ABCMdb

ABCG2 p.Thr402Leu

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PMID: 20088606 [PubMed] Polgar O et al: "Mutational analysis of threonine 402 adjacent to the GXXXG dimerization motif in transmembrane segment 1 of ABCG2."

No. Sentence Comment

4 Single mutations to leucine (T402L) or arginine (T402R) did not have a significant impact on the ABCG2 protein. Login to comment
5 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 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
45 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
92 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 The T402L mutation was designed to probe the perceived involvement of this residue in hydrogen bonding. Login to comment
101 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
106 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 The G406L/G410L and T402L/G406L/G410L mutants displayed substantially lower surface expression levels with the 5D3 antibody. Login to comment
114 The control and the T402L and T402Rmutants wereable toefflux MX,pheophorbide a, and BODIDY-prazosin from the cells. Login to comment
122 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
130 (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 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 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
142 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
144 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
147 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 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
151 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
155 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 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 (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
171 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
190 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 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 Cross-linking is observed in the control, T402L, T402R, and the triple mutants as suggested by higher-molecular mass species. Login to comment
204 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 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 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 The wild-type phenotype of the T402L mutant argues against H-bonding at this residue. Login to comment
228 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
233 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 (D) Close-up view of the T402L mutation. Login to comment
246 (F) Close-up view of the T402L, G406L, and G410L mutations. Login to comment
256 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

PMID: 20739628 [PubMed] Ni Z et al: "Transmembrane helices 1 and 6 of the human breast cancer resistance protein (BCRP/ABCG2): identification of polar residues important for drug transport."

No. Sentence Comment

287 Two mutants of Thr402 , T402L and T402R, were generated in that study. Login to comment

PMID: 21991363 [PubMed] Haider AJ et al: "Dimerization of ABCG2 analysed by bimolecular fluorescence complementation."

No. Sentence Comment

164 However, it remains possible that the effects of multiple mutations to a predicted interfacial site could be detected by BiFC, e.g. the surface of helix TM1 which contains a T402L/G406L/G410L dimerization motif [17]. Login to comment

PMID: 23307580 [PubMed] George RL et al: "Genetics of hyperuricemia and gout: implications for the present and future."

No. Sentence Comment

198 In the context of gout, the ABCG2 ligand mitoxantrone (an ABCG2 ligand) improved the altered protein structure of the T402L-G404L-G406L-G410L mutant in vitro [66]. Login to comment

PMID: 24021215 [PubMed] Stacy AE et al: "Molecular pharmacology of ABCG2 and its role in chemoresistance."

No. Sentence Comment

73 Mutational analysis of Thr402, which is located near the GXXXG motif (TXXGXXXG), in combination with mutations of the GXXXG motif (T402L or T402R and G406L or G410L; Fig. 3) resulted in a reduction in protein expression and drug efflux, alterations in glycosylation, and retention of ABCG2 in the ER (Polgar et al., 2010). Login to comment