ABCG2 p.Arg482His
| Predicted by SNAP2: | A: D (91%), C: D (85%), D: D (95%), E: D (95%), F: D (91%), G: D (95%), H: D (95%), I: D (85%), K: D (85%), L: D (91%), M: D (85%), N: D (95%), P: D (95%), Q: D (95%), S: D (91%), T: D (91%), V: D (91%), W: D (95%), Y: D (95%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, S: D, T: D, V: D, W: D, Y: D, |
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[hide] Single amino acid substitutions in the transmembra... Int J Cancer. 2003 Dec 10;107(5):757-63. Miwa M, Tsukahara S, Ishikawa E, Asada S, Imai Y, Sugimoto Y
Single amino acid substitutions in the transmembrane domains of breast cancer resistance protein (BCRP) alter cross resistance patterns in transfectants.
Int J Cancer. 2003 Dec 10;107(5):757-63., 2003-12-10 [PMID:14566825]
Abstract [show]
Breast cancer resistance protein (BCRP) is a member of ATP-binding cassette transporters that has an N-terminal ATP binding domain and a C-terminal transmembrane domain (TM). Expression of wild-type BCRP confers resistance to multiple chemotherapeutic agents such as mitoxantrone, SN-38 and topotecan, but not to doxorubicin. We made 32 BCRP mutants with an amino acid substitution in the TMs (7 E446-mutants in TM2, 15 R482-mutants in TM3, 4 N557-mutants in TM5 and 6 H630-mutants in TM6) and examined the effect of the substitutions on cellular drug resistance. PA317 cells transfected with any one of the 7 E446-mutant BCRP cDNAs did not show drug resistance. Cells transfected with any one of the 13 R482X2-BCRP cDNAs (X2 = N, C, M, S, T, V, A, G, E, W, D, Q and H, but not Y and K) showed higher resistance to mitoxantrone and doxorubicin than the wild-type BCRP-transfected cells. Cells transfected with N557D-BCRP cDNA showed similar resistance to mitoxantrone but lower resistance to SN-38 than the wild-type BCRP-transfected cells. Cells transfected with N557E-, H630E- or H630L-BCRP cDNA showed similar degrees of resistance to mitoxantrone and SN-38. Estrone and fumitremorgin C reversed the drug resistance of cells transfected with R482-, N557- or H630-mutant BCRP cDNA. Cells transfected with R482G- or R482S-BCRP cDNA showed less intracellular accumulation of [3H]mitoxantrone than the wild-type BCRP-transfected cells. These results suggest that E446 in TM2, R482 in TM3, N557 in TM5 and H630 in TM6 play important roles in drug recognition of BCRP.
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No. Sentence Comment
66 PA/R482Q and PA/R482H (Group 3) showed similar degrees of resistance to mitoxantrone and SN-38.
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ABCG2 p.Arg482His 14566825:66:16
status: VERIFIED165 Group 3 members (PA/R482Q and PA/R482H) showed similar degrees of resistance to mitoxantrone and SN-38.
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ABCG2 p.Arg482His 14566825:165:33
status: VERIFIED[hide] The nature of amino acid 482 of human ABCG2 affect... Protein Sci. 2006 Jul;15(7):1597-607. Ejendal KF, Diop NK, Schweiger LC, Hrycyna CA
The nature of amino acid 482 of human ABCG2 affects substrate transport and ATP hydrolysis but not substrate binding.
Protein Sci. 2006 Jul;15(7):1597-607., [PMID:16815914]
Abstract [show]
Several members of the ATP-binding cassette (ABC) transporter superfamily, including P-glycoprotein and the half-transporter ABCG2, can confer multidrug resistance to cancer cells in culture by functioning as ATP-dependent efflux pumps. ABCG2 variants harboring a mutation at arginine 482 have been cloned from several drug-resistant cell lines, and these variants differ in their substrate transport phenotype. In this study, we changed the wild-type arginine 482 in human ABCG2 to each one of the 19 other standard amino acids and expressed each one transiently in HeLa cells. Using the 5D3 antibody that recognizes a cell surface epitope of ABCG2, we observed that all the mutants were expressed at the cell surface. However, the mutant ABCG2 proteins differed markedly in transport activity. All of the variants were capable of transporting one or more of the substrates used in this study, with the exception of the R482K mutant, which is completely devoid of transport ability. Six of the mutants (R482G, R482H, R482K, R482P, R482T, and R482Y) and the wild-type protein (R482wt) were selected for studies of basal and stimulated ATPase activity and photoaffinity labeling with the substrate analog [125I]iodoarylazidoprazosin. Whereas these seven ABCG2 variants differed markedly in ATPase activity, all were able to specifically bind the substrate analog [125I]iodoarylazidoprazosin. These data suggest that residue 482 plays an important role in substrate transport and ATP turnover, but that the nature of this amino acid may not be important for substrate recognition and binding.
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No. Sentence Comment
7 Six of the mutants (R482G, R482H, R482K, R482P, R482T, and R482Y) and the wild-type protein (R482wt) were selected for studies of basal and stimulated ATPase activity and photoaffinity labeling with the substrate analog [125 I]iodoarylazidoprazosin.
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ABCG2 p.Arg482His 16815914:7:27
status: VERIFIED65 Like R482wt, the R482K mutant was also incapable of rhodamine 123 transport, and R482H, which also contains a basic side chain at position 482, was also impaired in rhodamine 123 transport.
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ABCG2 p.Arg482His 16815914:65:81
status: VERIFIED69 Transport of the fluorescent compound Bodipy FL prazosin followed a similar pattern to that observed for rhodamine 123, where the variants R482wt, R482K, R482H, and R482Y show the least transport (Fig. 3).
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ABCG2 p.Arg482His 16815914:69:154
status: VERIFIED71 Analysis of the substrate binding properties of wild-type and six mutant ABCG2 proteins In order to further investigate the effects of the R482X mutations, we studied the drug-binding ability of a selection of ABCG2 mutants (R482G, R482H, R482K, R482P, R482T, and R482Y) and the wild-type ABCG2 (R482wt).
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ABCG2 p.Arg482His 16815914:71:232
status: VERIFIED73 We selected mutants R482H, R482P, and R482Y because they are partially deficient in rhodamine 123 transport, whereas mitoxantrone transport is intact.
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ABCG2 p.Arg482His 16815914:73:20
status: VERIFIED75 Of these six ABCG2 mutants plus the wild-type protein, biochemical analyses of R482H and R482P have not been described previously.
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ABCG2 p.Arg482His 16815914:75:79
status: VERIFIED86 We analyzed expression of ABCG2 in the membranes using the monoclonal antibody BXP-21 (Fig. 5A), which shows that the R482G, R482wt, and R482T membranes used here express less ABCG2, compared with the membranes expressing the R482H, R482K, R482P, and R482Y variants.
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ABCG2 p.Arg482His 16815914:86:226
status: VERIFIED90 In contrast, the R482H, R482K, R482Y, and R482wt variants are not markedly affected by the addition of 20 mM prazosin.
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ABCG2 p.Arg482His 16815914:90:17
status: VERIFIED96 Specific [125 I]IAAP photoaffinity labeling of crude membranes derived from HeLa cells expressing wild-type ABCG2 (R482wt) and the ABCG2 variants R482G, R482H, R482K, R482P, R482T, and R482Y.
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ABCG2 p.Arg482His 16815914:96:153
status: VERIFIED106 Basal and drug-stimulated ATPase activity of wild-type ABCG2 (R482wt) and ABCG2 variants R482G, R482H, R482K, R482P, R482T, and R482Y.
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ABCG2 p.Arg482His 16815914:106:96
status: VERIFIED121 Our data demonstrate that the R482H mutant is able to efflux mitoxantrone to the same extent as the wild-type protein (data not shown), although it is somewhat deficient in rhodamine 123 and Bodipy FL prazosin transport.
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ABCG2 p.Arg482His 16815914:121:30
status: VERIFIED140 This hypothesis may also explain the differences in the ATPase activity seen for R482H and R482K mutants and the wild-type R482 protein.
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ABCG2 p.Arg482His 16815914:140:81
status: VERIFIED141 Moreover, several variants (R482H, R482K, R482wt, and R482Y) showed no prazosin-stimulated ATPase activity.
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ABCG2 p.Arg482His 16815914:141:28
status: VERIFIED