ala ABCG2 p.Leu555Ala We have generated the Leu to Ala mutations L555A, L558A, and L555A/L558A in this motif. Login to comment 122 ABCG2 p.Leu558Ala ABCG2 p.Leu558Ala ABCG2 p.Leu555Ala ABCG2 p.Leu555Ala As shown in Fig. 2B, the L555A, L558A, and L555A/L558A mutants exhibited a well-measurable vanadate-sensitive ATPase activity. Login to comment 123 ABCG2 p.Leu558Ala ABCG2 p.Leu555Ala ABCG2 p.Leu555Ala 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. Login to comment 124 ABCG2 p.Leu558Ala ABCG2 p.Leu558Ala ABCG2 p.Leu555Ala ABCG2 p.Leu555Ala 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. Login to comment 133 ABCG2 p.Leu558Ala ABCG2 p.Leu558Ala ABCG2 p.Leu555Ala ABCG2 p.Leu555Ala 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. Login to comment 136 ABCG2 p.Leu558Ala ABCG2 p.Leu555Ala ABCG2 p.Leu555Ala 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. Login to comment 137 ABCG2 p.Leu558Ala 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. Login to comment 139 ABCG2 p.Leu558Ala 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). Login to comment 145 ABCG2 p.Arg482Gly Effect of Cholesterol on the Function of Isolated and Reconstituted ABCG2-SBE Mutants It was shown earlier that an increase in the cholesterol levels of the insect membranes did not significantly modulate the function of the ABCG2 R482G mutant, which would imply its apparent cholesterol insensitivity. Login to comment 146 ABCG2 p.Arg482Gly However, reconstitution of the purified ABCG2 R482G variant revealed that the presence of cholesterol was also essential for the function of this mutant variant; however, lower cholesterol levels (amounts that are most probably present in native insect membranes) were sufficient to achieve its full activity as compared with wtABCG2 (Telbisz et al., 2013). Login to comment 147 ABCG2 p.Leu558Ala 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. Login to comment 148 ABCG2 p.Leu558Ala ABCG2 p.Leu558Ala ABCG2 p.Leu555Ala 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). Login to comment 149 ABCG2 p.Leu558Ala The membrane isolation as well as the purification and reconstitution of the L558A variant were successful. Login to comment 159 ABCG2 p.Leu558Ala ABCG2 p.Leu555Ala 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). Login to comment 160 ABCG2 p.Leu558Ala We analyzed the ATPase activity of the purified L558A variant, reconstituted in E. coli lipids in the absence and in the presence of cholesterol. Login to comment 187 ABCG2 p.Lys86Met However, the most pronounced effect of CA is observed in cholesterol-loaded membranes (Fig. 4B, right columns): baseline ATPase activity is strongly reduced (almost to the level of Sf9 membranes expressing the inactive ABCG2-K86M mutant; see Fig. 2B), while drug-stimulated ATPase activity is unchanged. Login to comment 204 ABCG2 p.Leu558Ala (A) ATPase activity of purified wtABCG2, ABCG2-L558A, and L555/558A in proteoliposomes. Login to comment 225 ABCG2 p.Arg482Gly ABCG2 p.Arg482Ser ABCG2 p.Arg482Lys ABCG2 p.Arg482Ile These were the R482G and R482S variants, which are fully active already at low membrane cholesterol levels, and the R482K and R482I mutants, which show similar cholesterol-sensing capability to the wtABCG2 (see earlier). Login to comment 237 ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly ABCG2 p.Arg482Ser 4, C and D, and 6A), in the case of the R482G or S variants low concentrations of CA did not significantly alter ABCG2-ATPase activity; however, when we used higher bile acid concentrations (above 0.5 mM), both baseline and substrate-stimulated ATPase activities decreased (see Fig. 6B for the R482G mutant; R482S is not shown). Login to comment 239 ABCG2 p.Arg482Lys In the case of the R482K and I mutants, a variable alteration in the substrate stimulation was observed for different bile acids (Fig. 6D). Login to comment 240 ABCG2 p.Leu558Ala ABCG2 p.Leu555Ala 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. Login to comment 241 ABCG2 p.Leu558Ala ABCG2 p.Leu555Ala GC and TC also did not influence the activity of the L555A/L558A mutant (data not shown). Login to comment 242 ABCG2 p.Leu555Ala Similarly, the activity of the L555A was not altered by the presence of bile acids. Login to comment 243 ABCG2 p.Arg482Gly ABCG2 p.Leu558Ala 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). Login to comment 244 ABCG2 p.Leu558Ala GC had no effect on either baseline or substrate-stimulated activity of the L558A mutant (Supplemental Fig. 5). Login to comment 252 ABCG2 p.Arg482Ile Members of the first cluster, also including the wild-type protein (R), contain large (hydrophobic or positively charged) amino acids, represented by R482I, M, K, and Y. Login to comment 254 ABCG2 p.Arg482Lys Interestingly, the activating effect of cholesterol is the most pronounced in the case of the R482K and Y mutants, as these variants are practically unable to transport Hoechst 33342 unless high levels of cholesterol are present in the cell membranes (Fig. 1B). Login to comment 258 ABCG2 p.Leu558Ala ABCG2 p.Leu555Ala 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. Login to comment 259 ABCG2 p.Leu558Ala ABCG2 p.Leu555Ala In our present work, we expressed and analyzed in detail the SBE (or LxxL motif) mutants L555A, L558A, and L555/558A of human ABCG2. Login to comment 262 ABCG2 p.Leu558Ala ABCG2 p.Leu555Ala 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). Login to comment 264 ABCG2 p.Leu558Ala ABCG2 p.Leu555Ala Moreover, the L555A/L558A mutant was absolutely insensitive to cholesterol loading in the ATPase-activity measurements (Fig. 2B). Login to comment 265 ABCG2 p.Leu558Ala ABCG2 p.Leu555Ala This apparent cholesterol independence of the L555A/L558A Fig. 5. Login to comment 271 ABCG2 p.Arg482Gly ABCG2 p.Leu558Ala ABCG2 p.Leu555Ala 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. Login to comment 272 ABCG2 p.Arg482Gly As demonstrated elsewhere and in this report as well, the R482G variant is already fully active in cholesterol-deficient Sf9 membranes. Login to comment 273 ABCG2 p.Arg482Gly ABCG2 p.Leu558Ala ABCG2 p.Leu555Ala To solve this contradiction, we also generated the triple mutant R482G/L555A/L558A of ABCG2 and expressed this protein in insect cells. Login to comment 275 ABCG2 p.Leu558Ala ABCG2 p.Leu558Ala ABCG2 p.Leu555Ala 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. Login to comment 276 ABCG2 p.Leu558Ala Surprisingly, we found that the L558A mutant also needs cholesterol for its full activity. Login to comment 295 ABCG2 p.Arg482Gly Membranes were incubated with or without 1 mM bile acid; the ratio of ATP hydrolysis in the presence of 1 mM quercetin (wt, R482G, S, K, and I) or 1 mM nilotinib (SBE mutants) and the baseline ATPase activity were calculated. Login to comment 299 ABCG2 p.Lys86Met Moreover, we observed that when membranes were loaded with cholesterol, CA decreased the baseline ATP hydrolysis down to the background level, that is, to ATP hydrolysis in membranes expressing the inactive mutant ABCG2-K86M (see Fig. 2B). Login to comment