ABCMdb

ABCG2 p.Gly410Leu

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Publications

PMID: 15260487 [PubMed] Polgar O et al: "Mutational analysis of ABCG2: role of the GXXXG motif."

No. Sentence Comment

4 The mutations resulted in lost transport for rhodamine 123 and impaired mitoxantrone, pheophorbide a, and BODIPY-prazosin transport, particularly in the double leucine mutant (G406L/G410L). Login to comment
5 Basal ATPase activity of the G406L/G410L mutant was comparable to the empty vector transfected cells with no substrate induction. Login to comment
55 The following ABCG2 mutants were generated: G406L, G410L, G406L/G410L, G406A, G410A, and G406A/ G410A. Login to comment
58 MXR406/410 5' and MXR406/ 410 3' contain the mutation sites of amino acids 406 and 410 (glycine to leucine or alanine at either or both sites). Login to comment
131 This was particularly true for the G406L/G410L double mutants. Login to comment
141 As indicated by almost no change in the level of fluorescence for mitoxantrone in the G406L/G410L mutant, this mutation impairs mitoxantrone transport more than the single mutations. Login to comment
143 As shown in Figure 3 the double leucine mutant does not transport this compound, while the G406L and G410L mutants show impaired transport of pheophorbide a. Login to comment
151 Using DSS, which cross-links proteins 11.4 Å apart, a dimer or higher order multimer was observed by immunoblot analysis in the G406L and G406L/G410L mutants and in the R482G control cells, as shown in Figure 4A. Next, cross-linking studies were performed with DSP, an agent that can be cleaved with the addition of DTT or -mercaptoethanol. Login to comment
155 (C) RNA levels of one representative clone of each of the leucine and alanine mutants on Northern blot with one of the highest levels in the G406L/G410L mutant. Login to comment
161 Transport capacity for BODIPY-prazosin, rhodamine 123, mitoxantrone, and pheohorbide A in the R482G, G406A/G410A, G406L/G410L, G406L, and G410L transfected cells. Plots: Accumulation without FTC (s) and with FTC (---). Login to comment
162 Note lost BODIPY-prazosin and pheophorbide a transport with greatly impaired mitoxantrone transport in the double leucine mutant (G406L/G410L) and almost completely lost rhodamine 123 transport in all leucine mutants. Login to comment
163 The single leucine mutants (G406L, G410L) show impaired transport for BODIPY-prazosin, mitoxantrone, and pheophorbide a when compared to the fully functional R482G control cell line. Login to comment
168 (C) Dimerization is suggested in both G406A and G410L mutants by high molecular weight bands detected following either exposure to DSP or harvesting under nonreducing conditions. Login to comment
174 Identical results were obtained with all the leucine and alanine mutants (Figure 4C showing results for the G406A and G410L mutants and Figure 4D for G406L and G410A; similar data not shown for the G406L/G410L and G406A/G410A mutants), implying that even if dimerization is impaired in these mutants, it has not prevented their close association on the cell surface. Login to comment
179 We determined the vanadate-sensitive component of the ATPase activity of the G406L/G410L mutant in the presence of 0, 1, 10, and 100 µM prazosin and compared it to the R482G and empty pcDNA3.1 vector transfected cells. Login to comment
181 Significantly, the G406L/G410L mutant, which previously showed the greatest impairment in transport capacity by flow cytometry (Figure 3), revealed ATPase levels identical to the empty vector transfected cell line with no stimulation with prazosin. Login to comment
186 The increased ABCG2 expression after overnight treatment with mitoxantrone in the G406L and G406L/G410L mutants was also confirmed with confocal microscopy (Figure 6B). Login to comment
192 While the double leucine mutant G406L/G410L was found on the cell surface, protein levels were reduced on immunoblot, and transport function and ATP hydrolysis were markedly impaired. Login to comment
201 The vanadate-sensitive ATP hydrolysis in the presence of the indicated concentrations of prazosin for crude membranes of HEK293 cells expressing ABCG2 R482G (0), G406L/G410L mutants (]), and empty vector transfected cells (pcDNA; O) is shown. Login to comment
202 The G406L/G410L mutant demonstrates basal ATPase levels identical to those of the pcDNA with no substrate induction observed. Login to comment
232 (B) Confocal microscopy of HEK293 cells transfected with ABCG2 mutants shows a significant increase in ABCG2 levels for the G406L and G406L/G410L mutants after overnight treatment with 5 µM mitoxantrone. Login to comment
242 Remarkably, both the basal and prazosin-stimulated ATPase activity was found to be severely impaired in the G406L/G410L mutant. Login to comment
247 Since both the basal and the prazosin-stimulated ATP hydrolyses were impaired in the G406L/G410L mutant, it is conceivable that the GXXXG motif could play a similar role in ABCG2. Login to comment

PMID: 15743976 [PubMed] Vethanayagam RR et al: "Functional analysis of the human variants of breast cancer resistance protein: I206L, N590Y, and D620N."

No. Sentence Comment

268 The ATPase activities of the G406L/G410L mutant, which is predicted in the first TM segment of BCRP, were found to be completely abolished (Polgar et al., 2004). Login to comment
269 These data suggest that, whereas ATPase activities of BCRP could be affected by mutations in the NBD (e.g., Q141K and I206L), mutations in other regions including the TM segments (e.g., G406L/G410L) and extracellular loops (e.g., N590Y and D620N) can also influence ATP hydrolysis. Login to comment

PMID: 16146333 [PubMed] Mao Q et al: "Role of the breast cancer resistance protein (ABCG2) in drug transport."

No. Sentence Comment

181 The mutants G406L, G410L, and G406L/G410L, particularly the double mutant, lost transport activity for rhodamine 123 and displayed reduced transport for mitoxantrone, pheophorbide a, and BODIPY-prazosine. Login to comment

PMID: 17027309 [PubMed] Li YF et al: "Towards understanding the mechanism of action of the multidrug resistance-linked half-ABC transporter ABCG2: a molecular modeling study."

No. Sentence Comment

175 It is a splicing variant with a somewhat lower expression and lower resistance [23] A347Ta Linker Low drug resistance [42] G406L, G410L, G406L/G410L GXXXG motif TM1 Dimerization in related proteins. Login to comment

PMID: 18249138 [PubMed] Hazai E et al: "Homology modeling of breast cancer resistance protein (ABCG2)."

No. Sentence Comment

227 406-410) sequence motif in ABCG2 was suggested to form a symmetrical interaction in the TM dimer (Polgar et al., 2004) based on site-directed mutagenesis data (Table 3 and Fig. 6), G406L and G410L substitutions resulted in impaired ATP hydrolysis and substrate transport. Login to comment

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

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
7 The T402R/G406L/G410L mutant carrying the more drastic substitution was found to primarily localize intracellularly. 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
47 The R482G control and G406L/G410L mutant were previously generated and characterized (19, 23). 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
95 We used the previously generated G406L/G410L and R482G transfectants in HEK 293 cells as controls (19). 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
103 In the case of T402R/G406L/G410L, the lower band comprised the majority of the detected protein. 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
108 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
117 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
125 Minimal change was observed in the molecular mass of ABCG2 in the G406L/G410L mutant after Endo H digestion. 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
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
141 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
143 As previously shown, a significant increase in protein levels was observed for the G406L/ G410L mutant (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
145 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
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
149 In contrast, after treatment with MX, the T402R/G406L/G410L mutant still primarily displayed intracellular localization. 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
152 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 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
170 Figure 7 demonstrates increased molecular mass bands consistent with cross-linking of the control, T402R, and T402R/ G406L/G410L proteins. 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
180 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
226 (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 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
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
258 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

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

PMID: 25036722 [PubMed] Szafraniec MJ et al: "Determinants of the activity and substrate recognition of breast cancer resistance protein (ABCG2)."

No. Sentence Comment

209 Position Type of mutation Effect on the transporter References NBD Lys 86 Met (i) No stimulation of the ATPase activity by prazosin; (ii) no influence on the transport of mitoxantrone Henriksen et al. (2005b) Glu 126 stop, Phe 208 Ser, Ser 248 Phe, Glu 334 stop Inability to transport hematoporphyrin Tamura et al. (2006) Glu 211 Gln Complete abolishment of the ATPase activity and methotrexate transport Hou et al. (2009) Pro 392 Ala Significant reduction in the efflux activity of mitoxantrone, BODIPY-prazosin and Hoechst 33342 Ni et al. (2011) TM1 Gly 406 Ala Gly 410 Ala No influence on the activity of the transporter Polgar et al. (2004) Gly 406 Leu Gly 410 Leu (i) Loss of the ability to transport rhodamine123; (ii) impaired transport of mitoxantrone, Pheide and BODIPY-prazosin Polgar et al. (2004) Extracellular loop 1 Phe 431 Leu (i) Loss of the ability to transport methotrexate; (ii) 10% level of hematoporphyrin transport compared to the WT protein Tamura et al. (2006) Ser 441 Asn Inability to transport hematoporphyrin Tamura et al. (2006) Ser 441 Asn Loss of the ability to transport methotrexate Tamura et al. (2006) TM2 Lys 452 Ala His 457 Ala Increase in transport of mitoxantrone, BODIPY-prazosin and Hoechst 33342 Cai et al. (2010) Lys 453 Ala Arg 465 Ala Decrease in transport of mitoxantrone, BODIPY-prazosin, Hoechst 33342, doxorubicin, SN-38 and rhodamine 123 Cai et al. (2010) TM3 Arg 482 Gly Arg 482 Thr (i) No change in the inhibitory activity of lapatinib; (ii) about two times greater inhibition by ritonavir, saquinavir and nalfinavir than in the WT variant; (iii) gaining the ability to transport rhodamine123 and doxorubicin; (iv) no influence on the transport of mitoxantrone; (v) loss of the ability to transport methotrexate Dai et al. (2008), Gupta et al. (2004), Honjo et al. (2001), Mitomo et al. (2003) Arg 482 Thr (i) Lower IC 50 of cyclosporine A for mutant than for WT variant; (ii) lower elacridar inhibition potency Xia et al. (2007) Arg 482 Lys Complete loss of transport activity Ejendal et al. (2006) Phe 489 Leu Impaired transport of porphyrins, no transport of methotrexate Tamura et al. (2006) Extracellular loop 3 Asn 590 Tyr Over twice reduced transport of mitoxantrone, topotecan, daunorubicin and rhodamine 123 Vethanayagam et al. (2005) Cys 592 Ala/Cys 608 Ala (i) Transport of mitoxantrone almost unchanged; (ii) transport of BODIPY-prazosin significantly impaired Henriksen et al. (2005a) Extracellular loop 3 Cys 603 Ser Cys 592 Ser/Cys 608 Ser Cys 592 Ser/Cys 603 Ser/Cys 608 Ser Diminished susceptibility to the inhibitory activity of fumitremorgin C Shigeta et al. (2010) Cys-less Arg 482 Gly-BCRP Complete loss of the ability to efflux mitoxantrone Liu et al. (2008b) The positions of the amino acid residues refer to the topological model of BCRP proposed by Wang et al. (2009). Login to comment
221 The substitution of a single or both Gly residues by Leu in this motif resulted in a loss of transport for rhodamine 123, and impaired transport of mitoxantrone, Pheide and BODIPY-prazosin, in particular, in the double mutant (Gly406 Leu/ Gly410 Leu). Login to comment