ABCMdb

ABCG2 p.Thr402Arg

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,

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[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.

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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
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
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
94 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
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
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
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
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
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
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
149 In contrast, after treatment with MX, the T402R/G406L/G410L mutant still primarily displayed intracellular localization. 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
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
170 Figure 7 demonstrates increased molecular mass bands consistent with cross-linking of the control, T402R, and T402R/ G406L/G410L proteins. Login to comment
178 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 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 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
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
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
229 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 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
245 (E) Close-up view of the T402R mutation. 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

[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.

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No. Sentence Comment
11 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. Login to comment
82 Generation of Flp-In-293 cells stably expressing wild-type BCRP and the mutants T402A and T402R. Login to comment
85 The resulting pcDNA5/FRT/BCRP plasmid containing full-length human BCRP cDNA was used as a template to generate T402A and T402R, as described above. Login to comment
86 The forward primer used to generate T402R was 5=-GCT CAG ATC ATT GTC AGA GTC GTA CTG GGA CTG-3=. Login to comment
88 One microgram of the pcDNA5/ FRT plasmid containing wild-type BCRP, T402A, or T402R cDNA or the empty vector were used to transfect Flp-In-293 cells, in combination with 2 ␮g of pOG44 using the FuGENE HD transfection reagent, according to the manufacturer`s instruction. Login to comment
90 The cells expressing wild-type BCRP, T402A, or T402R were then maintained in complete MEM medium containing 10% FBS, L-glutamine, and 125 ␮g/ml of hygromycin for subsequent experiments. Login to comment
255 Efflux activities of the mutants T402A and T402R expressed in Flp-In-293 cells. Login to comment
257 To investigate whether the decreased efflux activity of T402A was caused by altered pattern of oligomerization resulting from the higher level of protein expression that could subsequently affect BCRP activity, we generated Flp-In-293 cells stably expressing wild-type BCRP, T402A, or T402R. Login to comment
258 T402R was also analyzed, because a recent study revealed that this mutant could possibly affect BCRP activity (19). Login to comment
261 Indeed, as shown in Fig. 6A, wild-type BCRP, T402A, and T402R were expressed at comparable protein levels. Login to comment
262 Immunofluorescent confocal microscopy analysis indicated that T402A and T402R were predominantly localized on the plasma membrane of Flp-In-293 cells as was wild-type BCRP (Fig. 6B). Login to comment
264 Likewise, T402R also exhibited significantly lower activities compared with wild-type BCRP. Login to comment
265 Interestingly, efflux activities of T402R were further decreased compared with T402A, suggesting that Arg substitution of Thr402 caused a greater effect on BCRP function than Ala substitution, possibly due to more drastic changes in both size and polarity at position 402. Login to comment
287 Two mutants of Thr402 , T402L and T402R, were generated in that study. Login to comment
289 For example, a particularly strong reduction in efflux of BODIPY-prazosin was associated with T402R. Login to comment
290 We verified that T402A and T402R, which were expressed in Flp-In-293 cells at comparable protein levels to wild-type BCRP, also exhibited significantly decreased efflux activities (Fig. 6). Login to comment
300 Expression and efflux activities of T402A and T402R in Flp-In-293 cells. Login to comment
301 A: representative immunoblot of whole cell lysates for wild-type BCRP and its mutants T402A and T402R expressed in Flp-In-293 cells. Login to comment
303 B: confocal microscopy analysis of Flp-In-293 cells expressing wild-type BCRP and its mutants T402A and T402R. Login to comment
305 C: fumitremorgin C (FTC)-inhibitable efflux activities of Flp-In-293 cells expressing wild-type BCRP and its mutants T402A and T402R for mitoxantrone, BODIPY-prazosin, and Hoechst 33342. 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.

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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

[hide] Role of the breast cancer resistance protein (BCRP... AAPS J. 2015 Jan;17(1):65-82. doi: 10.1208/s12248-014-9668-6. Epub 2014 Sep 19. Mao Q, Unadkat JD
Role of the breast cancer resistance protein (BCRP/ABCG2) in drug transport--an update.
AAPS J. 2015 Jan;17(1):65-82. doi: 10.1208/s12248-014-9668-6. Epub 2014 Sep 19., [PMID:25236865]

Abstract [show]
The human breast cancer resistance protein (BCRP, gene symbol ABCG2) is an ATP-binding cassette (ABC) efflux transporter. It was so named because it was initially cloned from a multidrug-resistant breast cancer cell line where it was found to confer resistance to chemotherapeutic agents such as mitoxantrone and topotecan. Since its discovery in 1998, the substrates of BCRP have been rapidly expanding to include not only therapeutic agents but also physiological substances such as estrone-3-sulfate, 17beta-estradiol 17-(beta-D-glucuronide) and uric acid. Likewise, at least hundreds of BCRP inhibitors have been identified. Among normal human tissues, BCRP is highly expressed on the apical membranes of the placental syncytiotrophoblasts, the intestinal epithelium, the liver hepatocytes, the endothelial cells of brain microvessels, and the renal proximal tubular cells, contributing to the absorption, distribution, and elimination of drugs and endogenous compounds as well as tissue protection against xenobiotic exposure. As a result, BCRP has now been recognized by the FDA to be one of the key drug transporters involved in clinically relevant drug disposition. We published a highly-accessed review article on BCRP in 2005, and much progress has been made since then. In this review, we provide an update of current knowledge on basic biochemistry and pharmacological functions of BCRP as well as its relevance to drug resistance and drug disposition.

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No. Sentence Comment
183 Ala or Arg substitution of Thr402 caused a significant reduction by 50-90% in efflux of mitoxantrone, BODIPY-prazosin, and Hoechst33342 as well as its ability to confer resistance to mitoxantrone and SN-38 (92). Login to comment