ABCMdb

ABCG2 p.Thr402Leu

Predicted by SNAP2:	A: D (53%), C: D (71%), D: D (53%), E: D (66%), F: D (63%), G: D (63%), H: D (53%), I: D (59%), K: D (75%), L: D (71%), M: N (53%), N: N (66%), P: D (59%), Q: D (63%), R: D (75%), S: N (66%), V: D (59%), W: D (85%), Y: D (63%),
Predicted by PROVEAN:	A: N, C: D, D: D, E: D, F: D, G: D, H: D, I: N, K: D, L: N, M: N, N: N, P: D, Q: D, R: D, S: N, V: N, W: D, Y: D,

[switch to compact view]
Comments [show]

None has been submitted yet.

Publications
[hide] Mutational analysis of threonine 402 adjacent to t... Biochemistry. 2010 Mar 16;49(10):2235-45. 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 [PMID:20088606]

Abstract [show]
ABCG2 is an ATP-binding cassette half-transporter important in normal tissue protection, drug distribution, and excretion. ABCG2 requires homodimerization for function, though the mechanism for dimerization has not been elucidated. We conducted mutational analysis of threonine 402, three residues from the GXXXG motif in TM1, to study its potential role in ABCG2 dimerization (TXXXGXXXG). Single mutations to leucine (T402L) or arginine (T402R) did not have a significant impact on the ABCG2 protein. 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. 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. The T402R/G406L/G410L mutant carrying the more drastic substitution was found to primarily localize intracellularly. The same set of mutations also displayed impaired dimerization in the TOXCAT assay for TM1 compared to that of the wild type. Homology modeling of ABCG2 places the TXXXGXXXG motif at the dimer interface. These studies are consistent with a role for the extended TXXXGXXXG motif in ABCG2 folding, processing, and/or dimerization.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] Transmembrane helices 1 and 6 of the human breast ... Am J Physiol Cell Physiol. 2010 Nov;299(5):C1100-9. Epub 2010 Aug 25. Ni Z, Bikadi Z, Cai X, Rosenberg MF, Mao Q
Transmembrane helices 1 and 6 of the human breast cancer resistance protein (BCRP/ABCG2): identification of polar residues important for drug transport.
Am J Physiol Cell Physiol. 2010 Nov;299(5):C1100-9. Epub 2010 Aug 25., [PMID:20739628]

Abstract [show]
The human breast cancer resistance protein (BCRP/ABCG2) mediates efflux of drugs and xenobiotics. In this study, we investigated the role of polar residues within or near the predicted transmembrane alpha-helices 1 and 6 of BCRP in drug transport. We substituted Asn(387), Gln(398), Asn(629), and Thr(642) with Ala, Thr(402) with Ala and Arg, and Tyr(645) with Phe, and the mutants were stably expressed in human embryonic kidney-293 or Flp-In-293 cells. Immunoblotting and confocal microscopy analysis revealed that all of the mutants were well expressed and predominantly targeted to the plasma membrane. While T402A and T402R showed a significant global reduction in the efflux of mitoxantrone, Hoechst 33342, and BODIPY-prazosin, N629A exhibited significantly increased efflux activities for all of the substrates. N387A and Q398A displayed significantly impaired efflux for mitoxantrone and Hoechst 33342, but not for BODIPY-prazosin. In contrast, T642A and Y645F showed a moderate reduction in Hoechst 33342 efflux only. Drug resistance profiles of human embryonic kidney-293 cells expressing the mutants generally correlated with the efflux data. Furthermore, N629A was associated with a marked increase, and N387A and T402A with a significant reduction, in BCRP ATPase activity. Mutations of some of the polar residues may cause conformational changes, as manifested by the altered binding of the 5D3 antibody to BCRP in the presence of prazosin. The inward-facing homology model of BCRP indicated that Thr(402) within transmembrane 1 may be important for helical interactions, and Asn(629) may be involved in BCRP-substrate interaction. In conclusion, we have demonstrated the functional importance of some of these polar residues in BCRP activity.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
287 Two mutants of Thr402 , T402L and T402R, were generated in that study. Login to comment

[hide] Dimerization of ABCG2 analysed by bimolecular fluo... PLoS One. 2011;6(10):e25818. Epub 2011 Oct 3. Haider AJ, Briggs D, Self TJ, Chilvers HL, Holliday ND, Kerr ID
Dimerization of ABCG2 analysed by bimolecular fluorescence complementation.
PLoS One. 2011;6(10):e25818. Epub 2011 Oct 3., [PMID:21991363]

Abstract [show]
ABCG2 is one of three human ATP binding cassette transporters that are functionally capable of exporting a diverse range of substrates from cells. The physiological consequence of ABCG2 multidrug transport activity in leukaemia, and some solid tumours is the acquisition of cancer multidrug resistance. ABCG2 has a primary structure that infers that a minimal functional transporting unit would be a homodimer. Here we investigated the ability of a bimolecular fluorescence complementation approach to examine ABCG2 dimers, and to probe the role of individual amino acid substitutions in dimer formation. ABCG2 was tagged with fragments of venus fluorescent protein (vYFP), and this tagging did not perturb trafficking or function. Co-expression of two proteins bearing N-terminal and C-terminal fragments of YFP resulted in their association and detection of dimerization by fluorescence microscopy and flow cytometry. Point mutations in ABCG2 which may affect dimer formation were examined for alterations in the magnitude of fluorescence complementation signal. Bimolecular fluorescence complementation (BiFC) demonstrated specific ABCG2 dimer formation, but no changes in dimer formation, resulting from single amino acid substitutions, were detected by BiFC analysis.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] Genetics of hyperuricemia and gout: implications f... Curr Rheumatol Rep. 2013 Feb;15(2):309. doi: 10.1007/s11926-012-0309-8. George RL, Keenan RT
Genetics of hyperuricemia and gout: implications for the present and future.
Curr Rheumatol Rep. 2013 Feb;15(2):309. doi: 10.1007/s11926-012-0309-8., [PMID:23307580]

Abstract [show]
Gout is the most common inflammatory arthropathy and occurs in the setting of elevated serum urate levels. Gout is also known to be associated with multiple comorbidities including cardiovascular disease and the metabolic syndrome. Recent advances in research have increased our understanding and improved our knowledge of the pathophysiology of gout. Genome-wide association studies have permitted the identification of several new and common genetic factors that contribute to hyperuricemia and gout. Most of these are involved with the renal urate transport system (the uric acid transportasome), generally considered the most influential regulator of serum urate homeostasis. Thus far, SCL22A12, SCL2A9, and GLUT9 have been found to have the greatest variation and most influence on serum urate levels. However, genetics are only a part of the explanation in the development of hyperuricemia and gout. As results have been mixed, the role of known urate influential genes in gout's associated comorbidities remains unclear. Regardless, GWAS findings have expanded our understanding of the pathophysiology of hyperuricemia and gout, and will likely play a role in the development of future therapies and treatment of this ancient disease.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] Molecular pharmacology of ABCG2 and its role in ch... Mol Pharmacol. 2013 Nov;84(5):655-69. doi: 10.1124/mol.113.088609. Epub 2013 Sep 10. Stacy AE, Jansson PJ, Richardson DR
Molecular pharmacology of ABCG2 and its role in chemoresistance.
Mol Pharmacol. 2013 Nov;84(5):655-69. doi: 10.1124/mol.113.088609. Epub 2013 Sep 10., [PMID:24021215]

Abstract [show]
The ATP-binding cassette, subfamily G, isoform 2 protein (ABCG2) is an important member of the ABC transporter superfamily, which has been suggested to be involved in multidrug resistance (MDR) in cancer. Its diverse range of substrates includes many common chemotherapeutics such as imatinib, doxorubicin, and mitoxantrone. Physiologically, ABCG2 is highly expressed in areas such as the blood-brain barrier and gastrointestinal tract, where it is thought to play a role in protection against xenobiotic exposure. High ABCG2 expression has also been found in a variety of solid tumors and in hematologic malignancies and has been correlated with poorer clinical outcomes. Furthermore, ABCG2 expression is a characteristic feature of cancer stem cells, which are able to self-renew and differentiate. These cancer stem cells have been postulated to play an important role in MDR, where their inherent ABCG2 expression may allow them to survive chemotherapy and repopulate the tumor after exposure to chemotherapeutics. This observation raises the exciting possibility that by inhibiting ABCG2, cancer stem cells and other cancers may be targeted and eradicated, at which point conventional chemotherapeutics would be sufficient to eliminate the remaining tumor cells. Inhibitors of ABCG2, such as tyrosine kinase inhibitors, phosphodiesterase-5 inhibitors, and the fumitremorgin-type indolyl diketopiperazine, Ko143 [(3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxo pyrazino[1',2':1,6]pyrido[3,4-b]indole-3-propanoic acid 1,1-dimethylethyl ester], could potentially be used for this purpose. However, these agents are still awaiting comprehensive clinical assessment.

Comments [show]

None has been submitted yet.

Sentences [show]
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