ABCMdb

ABCG2 p.Arg482Lys

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Publications

PMID: 14566825 [PubMed] Miwa M et al: "Single amino acid substitutions in the transmembrane domains of breast cancer resistance protein (BCRP) alter cross resistance patterns in transfectants."

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. Login to comment
43 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). Login to comment
48 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. Login to comment
61 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. Login to comment
67 PA/R482K (Group 4) did not show drug resistance. Login to comment
148 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). Login to comment
150 PA/R482K did not show drug resistance. Login to comment
151 The low-level drug resistance of the transfectants would explain why R482W- and R482K-BCRP were not expressed in drug-selected cell lines. Login to comment
167 PA/R482K (Group 4) did not show drug resistance. Login to comment

PMID: 15670731 [PubMed] Ozvegy-Laczka C et al: "Single amino acid (482) variants of the ABCG2 multidrug transporter: major differences in transport capacity and substrate recognition."

No. Sentence Comment

5 In intact cells, mitoxantrone was transported by all ABCG2 variants, except by R482K. Login to comment
6 Rhodamine 123 was extruded by most of the mutants, except by R482K, Y and by wild-type ABCG2. Login to comment
7 Hoechst 33342 was pumped out from cells expressing the wild-type and all Arg-482 variants, but not from those expressing R482K and Y. Login to 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). Login to comment
118 In contrast, the basal ATPase activity of R482K and Y mutants was rather inhibited than stimulated by prazosin Fig. 3. Login to comment
123 Panel B: Concentration dependence of MTX uptake by wtABCG2, R482I, R482K and K86M. Login to comment
124 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. Login to comment
145 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). Login to comment
157 These data indicate that most of the ABCG2-R482 mutant variants can actively extrude mitoxantrone, while the R482K mutant is inactive in this regard. Login to comment
159 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. Login to comment
169 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). Login to comment
195 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). Login to comment
198 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. Login to comment
209 The exceptions were the R482Y mutant, effective only in mitoxantrone transport, and the R482K mutant, showing no transport activity with any of these substrates. Login to comment
216 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). Login to comment
227 Only low level of expression of the R482K mutant was achieved in murine cells, nevertheless, the cells were sensitive to MX. Login to comment

PMID: 16815914 [PubMed] Ejendal KF et al: "The nature of amino acid 482 of human ABCG2 affects substrate transport and ATP hydrolysis but not substrate binding."

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. Login to 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. Login to comment
45 One mutant, R482K, did not transport any of the substrates tested, and the addition of prazosin had little effect on its ATPase activity. Login to comment
46 In contrast, all the variants analyzed here, including R482K, specifically bound the substrate analog [125 I]IAAP. Login to comment
65 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. Login to comment
69 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). Login to comment
70 All the variants of ABCG2, except the R482K variant, were able to efflux the substrate mitoxantrone at comparable levels (data not shown). Login to comment
71 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). Login to comment
74 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. Login to comment
86 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. Login to comment
90 In contrast, the R482H, R482K, R482Y, and R482wt variants are not markedly affected by the addition of 20 mM prazosin. Login to comment
96 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. Login to comment
106 Basal and drug-stimulated ATPase activity of wild-type ABCG2 (R482wt) and ABCG2 variants R482G, R482H, R482K, R482P, R482T, and R482Y. Login to comment
116 However, the R482K mutant is devoid of substrate transport and shows inhibition of ATPase activity in the presence of substrate. Login to comment
117 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). Login to comment
119 The R482K mutant is completely devoid of any transport, even though lysine is generally considered a conservative substitution for arginine. Login to comment
128 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. Login to comment
140 This hypothesis may also explain the differences in the ATPase activity seen for R482H and R482K mutants and the wild-type R482 protein. Login to comment
141 Moreover, several variants (R482H, R482K, R482wt, and R482Y) showed no prazosin-stimulated ATPase activity. Login to comment
144 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. Login to comment
146 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). Login to comment
153 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. Login to comment

PMID: 17015488 [PubMed] Sarkadi B et al: "Human multidrug resistance ABCB and ABCG transporters: participation in a chemoimmunity defense system."

No. Sentence Comment

810 Mitoxantrone was transported by all ABCG2 variants, except by R482K. Login to comment
811 Rhodamine-123 was extruded by most of the mutants, except by R482K, R482Y, and the wild-type ABCG2. Login to comment
812 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. Login to comment

PMID: 20812902 [PubMed] Ni Z et al: "Structure and function of the human breast cancer resistance protein (BCRP/ABCG2)."

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]. Login to comment

PMID: 24384916 [PubMed] Telbisz A et al: "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."

No. Sentence Comment

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). Login to comment
110 Moreover, significant Hst transport activity occurred in the case of the R482K and Y variants. Login to comment
116 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. Login to comment
225 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). Login to comment
239 In the case of the R482K and I mutants, a variable alteration in the substrate stimulation was observed for different bile acids (Fig. 6D). Login to comment
254 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). Login to comment

PMID: 25036722 [PubMed] Szafraniec MJ et al: "Determinants of the activity and substrate recognition of breast cancer resistance protein (ABCG2)."

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. Login to comment
172 Rhodamine was effluxed by most of these variants except for Arg482 Lys, Arg482 Tyr and WT protein. Login to comment
173 Arg482 Lys and Arg482 Tyr did not drive out Hoechst 33342 either (O &#a8; zvegy-Laczka et al., 2005). Login to comment
175 Out of 19 BCRP variants, with different standard amino acids at position 482, only Arg482 Lys-BCRP is completely devoid of transport capability. Login to comment
209 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). Login to comment
241 Because the Arg482 Lys variant is completely inactive, it seems likely that residue 482 confers the transport activity on BCRP. Login to comment