ABCG2 p.Arg482Lys
| 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
42 Because FACS analysis showed that certain populations of the methotrexate-resistant PA/R482A and PA/R482W cells did not express BCRP (data not shown), all the R482-mutant BCRP transfectants (except R482K-BCRP transfectant) were further selected with 1 ng/ml of mitoxantrone for 5 days to eliminate untransfected cells.
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ABCG2 p.Arg482Lys 14566825:42:198
status: VERIFIED43 These mitoxantrone-selected cells (or R482K-BCRP transfectant) were termed PA/R482X2 (X2 ϭ Y, N, C, M, S, T, V, A, G, E, W, D, Q, H, or K).
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ABCG2 p.Arg482Lys 14566825:43:38
status: VERIFIED48 Among PA/E446X1 transfectants, PA/E446D expressed a small amount of BCRP. Among PA/R482X2, PA/R482D and PA/R482K expressed lesser amounts of BCRP than the other transfectants.
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ABCG2 p.Arg482Lys 14566825:48:107
status: VERIFIED61 Most of the PA/ R482X2 cells (except for PA/R482D and PA/R482K) showed similar or somewhat lower levels of SN-38 resistance as compared to PA/WT2.
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ABCG2 p.Arg482Lys 14566825:61:57
status: VERIFIED67 PA/R482K (Group 4) did not show drug resistance.
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ABCG2 p.Arg482Lys 14566825:67:3
status: VERIFIED148 As described above, human drug-resistant cell lines MCF-7 AdVp3000, S1-M1-80, MT-4/DOX500 and a mouse drug-resistant line 88.6/D800-B overexpressed R482T- (ACG), R482G- (GGG), R482M- (ATG) and R482S- (AGT) BCRP, respectively.5,6,13,14,23 The other possible mutations are R482W (TGG) and R482K (AAG).
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ABCG2 p.Arg482Lys 14566825:148:287
status: VERIFIED150 PA/R482K did not show drug resistance.
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ABCG2 p.Arg482Lys 14566825:150:3
status: VERIFIED151 The low-level drug resistance of the transfectants would explain why R482W- and R482K-BCRP were not expressed in drug-selected cell lines.
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ABCG2 p.Arg482Lys 14566825:151:80
status: VERIFIED167 PA/R482K (Group 4) did not show drug resistance.
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ABCG2 p.Arg482Lys 14566825:167:3
status: VERIFIED[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
5 In intact cells, mitoxantrone was transported by all ABCG2 variants, except by R482K.
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ABCG2 p.Arg482Lys 15670731:5:79
status: VERIFIED6 Rhodamine 123 was extruded by most of the mutants, except by R482K, Y and by wild-type ABCG2.
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ABCG2 p.Arg482Lys 15670731:6:61
status: VERIFIED7 Hoechst 33342 was pumped out from cells expressing the wild-type and all Arg-482 variants, but not from those expressing R482K and Y.
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ABCG2 p.Arg482Lys 15670731:7:121
status: VERIFIED48 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.Arg482Lys 15670731:48:594
status: VERIFIED118 In contrast, the basal ATPase activity of R482K and Y mutants was rather inhibited than stimulated by prazosin Fig. 3.
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ABCG2 p.Arg482Lys 15670731:118:42
status: VERIFIED123 Panel B: Concentration dependence of MTX uptake by wtABCG2, R482I, R482K and K86M.
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ABCG2 p.Arg482Lys 15670731:123:67
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.Arg482Lys 15670731:124:106
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.Arg482Lys 15670731:145:150
status: VERIFIED157 These data indicate that most of the ABCG2-R482 mutant variants can actively extrude mitoxantrone, while the R482K mutant is inactive in this regard.
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ABCG2 p.Arg482Lys 15670731:157:109
status: VERIFIED159 We found that while the R482K, R482Y, the wtABCG2, and the inactive K86M mutant had no R123 extrusion activity, several ABCG2 variants were highly active in R123 extrusion.
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ABCG2 p.Arg482Lys 15670731:159:24
status: VERIFIED169 On the other hand, mutants R482K and Y showed no Hst-transport capacity, although their expression levels were similar to that of the wild-type ABCG2 (not shown).
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ABCG2 p.Arg482Lys 15670731:169:27
status: VERIFIED195 On the other hand, mutants R482K and Y showed inhibition in the presence of prazosin, as well as in the presence of all other potential substrates tested (e.g., mitoxantrone, rhodamine 123; not shown here).
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ABCG2 p.Arg482Lys 15670731:195:27
status: VERIFIED198 While the wtABCG2, the R482K and R482Y mutants are already fully activated, and prazosin either does not affect or reduces the ATPase activity, the other variants can be further stimulated by exogenously added substrates.
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ABCG2 p.Arg482Lys 15670731:198:23
status: VERIFIED209 The exceptions were the R482Y mutant, effective only in mitoxantrone transport, and the R482K mutant, showing no transport activity with any of these substrates.
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ABCG2 p.Arg482Lys 15670731:209:88
status: VERIFIED216 Actually, the R482K mutant showed no measurable transport activity in any of the assays applied here, while the R482Y was found to be active only in the whole-cell mitoxantrone extrusion assay (see Fig. 4A).
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ABCG2 p.Arg482Lys 15670731:216:14
status: VERIFIED227 Only low level of expression of the R482K mutant was achieved in murine cells, nevertheless, the cells were sensitive to MX.
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ABCG2 p.Arg482Lys 15670731:227:36
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
6 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.
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ABCG2 p.Arg482Lys 16815914:6:125
status: VERIFIED7 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.Arg482Lys 16815914:7:34
status: VERIFIED45 One mutant, R482K, did not transport any of the substrates tested, and the addition of prazosin had little effect on its ATPase activity.
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ABCG2 p.Arg482Lys 16815914:45:12
status: VERIFIED46 In contrast, all the variants analyzed here, including R482K, specifically bound the substrate analog [125 I]IAAP.
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ABCG2 p.Arg482Lys 16815914:46:55
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.Arg482Lys 16815914:65:17
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.Arg482Lys 16815914:69:147
status: VERIFIED70 All the variants of ABCG2, except the R482K variant, were able to efflux the substrate mitoxantrone at comparable levels (data not shown).
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ABCG2 p.Arg482Lys 16815914:70:38
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.Arg482Lys 16815914:71:239
status: VERIFIED74 R482K was chosen because it is completely devoid of any transport, and the R482G and R482T mutants were chosen because these two mutants have been used in many different studies and would therefore be of broader interest.
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ABCG2 p.Arg482Lys 16815914:74:0
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.Arg482Lys 16815914:86:233
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.Arg482Lys 16815914:90:24
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.Arg482Lys 16815914:96:160
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.Arg482Lys 16815914:106:103
status: VERIFIED116 However, the R482K mutant is devoid of substrate transport and shows inhibition of ATPase activity in the presence of substrate.
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ABCG2 p.Arg482Lys 16815914:116:13
status: VERIFIED117 The ABCG2-mediated transport of rhodamine 123 and Bodipy FL prazosin varies greatly between the mutants described here (Figs. 2, 3), whereas wild-type ABCG2 (R482) and all but one of the ABCG2 mutants (R482K) are able to transport mitoxantrone (data not shown).
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ABCG2 p.Arg482Lys 16815914:117:202
status: VERIFIED119 The R482K mutant is completely devoid of any transport, even though lysine is generally considered a conservative substitution for arginine.
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ABCG2 p.Arg482Lys 16815914:119:4
status: VERIFIED128 Importantly, the R482K mutant, which is unable to transport any of the substrates tested here, including Bodipy FL prazosin, and the R482 wild-type protein, which only transports Bodipy FL prazosin to a low extent, are labeled with [125 I]IAAP.
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ABCG2 p.Arg482Lys 16815914:128:17
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.Arg482Lys 16815914:140:91
status: VERIFIED141 Moreover, several variants (R482H, R482K, R482wt, and R482Y) showed no prazosin-stimulated ATPase activity.
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ABCG2 p.Arg482Lys 16815914:141:35
status: VERIFIED144 Based on these findings, it is possible that the transport-deficient ABCG2 variant R482K, which specifically binds [125 I]IAAP, is unable to undergo these necessary conformational changes.
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ABCG2 p.Arg482Lys 16815914:144:83
status: VERIFIED146 The R482K mutation may also play an analogous role to the allosteric effect mediated by the P-gp inhibitor cis(Z)-flupentixol, which allows P-gp to bind substrates but not transport them (Maki et al. 2003).
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ABCG2 p.Arg482Lys 16815914:146:4
status: VERIFIED153 Among the 20 R482X mutants analyzed, R482K may show to be an interesting ABCG2 variant to study structurally since finding a transporter locked in a rigid conformation may aid in three-dimensional structure solving.
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ABCG2 p.Arg482Lys 16815914:153:37
status: VERIFIED[hide] Human multidrug resistance ABCB and ABCG transport... Physiol Rev. 2006 Oct;86(4):1179-236. Sarkadi B, Homolya L, Szakacs G, Varadi A
Human multidrug resistance ABCB and ABCG transporters: participation in a chemoimmunity defense system.
Physiol Rev. 2006 Oct;86(4):1179-236., [PMID:17015488]
Abstract [show]
In this review we give an overview of the physiological functions of a group of ATP binding cassette (ABC) transporter proteins, which were discovered, and still referred to, as multidrug resistance (MDR) transporters. Although they indeed play an important role in cancer drug resistance, their major physiological function is to provide general protection against hydrophobic xenobiotics. With a highly conserved structure, membrane topology, and mechanism of action, these essential transporters are preserved throughout all living systems, from bacteria to human. We describe the general structural and mechanistic features of the human MDR-ABC transporters and introduce some of the basic methods that can be applied for the analysis of their expression, function, regulation, and modulation. We treat in detail the biochemistry, cell biology, and physiology of the ABCB1 (MDR1/P-glycoprotein) and the ABCG2 (MXR/BCRP) proteins and describe emerging information related to additional ABCB- and ABCG-type transporters with a potential role in drug and xenobiotic resistance. Throughout this review we demonstrate and emphasize the general network characteristics of the MDR-ABC transporters, functioning at the cellular and physiological tissue barriers. In addition, we suggest that multidrug transporters are essential parts of an innate defense system, the "chemoimmunity" network, which has a number of features reminiscent of classical immunology.
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No. Sentence Comment
810 Mitoxantrone was transported by all ABCG2 variants, except by R482K.
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ABCG2 p.Arg482Lys 17015488:810:62
status: VERIFIED811 Rhodamine-123 was extruded by most of the mutants, except by R482K, R482Y, and the wild-type ABCG2.
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ABCG2 p.Arg482Lys 17015488:811:61
status: VERIFIED812 Interestingly, the R482K variant had relatively low activity in all assays, although this mutation (Arg to Lys) represents only a minor change without charge alteration.
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ABCG2 p.Arg482Lys 17015488:812:19
status: VERIFIED[hide] Structure and function of the human breast cancer ... Curr Drug Metab. 2010 Sep;11(7):603-17. Ni Z, Bikadi Z, Rosenberg MF, Mao Q
Structure and function of the human breast cancer resistance protein (BCRP/ABCG2).
Curr Drug Metab. 2010 Sep;11(7):603-17., [PMID:20812902]
Abstract [show]
The human breast cancer resistance protein (BCRP/ABCG2) is the second member of the G subfamily of the large ATP-binding cassette (ABC) transporter superfamily. BCRP was initially discovered in multidrug resistant breast cancer cell lines where it confers resistance to chemotherapeutic agents such as mitoxantrone, topotecan and methotrexate by extruding these compounds out of the cell. BCRP is capable of transporting non-chemotherapy drugs and xenobiotiocs as well, including nitrofurantoin, prazosin, glyburide, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. BCRP is frequently detected at high levels in stem cells, likely providing xenobiotic protection. BCRP is also highly expressed in normal human tissues including the small intestine, liver, brain endothelium, and placenta. Therefore, BCRP has been increasingly recognized for its important role in the absorption, elimination, and tissue distribution of drugs and xenobiotics. At present, little is known about the transport mechanism of BCRP, particularly how it recognizes and transports a large number of structurally and chemically unrelated drugs and xenobiotics. Here, we review current knowledge of structure and function of this medically important ABC efflux drug transporter.
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No. Sentence Comment
265 The positive charge of Arg482 does not seem to be a key determinant because the substitution of Arg482 with Lys of the same charge resulted in a complete loss of drug resistance [109].
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ABCG2 p.Arg482Lys 20812902:265:96
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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108 Similar to earlier findings, there was a well-measurable Ko143-sensitive Hst dye transport both in the cells expressing wtABCG2 and in those expressing most R482 mutants, with only very low activity in the case of the R482K and R482Y variants (Fig. 1B).
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ABCG2 p.Arg482Lys 24384916:108:218
status: NEW110 Moreover, significant Hst transport activity occurred in the case of the R482K and Y variants.
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ABCG2 p.Arg482Lys 24384916:110:73
status: NEW116 In the case of the wild-type protein and the R482K and Y variants, there was no detectable R123 extrusion in either the absence or presence of cholesterol.
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ABCG2 p.Arg482Lys 24384916:116:45
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.Arg482Lys 24384916:225:116
status: NEW239 In the case of the R482K and I mutants, a variable alteration in the substrate stimulation was observed for different bile acids (Fig. 6D).
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ABCG2 p.Arg482Lys 24384916:239:19
status: NEW254 Interestingly, the activating effect of cholesterol is the most pronounced in the case of the R482K and Y mutants, as these variants are practically unable to transport Hoechst 33342 unless high levels of cholesterol are present in the cell membranes (Fig. 1B).
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ABCG2 p.Arg482Lys 24384916:254:94
status: NEW[hide] Determinants of the activity and substrate recogni... Drug Metab Rev. 2014 Nov;46(4):459-74. doi: 10.3109/03602532.2014.942037. Epub 2014 Jul 18. Szafraniec MJ, Szczygiel M, Urbanska K, Fiedor L
Determinants of the activity and substrate recognition of breast cancer resistance protein (ABCG2).
Drug Metab Rev. 2014 Nov;46(4):459-74. doi: 10.3109/03602532.2014.942037. Epub 2014 Jul 18., [PMID:25036722]
Abstract [show]
The xenobiotic transporters are among the most important constituents of detoxification system in living organisms. Breast cancer resistance protein (BCRP/ABCG2) is one of the major transporters involved in the efflux of xenobiotics. To understand its role in chemotherapeutic and multidrug resistance, it is crucial to establish the determinants of its substrate specificity, which obviously is of high relevance for successful therapy of many diseases. This article summarizes the current knowledge about the substrate preferences of BCRP. We overview the factors which determine its activity, inhibition and substrate recognition, focusing on the structural features of the transporter. BCRP substrate specificity is quite low as it interacts with a spectrum of substances with only a few common features: hydrophobic and aromatic regions, possibly a flat conformation and the metal ion-, oxygen- and nitrogen-containing functionalities, most of which may be the donors/acceptors of H-bonds. Several amino acid residues and structural motifs are responsible for BCRP activity and substrate recognition. Thus, the active form of BCRP, at least a dimer or a larger oligomer is maintained by intramolecular disulfide bridge that involves Cys(603) residues. The GXXXG motif in transmembrane helix 1, Cys residues, Arg(482) and Lys(86) are responsible for maintaining the protein structure, which confers transport activity, and the His(457) or Arg(456) residues are directly involved in substrate binding. Arg(482) does not directly bind substrates, but electrostatically interacts with charged molecules, which initiates the conformational changes that transmit the signal from the transmembrane regions to the ABC domain.
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No. Sentence Comment
171 Many other amino acids, i.e. Gly, Ile, Met, Ser, Thr, Asp, Asn, Lys and Tyr, have been substituted at position 482, and all of these variants, except for Arg482 Lys, transported mitoxantrone, but only the WT protein transported methotrexate.
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ABCG2 p.Arg482Lys 25036722:171:154
status: NEW172 Rhodamine was effluxed by most of these variants except for Arg482 Lys, Arg482 Tyr and WT protein.
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ABCG2 p.Arg482Lys 25036722:172:60
status: NEW173 Arg482 Lys and Arg482 Tyr did not drive out Hoechst 33342 either (O &#a8; zvegy-Laczka et al., 2005).
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ABCG2 p.Arg482Lys 25036722:173:0
status: NEW175 Out of 19 BCRP variants, with different standard amino acids at position 482, only Arg482 Lys-BCRP is completely devoid of transport capability.
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ABCG2 p.Arg482Lys 25036722:175:83
status: NEW209 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).
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ABCG2 p.Arg482Lys 25036722:209:1981
status: NEW241 Because the Arg482 Lys variant is completely inactive, it seems likely that residue 482 confers the transport activity on BCRP.
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ABCG2 p.Arg482Lys 25036722:241:12
status: NEW