ABCG2 p.Arg482Ile
| 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 (482) variants of the ABCG2 mult... Biochim Biophys Acta. 2005 Feb 1;1668(1):53-63. Ozvegy-Laczka C, Koblos G, Sarkadi B, Varadi A
Single amino acid (482) variants of the ABCG2 multidrug transporter: major differences in transport capacity and substrate recognition.
Biochim Biophys Acta. 2005 Feb 1;1668(1):53-63., 2005-02-01 [PMID:15670731]
Abstract [show]
The human ABCG2 protein is an ATP binding cassette half-transporter, which protects our cells and tissues against various xenobiotics, while overexpression of ABCG2 in tumor cells confers multidrug resistance. It has been documented that single amino acid changes at position 482 resulted in altered drug resistance and transport capacity. In this study, we have generated nine Arg-482 mutants (G, I, M, S, T, D, N, K, Y) of ABCG2, and expressed them in insect cells. All ABCG2 variants showed cell surface expression and, in isolated membranes, an ABCG2-specific ATPase activity. When methotrexate accumulation was measured in inside-out membrane vesicles, this transport was supported only by the wild-type ABCG2. In intact cells, mitoxantrone was transported by all ABCG2 variants, except by R482K. Rhodamine 123 was extruded by most of the mutants, except by R482K, Y and by wild-type ABCG2. Hoechst 33342 was pumped out from cells expressing the wild-type and all Arg-482 variants, but not from those expressing R482K and Y. Our study demonstrates that the substrate specificity of the Arg (wild-type) form is unique and that amino acid replacements at position 482 induce major alterations in both the transport activity and substrate specificity of this protein.
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No. Sentence Comment
48 The two internal complementary primer pairs containing the specific mutation were: 5V-tta tta cca atg atc atg tta cc-3Vand 5-Vgg taa cat gat cat tgg taa taa-3V (R482I), 5V-tta tca gat cta tta ccc atg-3Vand 5V-gg taa cat cat cat ggg taa t-3V(R482M), 5V-ta ccc atg tcg atg tta cca a-3Vand 5V-t tgg taa cat cga cat ggg ta-3V(R482S), 5V-cc atg gac atg tta cca tcg att ata-3V and 5V-tat aat cga tgg taa cat gtc cat gg-3V (R482D), 5V-atg tta cca tcg att ata ttt acc-3Vand 5V-cc atg aat atg tta cca tcg att ata-3V (R482N), 5V-tta tta cct atg aag atg tta-3V cc and 5V-gg taa cat ctt cat agg taa taa-3V(R482K) and 5V-tta tta cct atg tac atg tta cc-3Vand 5V-gg taa cat gta cat agg taa taa-3V (R482Y).
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ABCG2 p.Arg482Ile 15670731:48:161
status: VERIFIED82 The expression levels of the R482I and the R482D variants were usually somewhat lower than those for the other mutants.
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ABCG2 p.Arg482Ile 15670731:82:29
status: VERIFIED117 When tested in the ATPase assay, prazosin was found to stimulate the activity of the R482I, M, S, D and N mutants by 1.3-to 1.6-fold.
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ABCG2 p.Arg482Ile 15670731:117:85
status: VERIFIED123 Panel B: Concentration dependence of MTX uptake by wtABCG2, R482I, R482K and K86M.
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ABCG2 p.Arg482Ile 15670731:123:60
status: VERIFIED124 Sf9 membrane vesicles (90 Ag) containing wtABCG2 (solid square), K86M (open square), R482I (diamond), and R482K (cross) were incubated in the presence or absence of 4 mM MgATP, with (up-triangle) or without 1 AM Ko143, with different MTX concentrations (10-3000 AM in a final volume of 150 Al) at 37 8C for 5 min.
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ABCG2 p.Arg482Ile 15670731:124:85
status: VERIFIED145 We found that none of the Arg-482 mutants had any measurable MTX uptake, as compared to the inactive ABCG2-K86M mutant (see Fig. 3B for R482I and for R482K; the data for the other variants are not shown).
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ABCG2 p.Arg482Ile 15670731:145:136
status: VERIFIED168 The R482I mutant was also active in this assay, but it had a decreased Hst-transport activity when compared to the wild-type protein, which may be due to a lower expression level of this mutant (not shown).
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ABCG2 p.Arg482Ile 15670731:168:4
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
114 O¨ zvegy-Laczka et al. (2005a) selected eight of these 15 R482 variants and the R482I mutant to further study substrate transport and ATPase activity in insect cells.
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ABCG2 p.Arg482Ile 16815914:114:85
status: VERIFIED[hide] Regulation of the function of the human ABCG2 mult... Drug Metab Dispos. 2014 Apr;42(4):575-85. doi: 10.1124/dmd.113.055731. Epub 2014 Jan 2. Telbisz A, Hegedus C, Varadi A, Sarkadi B, Ozvegy-Laczka C
Regulation of the function of the human ABCG2 multidrug transporter by cholesterol and bile acids: effects of mutations in potential substrate and steroid binding sites.
Drug Metab Dispos. 2014 Apr;42(4):575-85. doi: 10.1124/dmd.113.055731. Epub 2014 Jan 2., [PMID:24384916]
Abstract [show]
ABCG2 (ATP-binding cassette, subfamily G, member 2) is a plasma membrane glycoprotein that actively extrudes xenobiotics and endobiotics from the cells and causes multidrug resistance in cancer. In the liver, ABCG2 is expressed in the canalicular membrane of hepatocytes and excretes its substrates into the bile. ABCG2 is known to require high membrane cholesterol content for maximal activity, and by examining purified ABCG2 reconstituted in proteoliposomes we have recently shown that cholesterol is an essential activator, while bile acids significantly modify the activity of this protein. In the present work, by using isolated insect cell membrane preparations expressing human ABCG2 and its mutant variants, we have analyzed whether certain regions in this protein are involved in sterol recognition. We found that replacing ABCG2-R482 with large amino acids does not affect cholesterol dependence, but changes to small amino acids cause altered cholesterol sensitivity. When leucines in the potential steroid-binding element (SBE, aa 555-558) of ABCG2 were replaced by alanines, cholesterol dependence of ABCG2 activity was strongly reduced, although the L558A mutant variant when purified and reconstituted still required cholesterol for full activity. Regarding the effect of bile acids in isolated membranes, we found that these compounds decreased ABCG2-ATPase in the absence of drug substrates, which did not significantly affect substrate-stimulated ATPase activity. These ABCG2 mutant variants also altered bile acid sensitivity, although cholic acid and glycocholate were not transported by the protein. We suggest that the aforementioned two regions in ABCG2 are important for sterol sensing and may represent potential targets for pharmacologic modulation of ABCG2 function.
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No. Sentence Comment
96 In contrast, in the case of the R482I, K, M, and Y variants, similarly to the wild-type ABCG2, cholesterol enrichment significantly improved the ratio of substrate stimulation, as examined after the addition of prazosin (Fig. 1A).
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ABCG2 p.Arg482Ile 24384916:96:32
status: NEW109 When the cells were loaded with cholesterol, this Hst dye uptake was significantly improved in the wild-type and the R482I and M variants.
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ABCG2 p.Arg482Ile 24384916:109:117
status: NEW115 Cholesterol loading significantly increased R123 extrusion in cells expressing the R482I and M variants, while there was no measurable effect in the D, G, N, S, or T variants.
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ABCG2 p.Arg482Ile 24384916:115:83
status: NEW225 These were the R482G and R482S variants, which are fully active already at low membrane cholesterol levels, and the R482K and R482I mutants, which show similar cholesterol-sensing capability to the wtABCG2 (see earlier).
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ABCG2 p.Arg482Ile 24384916:225:126
status: NEW252 Members of the first cluster, also including the wild-type protein (R), contain large (hydrophobic or positively charged) amino acids, represented by R482I, M, K, and Y.
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ABCG2 p.Arg482Ile 24384916:252:150
status: NEW