ABCMdb

PMID: 20088606

Polgar O, Ierano C, Tamaki A, Stanley B, Ward Y, Xia D, Tarasova N, Robey RW, Bates SE
Mutational analysis of threonine 402 adjacent to the GXXXG dimerization motif in transmembrane segment 1 of ABCG2.
Biochemistry. 2010 Mar 16;49(10):2235-45., 2010-03-16 [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCG2 p.Thr402Leu ABCG2 p.Thr402Arg Single mutations to leucine (T402L) or arginine (T402R) did not have a significant impact on the ABCG2 protein. Login to comment 5 ABCG2 p.Gly406Leu ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Gly410Leu ABCG2 p.Thr402Leu ABCG2 p.Thr402Arg On the other hand, combining the T402 mutations with the GXXXG glycine to leucine mutations (T402L/ G406L/G410L and T402R/G406L/G410L) resulted in a substantially reduced level of expression, altered glycosylation, degradation by a proteosome-independent pathway, and partial retention in the endoplasmic reticulum as suggested by immunostaining, Endo H sensitivity, and MG132 and bafilomycin failed effect. Login to comment 6 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Thr402Leu The T402L/G406L/G410L mutant when incubated with the ABCG2 substrate MX showed a shift on immunoblot analysis to the band representing the fully mature glycoprotein. Login to comment 7 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Thr402Arg The T402R/G406L/G410L mutant carrying the more drastic substitution was found to primarily localize intracellularly. Login to comment 45 ABCG2 p.Gly406Leu ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Gly410Leu ABCG2 p.Thr402Leu ABCG2 p.Thr402Leu ABCG2 p.Thr402Arg ABCG2 p.Thr402Arg The T402L, T402R, T402L/ G406L/G410L, and T402R/G406L/G410L mutants were generated by site-directed mutagenesis in the pcDNA3.1/Myc-HisA(-) vector (Invitrogen) as previously described (19). Login to comment 47 ABCG2 p.Arg482Gly ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu The R482G control and G406L/G410L mutant were previously generated and characterized (19, 23). Login to comment 92 ABCG2 p.Gly406Leu ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Gly410Leu ABCG2 p.Thr402Leu ABCG2 p.Thr402Leu ABCG2 p.Thr402Arg ABCG2 p.Thr402Arg To study the potential role of this threonine in ABCG2 dimerization, we performed substitutions with a leucine (T402L) or arginine (T402R) substitution or combined these substitutions with the glycine to leucine mutations at the GXXXG motif (T402L/ G406L/G410L and T402R/G406L/G410L) followed by stable transfections in HEK 293 cells. Login to comment 93 ABCG2 p.Thr402Leu The T402L mutation was designed to probe the perceived involvement of this residue in hydrogen bonding. Login to comment 94 ABCG2 p.Thr402Arg We expected the T402R substitution to be a more drastic substitution on the basis of the change in both size and polarity. Login to comment 95 ABCG2 p.Arg482Gly ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu We used the previously generated G406L/G410L and R482G transfectants in HEK 293 cells as controls (19). Login to comment 96 ABCG2 p.Arg482Gly The R482G mutation is considered a gain-of-function mutation, which enables the protein to transport a wider range of substrates (23). Login to comment 97 ABCG2 p.Arg482Gly As in the previous study (19), all the GXXXG and threonine 402 mutants described here carry the R482G mutation. Login to comment 101 ABCG2 p.Arg482Gly ABCG2 p.Gly406Leu ABCG2 p.Gly406Leu ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Gly410Leu ABCG2 p.Gly410Leu ABCG2 p.Thr402Leu ABCG2 p.Thr402Leu ABCG2 p.Thr402Arg ABCG2 p.Thr402Arg The T402L and T402R mutants exhibited levels comparable to the control (R482G), while the double (G406L/G410L) and triple mutants (T402L/G406L/G410L and T402R/G406L/G410L) had significantly decreased levels. Login to comment 103 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Thr402Arg In the case of T402R/G406L/G410L, the lower band comprised the majority of the detected protein. Login to comment 106 ABCG2 p.Arg482Gly ABCG2 p.Thr402Leu ABCG2 p.Thr402Arg As shown in the first column of Figure 1B, the control (R482G) and the T402L and T402R mutants displayed similar levels of surface expression. Login to comment 107 ABCG2 p.Gly406Leu ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Gly410Leu ABCG2 p.Thr402Leu The G406L/G410L and T402L/G406L/G410L mutants displayed substantially lower surface expression levels with the 5D3 antibody. Login to comment 108 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Thr402Arg In the T402R/G406L/G410L triple mutant, only a slight shift between the negative control(solid line) and the 5D3-labeled histograms (dashed line) was present. Login to comment 114 ABCG2 p.Thr402Leu The control and the T402L and T402Rmutants wereable toefflux MX,pheophorbide a, and BODIDY-prazosin from the cells. Login to comment 117 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu This was an expected result, given the low levels of expression and the fact that the GXXXG double leucine mutant (G406L/G410L) displayed almost no transport activity (19). Login to comment 121 ABCG2 p.Arg482Gly As shown in Figure 2A, a lower-molecular mass band was observed in the control (R482G) following digestion with N-glycosidase F to remove all N-linked glycans, consistent with removal of the polysaccharide chain from asparagine 596 of ABCG2 as shown following site-directed mutagenesis of N596 (31). Login to comment 122 ABCG2 p.Thr402Leu ABCG2 p.Thr402Arg As expected, no change in molecular mass was observed in the control, T402L, or T402R after Endo H digestion (Figure 2A), since Endo H resistance is consistent with a fully mature glycoprotein. Login to comment 125 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu Minimal change was observed in the molecular mass of ABCG2 in the G406L/G410L mutant after Endo H digestion. Login to comment 130 ABCG2 p.Arg482Gly ABCG2 p.Gly406Leu ABCG2 p.Gly406Leu ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Gly410Leu ABCG2 p.Gly410Leu ABCG2 p.Thr402Leu ABCG2 p.Thr402Leu ABCG2 p.Thr402Arg ABCG2 p.Thr402Arg (A) Cell lysates from stably transfected HEK 293 cells (20 μg for R482G, T402L, and T402R and 60 μg for G406L/G410L, T402L/G406L/G410L, and T402R/G406L/G410L) were separated via SDS-PAGE, transferred onto a PVDF membrane, and probed with monoclonal anti-ABCG2 antibody BXP-21. Login to comment 135 ABCG2 p.Thr402Leu ABCG2 p.Thr402Arg On the other hand, the protein level of the control, T402R, and T402L proteins increased more than 2-fold when cells were treated with bafilomycin, consistent with a previous report that fully processed ABCG2 undergoes lysosomal degradation as part of the protein turnover (33). Login to comment 136 ABCG2 p.Gly406Leu ABCG2 p.Gly406Leu ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Gly410Leu ABCG2 p.Gly410Leu ABCG2 p.Thr402Leu ABCG2 p.Thr402Arg In contrast, the protein levels of the G406L/G410L, T402L/G406L/G410L, and T402R/G406L/ G410L mutants were little affected by bafilomycin treatment (Figure 3). Login to comment 141 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu Previously, we reported that the levels of the G406L/G410L mutant significantly increased on immunoblot and on immunofluorescence following MX treatment (19). Login to comment 142 ABCG2 p.Thr402Leu ABCG2 p.Thr402Arg In the case of the control, T402L, and T402R proteins, no significant differences between the MX-treated and untreated samples were observed (Figure 4A). Login to comment 143 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu As previously shown, a significant increase in protein levels was observed for the G406L/ G410L mutant (Figure 4A). Login to comment 144 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Thr402Leu In contrast, overnight treatment with MX resulted in a majority of the T402L/G406L/G410L mutant being detected in the normal 72 kDa band as opposed to the double band observed without drug exposure, representing a shift from the lower to the upper molecular mass band (Figure 4A). Login to comment 145 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Thr402Arg The average percent of mutant protein detected in the lower and upper bands with and without MX is presented in Figure 4B. Notably, the results obtained with the arginine triple mutant (T402R/G406L/G410L) revealed little shift of the lower band, consistent with a more profound defect. Login to comment 147 ABCG2 p.Thr402Leu ABCG2 p.Thr402Arg No change was observed in the mainly plasma membrane staining for the control, T402L, and T402R proteins after treatment with MX by confocal microscopy (Figure 5). Login to comment 148 ABCG2 p.Gly406Leu ABCG2 p.Gly406Leu ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Gly410Leu ABCG2 p.Thr402Leu The G406L/G410L mutant exhibited primarily plasma membrane staining with little intracellular signal and after MX treatment displayed a slightly increased level of surfaceexpression.InthecaseoftheT402L/G406L/G410Lmutant, both intracellular and cell surface staining could be observed prior to treatment with MX, while after overnight MX treatment, we observed an increased level of ABCG2 plasma membrane localization in the T402L/G406L/G410L mutant (Figure 5). Login to comment 149 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Thr402Arg In contrast, after treatment with MX, the T402R/G406L/G410L mutant still primarily displayed intracellular localization. Login to comment 151 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Thr402Leu As demonstrated in Figure 6, following MX treatment, the T402L/G406L/G410L mutant was no longer sensitive to Endo H (Figure 6B), implying that its glycan has matured and that the protein has most likely moved out of the ER. Login to comment 152 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Thr402Arg In contrast, the lower-molecular mass form of the T402R/G406L/G410L mutant, which did not shift to the upper 72 kDa band in the presence of MX, was still sensitive to digestion with Endo H (Figure 6B). Login to comment 155 ABCG2 p.Arg482Gly ABCG2 p.Gly406Leu ABCG2 p.Gly406Leu ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Gly410Leu ABCG2 p.Gly410Leu ABCG2 p.Thr402Leu ABCG2 p.Thr402Leu ABCG2 p.Thr402Arg ABCG2 p.Thr402Arg Immunoblot analysis of cell lysates from the R482G, T402L, and T402R mutants [(A) 35 μg] and from the G406L/G410L, T402L/G406L/G410L, and T402R/G406L/G410L mutants [(B) 70 μg] with the BXP-21 antibody following overnight treatment with Endo H or N-glycosidase F. Login to comment 158 ABCG2 p.Arg482Gly ABCG2 p.Gly406Leu ABCG2 p.Gly406Leu ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Gly410Leu ABCG2 p.Gly410Leu ABCG2 p.Thr402Leu ABCG2 p.Thr402Leu ABCG2 p.Thr402Arg ABCG2 p.Thr402Arg Cells were harvested after overnight incubation with or without 10 nM bafilomycin followed by immunoblot analysis with the BXP-21 and GAPDH antibodies for the R482G, T402L, and T402R mutants (40 μg/lane) and the G406L/G410L, T402L/G406L/G410L, and T402R/G406L/G410L mutants (100 μg/lane). Login to comment 166 ABCG2 p.Arg482Gly ABCG2 p.Thr402Leu ABCG2 p.Thr402Arg (A) Following overnight incubation with or without 5 μM MX, cells were harvested, and the lysates (25 μg for R482G, T402R, and T402L and 50 μg for the other mutants) were subjected to immunoblot analysis with the BXP-21 and GAPDH antibodies as described in the legend of Figure 1. Login to comment 170 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Thr402Arg ABCG2 p.Thr402Arg Figure 7 demonstrates increased molecular mass bands consistent with cross-linking of the control, T402R, and T402R/ G406L/G410L proteins. Login to comment 171 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Thr402Leu ABCG2 p.Thr402Leu Similar results were obtained with the T402L and T402L/G406L/G410L mutants with DSG; furthermore, the triple mutants were also cross-linked by DSS at both 4 °C and room temperature (data not shown). Login to comment 178 ABCG2 p.Thr402Arg To evaluate the extended TXXXGXXXG motif in ABCG2, we substituted threonine 402 with arginine, since it had a greater impact on the protein relative to leucine (as suggested by the differences observed in localization and ability to be rescued by MX). Login to comment 179 ABCG2 p.Thr402Arg Figure 8B shows that the TM carrying the T402R mutation had CAT activity comparable to that of wild-type ABCG2 TM1. Login to comment 180 ABCG2 p.Gly406Leu ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Gly410Leu ABCG2 p.Thr402Arg In contrast, G406L/G410L and T402R/G406L/G410L demonstrated an approximately 50% or more decrease in their CAT activity, implying impaired dimerization of these mutant TMs. Login to comment 190 ABCG2 p.Thr402Leu When created insilico, the T402L mutation does not appear to interfere with interhelix packing between TM1 and TM5 and TM6 of its adjacent subunit (Figure 9D), which is consistent with the biochemical data for this mutant. Login to comment 196 ABCG2 p.Arg482Gly ABCG2 p.Thr402Leu ABCG2 p.Thr402Arg After overnight incubation with or without 5 μM MX, cells were harvested, and the lysates were subjected to overnight digestion with Endo H followed by immunoblot analysis with the BXP-21 antibody [(A) 35 μg for R482G, T402R, and T402L and (B) 70 μg for the double and triple mutants]. Login to comment 199 ABCG2 p.Thr402Leu ABCG2 p.Thr402Arg Cross-linking is observed in the control, T402L, T402R, and the triple mutants as suggested by higher-molecular mass species. Login to comment 204 ABCG2 p.Thr402Leu ABCG2 p.Thr402Arg Following incubation with the ABCG2 substrate, MX, T402L/T406L/T410L readily shifted to the plasma membrane while T402R/T406L/T410L did not. Login to comment 217 ABCG2 p.Thr402Leu Under the consideration that the GXXXG motif in TM1 may be involved in the dimer interface, or alternatively, in a transient interaction with the TM1 of a neighboring subunit, we chose to substitute threonine with leucine at position 402 in the mammalian system to replace a polar with a nonpolar residue, similar to the substitution disruptive of Thr87 with Val in glycophorin A (22), and in doing so also introduced a small increase in size. Login to comment 219 ABCG2 p.Thr402Leu More importantly, we wanted to use the T402L substitution to probe the existence of the perceived hydrogen bonding interaction by T402. Login to comment 220 ABCG2 p.Thr402Leu The wild-type phenotype of the T402L mutant argues against H-bonding at this residue. Login to comment 226 ABCG2 p.Gly406Leu ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Gly410Leu ABCG2 p.Thr402Arg ABCG2 p.Thr402Arg (B) CAT activity measured in the TOXCAT assay for the G406L/G410L, T402R, and T402R/G406L/G410L mutants compared to that of wild-type TM1 of ABCG2. Login to comment 228 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Thr402Leu ABCG2 p.Thr402Leu This point is supported by the efficient shift of the lower band in the MX rescue experiments for the T402L and T402L/G406L/ G410L mutants discussed below. Login to comment 229 ABCG2 p.Thr402Arg Although the substitution with the larger positively charged arginine was expected to be an unfavorable change in the membrane environment and likely to disrupt the association of the transmembrane helices, the T402R mutant appeared on the cell surface and is capable of efflux. Login to comment 233 ABCG2 p.Gly406Leu ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Gly410Leu ABCG2 p.Thr402Leu ABCG2 p.Thr402Arg ABCG2 p.Thr402Arg ABCG2 p.Thr402Arg Our results with the ABCG2 triple mutants carrying the threonine 402 to leucine or arginine substitutions (T402R/ G406L/G410L and T402R/G406L/G410L) are consistent with destabilization of the homodimer. Login to comment 244 ABCG2 p.Thr402Leu (D) Close-up view of the T402L mutation. Login to comment 245 ABCG2 p.Thr402Arg (E) Close-up view of the T402R mutation. Login to comment 246 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Thr402Leu (F) Close-up view of the T402L, G406L, and G410L mutations. Login to comment 256 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Thr402Leu As noted above, the T402L/G406L/G410L mutant could be rescued by overnight treatment with the substrate MX as suggested by its shift to the normal 72 kDa molecular mass band on immunoblot and its loss of Endo H sensitivity. Login to comment 258 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu ABCG2 p.Thr402Arg In contrast, the T402R/G406L/G410L mutant, carrying the more drastic substitution at residue 402, did not show the same phenomenon (Figures 4-6). Login to comment