ABCMdb

ABCG2 p.Cys603Ser

Predicted by SNAP2:	A: D (85%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (95%), I: D (91%), K: D (95%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), Q: D (95%), R: D (95%), S: D (91%), T: D (91%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: 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, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Role of Cys-603 in dimer/oligomer formation of the... Cancer Sci. 2005 Dec;96(12):866-72. Kage K, Fujita T, Sugimoto Y
Role of Cys-603 in dimer/oligomer formation of the breast cancer resistance protein BCRP/ABCG2.
Cancer Sci. 2005 Dec;96(12):866-72., [PMID:16367905]

Abstract [show]
Breast cancer resistance protein (BCRP/ABCG2) is a half-molecule ATP-binding cassette transporter that we have previously suggested might function as a homodimer, bridged by disulfide bonds. In the present study, we carried out cysteine-scanning mutagenesis, substituting Ser for Cys, and established 12 PA317 transfectants expressing BCRP mutants with possible disruptions to their S-S bonds. Western blot analysis of BCRP from the wild-type transfectants (PA/WT) confirmed that the wild-type protein migrates as a 140-kDa dimer under non-reducing conditions, but as a 70-kDa monomer under reducing conditions. However, under non-reducing conditions the BCRP-C603S mutant migrated both as a 70-kDa monomer and a 140-kDa dimer, whereas all other mutant BCRP migrated only as dimers. PA317 cells transfected with C603S-BCRP (PA/C603S) showed either similar or only marginally lower SN-38 resistance than PA/WT cells, despite the reduced levels of BCRP dimer in these cells. Moreover, the degree of SN-38 resistance in the mutant BCRP transfectants was found to be associated with the monomer expression levels under reducing conditions. Reverse transcription-polymerase chain reaction analysis showed that the BCRP mRNA levels were similar in the transfectants. We subsequently generated six C603X mutants of BCRP (X=D, H, R, Y, A and W) and carried out western blot analysis and drug sensitivity assays. The results were equivalent to those from the PA/C603S cells, with some variations that again corresponded to the monomer levels. Our findings suggest that Cys-603 is an important residue in the covalent bridge between BCRP monomers but that a functioning unit of BCRP may not necessarily require covalent linkages.

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5 However, under non-reducing conditions the BCRP-C603S mutant migrated both as a 70-kDa monomer and a 140-kDa dimer, whereas all other mutant BCRP migrated only as dimers. Login to comment
6 PA317 cells transfected with C603S-BCRP (PA/C603S) showed either similar or only marginally lower SN-38 resistance than PA/WT cells, despite the reduced levels of BCRP dimer in these cells. Login to comment
10 The results were equivalent to those from the PA/C603S cells, with some variations that again corresponded to the monomer levels. Login to comment
57 Under nonreducing conditions, however, the BCRP-C603S mutant was found to migrate as both a 70-kDa monomer and a 140-kDa dimer, whereas the BCRP-WT and each of the other mutant proteins migrated only as 140-kDa dimers (Fig. 2b). Login to comment
58 Also under reducing conditions, the BCRP protein levels in PA/ C43S, PA/C55S, PA/C284S and PA/C603S cells were slightly lower than the levels in the wild-type transfectants. Login to comment
63 Following this increased exposure time, these mutants could also be detected as both a 70-kDa monomer and a 140-kDa dimer, as is the case for BCRP-C603S, although their expression levels were less than 10% of wild-type. Login to comment
64 The ratio of monomer to dimer in these mutants was also far higher than PA/WT, as observed for PA/C603S. Login to comment
67 In contrast, the PA/C43S, PA/C55S, PA/C284S and PA/C603S mutants expressed intermediate amounts of BCRP and, in PA/C438S cells, little surface expression of BCRP was observed. Login to comment
71 Hence, the observed differences in BCRP expression, particularly in the PA/C438S, PA/C592S, PA/C603S and PA/C608S cells, are not attributable to low transcript levels. Login to comment
76 Protein expression and SN-38 sensitivity levels of PA317 cells transfected with mutant BCRP containing Cys-603 substitutions Because the monomeric form of BCRP was detectable in PA/ C603S cells under non-reducing conditions (Fig. 2b), we chose to further analyze this specific cysteine residue by generating additional amino acid substitutions at this position. Login to comment
77 The resulting PA/C603X mutants (X = D, H, R, S, Y, A and W) were also investigated by western blotting under the same conditions used for PA/C603S. Login to comment
89 intensities of the 70-kDa BCRP monomeric mutant bands were similar to wild-type with the exception of PA/C603S and PA/C603Y, which had somewhat lower expression levels (Fig. 6a). Login to comment
108 PA/C43S, PA/C55S, PA/C284S and PA/C603S transfectants acquired similar or somewhat lower degrees of SN-38 resistance than PA/WT cells. Login to comment
120 In contrast, considerable levels of the monomeric form of BCRP were observed for the PA/C603S mutant, which correspondingly showed decreased dimer levels (Fig. 2b). Login to comment
122 Under non-reducing conditions, small quantities of both monomeric and dimeric forms of BCRP could be observed for the PA/C438S, PA/C592S and PA/C608S transfectants as well as in PA/C603S cell extracts following overexposure of the blot (Fig. 2c). Login to comment
125 The intensity of the BCRP-C603S monomer band under non-reducing conditions was far stronger than the other mutants (Fig. 2b). Login to comment
131 The drug resistance levels induced by exogenous PA/ C603S were almost equivalent to PA/WT (Fig. 5b), although the intensity of the 140-kDa BCRP-C603S band under nonreducing conditions was far less than BCRP-WT (Fig. 2b). Login to comment
143 (c) SN-38 sensitivity of parental PA317, PA/WT, PA/C603D, PA/C603Y, PA/ C603S and PA/C603A. Login to comment

[hide] Interaction with the 5D3 monoclonal antibody is re... J Biol Chem. 2008 Sep 19;283(38):26059-70. Epub 2008 Jul 21. Ozvegy-Laczka C, Laczko R, Hegedus C, Litman T, Varady G, Goda K, Hegedus T, Dokholyan NV, Sorrentino BP, Varadi A, Sarkadi B
Interaction with the 5D3 monoclonal antibody is regulated by intramolecular rearrangements but not by covalent dimer formation of the human ABCG2 multidrug transporter.
J Biol Chem. 2008 Sep 19;283(38):26059-70. Epub 2008 Jul 21., 2008-09-19 [PMID:18644784]

Abstract [show]
Human ABCG2 is a plasma membrane glycoprotein working as a homodimer or homo-oligomer. The protein plays an important role in the protection/detoxification of various tissues and may also be responsible for the multidrug-resistant phenotype of cancer cells. In our previous study we found that the 5D3 monoclonal antibody shows a function-dependent reactivity to an extracellular epitope of the ABCG2 transporter. In the current experiments we have further characterized the 5D3-ABCG2 interaction. The effect of chemical cross-linking and the modulation of extracellular S-S bridges on the transporter function and 5D3 reactivity of ABCG2 were investigated in depth. We found that several protein cross-linkers greatly increased 5D3 labeling in ABCG2 expressing HEK cells; however, there was no correlation between covalent dimer formation, the inhibition of transport activity, and the increase in 5D3 binding. Dithiothreitol treatment, which reduced the extracellular S-S bridge-forming cysteines of ABCG2, had no effect on transport function but caused a significant decrease in 5D3 binding. When analyzing ABCG2 mutants carrying Cys-to-Ala changes in the extracellular loop, we found that the mutant C603A (lacking the intermolecular S-S bond) showed comparable transport activity and 5D3 reactivity to the wild-type ABCG2. However, disruption of the intramolecular S-S bridge (in C592A, C608A, or C592A/C608A mutants) in this loop abolished 5D3 binding, whereas the function of the protein was preserved. Based on these results and ab initio folding simulations, we propose a model for the large extracellular loop of the ABCG2 protein.

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28 Interestingly, mutation of Cys-603 to Ala, Gly, or Ser does not remarkably influence the expression and functionality of the transporter (9-11). Login to comment

[hide] BCRP/ABCG2 confers anticancer drug resistance with... Cancer Sci. 2010 Aug;101(8):1813-21. Epub 2010 Apr 28. Shigeta J, Katayama K, Mitsuhashi J, Noguchi K, Sugimoto Y
BCRP/ABCG2 confers anticancer drug resistance without covalent dimerization.
Cancer Sci. 2010 Aug;101(8):1813-21. Epub 2010 Apr 28., [PMID:20518788]

Abstract [show]
In previous studies, we demonstrated that the breast cancer resistance protein (BCRP, ABCG2) forms an S-S homodimer. The BCRP-C603S mutant substituting Ser for Cys-603 in the third extracellular domain formed both a 70-75-kDa monomer and 140-150-kDa dimer, suggesting that Cys-603 is an important residue in the covalent bridge. These results also suggested the involvement of other Cys residues in dimer formation. In the present study, we examined the possible involvement of the other extracellular Cys residues, Cys-592 and Cys-608, in the dimerization and transporter functions of BCRP using double and triple Cys-mutant BCRP transfectants. In SDS-PAGE under non-reducing conditions, BCRP-C592S.C603S and BCRP-C592S.C608S were detected as dimers whereas BCRP-C603S.C608S and BCRP-C592S.C603S.C608S were found only as monomers. This finding indicated that no Cys residues other than the three extracellular Cys are responsible for the dimer formation. The formation of BCRP-C592S.C603S dimer suggested the involvement of Cys-608 in the covalent linkage of this mutant BCRP. PA/C592S.C603S.C608S-cl.7 cells showed a significant level of multiple drug resistance and low-level accumulation of mitoxantrone. These results clearly demonstrate that BCRP functions as a drug resistance protein without covalent dimerization. Among drug-resistant Cys-mutant BCRP transfectants, PA/C603S, PA/C592S.C608S, and PA/C592S.C603S.C608S were found to be more resistant to the reversal effects of fumitremorgin C than PA/WT, suggesting some alteration in the substrate recognition in Cys-mutant BCRPs. In conclusion, Cys-mediated covalent dimerization is not required for BCRP to function as a transporter. In addition to Cys-603, Cys-608 may also be involved in BCRP dimer formation.

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1 The BCRP-C603S mutant substituting Ser for Cys-603 in the third extracellular domain formed both a 70-75-kDa monomer and 140-150-kDa dimer, suggesting that Cys-603 is an important residue in the covalent bridge. Login to comment
8 Among drug-resistant Cys-mutant BCRP transfectants, PA / C603S, PA / C592SÆC608S, and PA / C592SÆC603SÆC608S were found to be more resistant to the reversal effects of fumitremorgin C than PA / WT, suggesting some alteration in the substrate recognition in Cys-mutant BCRPs. Login to comment
21 Among these, BCRP-C603S mutant substituting Ser for Cys-603 was found to migrate both as a 70-75-kDa monomer and as a 140-150-kDa dimer in SDS-PAGE under non-reducing conditions. Login to comment
22 (16) The PA /C603S transfectant showed the drug resistance phenotype. Login to comment
25 However, the existence of a small amount of 150-kDa dimer in the PA/C603S transfectant suggested the involvement of other Cys residues in this process. Login to comment
29 In the present study, to elucidate the possible involvement of Cys-592 and Cys-608 in BCRP dimerization and protein activity, we established double (C592SÆC603S, C592SÆC608S, and C603SÆC608S) and triple (C592SÆC603SÆC608S) mutant BCRP transfectants, and compared the expression, dimer formation, and function of these BCRP mutants with wild-type and single (C592S, C603S, and C608S) mutant proteins. Login to comment
33 The wild-type and single (C592S, C603S, and C608S) mutant BCRP cDNAs in the same vector were also used as controls. Login to comment
63 PA317 cells were transfected with the wild-type, single (C592S, C603S, and C608S), double (C592SÆC603S, C592SÆC608S, and C603SÆC608S), and triple (C592SÆC603SÆC608S) mutant BCRP cDNAs in pHa-IRES-DHFR bicistronic expression vector as described previously. Login to comment
67 The resulting mixed populations of the drug-selected cells were designated as PA /WT-mix, PA / C592S-mix, PA /C603S-mix, PA /C608S-mix, PA /C592SÆC603S-mix, PA/ C592SÆC608S-mix, PA /C603SÆC608S-mix, and PA /C592SÆC603SÆC608S-mix. Login to comment
71 Wild-type BCRP and BCRP-C603S both migrated as a 75-kDa molecule. Login to comment
74 A high level of BCRP expression was observed in PA /WT-mix, PA /C603S-mix, and PA /C592SÆ C608S-mix cells (Fig. 2a). Login to comment
76 As reported previously, BCRP-C603S migrated as both a 150-kDa dimer and a 70-75-kDa monomer. Login to comment
82 The BCRP expression levels and monomer /dimer formation in PA/ WT and single mutant BCRP transfectants (PA /C592S, PA/C603S, and PA/C608S) were essentially the same as those in our previous report (Fig. 3a-d). Login to comment
94 PA /WT cells expressed 75-kDa BCRP only, and PA /C603S cells mostly expressed the 75-kDa molecule. Login to comment
97 +SH -SH PA/WT-mix PA/C592S-mix PA/C603S-mix PA/C608S-mix PA/C592S·C603S-mix PA/C592S·C608S-mix PA/C603S·C608S-mix PA/C592S·C603S·C608S-mix PA317 PA/WT-mix PA/WT-mix(0.5) PA/C592S-mix PA/C603S-mix PA/C608S-mix PA/C592S·C603S-mix PA/C592S·C608S-mix PA/C603S·C608S-mix PA/C592S·C603S·C608S-mix PA317 150 75 kDa 150 75 kDa GAPDH (b)(a) Fig. 2. Login to comment
102 -mix -cl.1 -cl.8 PA317 PA/WT PA/C592S PA/C603S PA/WT-mix -mix -cl.3 -cl.7 PA317 PA/WT-mix -mix -cl.5 -cl.7 PA317 PA/C608S PA/WT-mix -mix -cl.6 -cl.7 PA317 BCRP (-SH) 75 150 kDa GAPDH (+SH) BCRP (+SH) PA/WT-mix -mix -cl.1 -cl.4 PA317 PA/WT-mix -mix -cl.6 -cl.7 PA317 PA/WT-mix -mix -cl.6 -cl.7 PA317 PA/C592S ·C603S PA/C592S ·C608S PA/C603S ·C608S PA/C592S ·C603S·C608S PA/WT-mix -mix -cl.2 -cl.7 PA317 BCRP (-SH) 75 150 kDa GAPDH (+SH) BCRP (+SH) (a) (b) (c) (d) (e) (g)(f) (h) Fig. 3. Login to comment
105 (a) PA / WT cells; (b) PA / C592S cells; (c) PA / C603S cells; (d) PA / C608S cells; (e) PA / C592SÆC603S cells; (f) PA / C592SÆC608S cells; (g) PA / C603SÆC608S cells; (h) PA / C592SÆC603SÆ C608S cells. Login to comment
109 PA /WT, PA/C603S-cl.3, and PA /C603S-cl.7 transfectants showed high levels of resistance to SN-38. Login to comment
120 Based on the results of drug sensitivity assay, we selected PA /WT-mix, PA/C592S-cl.6, PA /C603S-cl.3, PA /C592SÆC608S-cl.7, and PA/C592SÆC603SÆC608S-cl.7 cells as representative drug-resistant transfectants for further study. Login to comment
122 As shown in Figure 7(a), intracellular mitoxantrone was at low levels in PA /WT-mix and PA /C603S-cl.3 cells and at intermediate levels in PA /C592S-cl.6, PA /C592SÆC608S-cl.7, and PA /C592SÆC603SÆC608S-cl.7 cells. Login to comment
134 Immunofluorescent microscopy analysis using an anti-BCRP antibody BXP-21 was performed to examine whether 250 200 70-kDa BCRP 75-kDa BCRP 100 150 0 50 NormalizedBCRPexpression(%ofPA/WT) PA317 -mix -cl.1 -cl.8 -mix -cl.6 -cl.7 -mix -cl.3 -cl.7 -mix -cl.5 -cl.7 -mix -cl.1 -cl.4 -mix -cl.2 -cl.7 -mix -cl.6 -cl.7 -mix -cl.6 -cl.7 PAC592S ·C603S PA/C592S ·C608S PA/C603S ·C608S PA/C592S ·C603S·C608S PA/WT PA/C592S PA/C603S PA/C608S Fig. 4. Login to comment
138 SN-38 resistance of mutant BCRP transfectants Cell line IC50 to SN-38† (nM) Degree of resistance‡ (x-fold) Relative expression level Total BCRP§ 75-kDa BCRP- PA317 3.7 ± 0.2 1 - - PA / WT-mix 90 ± 4.4 24 100 100 PA / WT-cl.1 172 ± 6.1 47 207 206 PA / WT-cl.8 77 ± 10.3 21 56 56 PA / C592S-mix 12.4 ± 0.8 3.4 34 24 PA / C592S-cl.6 28.5 ± 1.4 7.7 52 37 PA / C592S-cl.7 13.7 ± 0.5 3.7 16 11 PA / C603S-mix 9.7 ± 0.5 2.6 54 51 PA / C603S-cl.3 409 ± 15.9 110 146 132 PA / C603S-cl.7 158 ± 17.7 43 135 122 PA / C608S-mix 4.8 ± 0.3 1.3 27 3 PA / C608S-cl.5 5.6 ± 0.2 1.5 26 7 PA / C608S-cl.7 6.3 ± 0.6 1.7 43 12 PA / C592SÆC603S-mix 3.6 ± 0.2 1.0 27 10 PA / C592SÆC603S-cl.1 5.1 ± 1.1 1.4 27 10 PA / C592SÆC603S-cl.4 6.2 ± 0.8 1.7 43 26 PA / C592SÆC608S-mix 16.7 ± 1.8 4.6 92 67 PA / C592SÆC608S-cl.2 22.8 ± 2.0 6.2 163 111 PA / C592SÆC608S-cl.7 34.5 ± 4.9 9.4 151 106 PA / C603SÆC608S-mix 2.9 ± 0.2 0.8 21 3 PA / C603SÆC608S-cl.6 4.4 ± 0.4 1.2 57 3 PA / C603SÆC608S-cl.7 3.4 ± 0.1 0.9 51 4 PA / C592SÆC603SÆC608S-mix 4.1 ± 0.2 1.1 13 7 PA / C592SÆC603SÆC608S-cl.6 4.0 ± 0.4 1.1 25 13 PA / C592SÆC603SÆC608S-cl.7 11.1 ± 1.5 3.0 145 102 †The cells were incubated for 4 days at 37°C in the presence or absence of various concentrations of 7-ethyl-10-hydroxycamptothecin (SN-38). Login to comment
149 To examine the sensitivity of mutant BCRPs to a BCRP inhibitor, namely FTC, the reversal effect of FTC on the mutant BCRP transfectants was examined. PA /C603S-cl.7 was added to the panel of drug-resistant transfectants in this experiment. Login to comment
151 Fumitremorgin C (FTC) reversed the drug resistance of PA /C603S-cl.3 and PA /C603S-cl.7 cells, although these clones still showed 4-to 15-fold higher levels of resistance to SN-38 and mitoxantrone in the presence of FTC. Login to comment
162 Consistent with our previous study, PA/C603S cells expressed both monomeric and dimeric BCRP. Login to comment
170 Single mutation on Cys-603 loses this homo-dimerization site, and it can therefore be concluded that BCRP-C603S protein mainly existed as a monomer. Login to comment
171 The intramolecular disulfide bridge between Cys-592 and Cys-608 is partially disrupted for some reason, and small amount of the BCRP dimer could be detected in PA /C603S. Login to comment
172 Introduction of the C592S mutation into BCRP-C603S extinguishes the counter partner against Cys-608 in the intramolecular disulfide bridge. Login to comment
196 PA/C603S-cl.3 5. Login to comment
208 (1) PA317 cells; (2) PA / WT-mix cells; (3) PA / C592S-cl.6 cells; (4) PA / C603S-cl.3 cells; (5) PA / C592SÆC608S-cl.7 cells; (6) PA / C592SÆC603SÆC608S-cl.7 cells. Login to comment
226 PA/WT, PA /C592S, PA/C603S, PA /C592SÆC608S, and PA /C592SÆC603SÆC608S-cl.7 cells exhibited drug resistance and low accumulation of mitoxantrone. Login to comment
227 PA/WT and PA /C603S cells expressed a 75-kDa BCRP product (Fig. 2). Login to comment
234 Effect of FTC on the SN-38 and mitoxantrone resistance of mutant BCRP transfectants Cell line SN-38 Mitoxantrone Relative expression level of total BCRP§ Degree of resistance† RI‡ Degree of resistance† RI‡ )FTC +FTC )FTC + FTC PA317 1.0 ± 0.1 0.9 ± 0.1 - 1.0 ± 0.1 0.9 ± 0.1 - - PA / WT-mix 19.3 ± 1.7 1.4 ± 0.1 13.8 9.0 ± 0.3 1.2 ± 0.1 7.5 100 PA / C592S-cl.6 4.2 ± 0.3 1.2 ± 0.1 3.5 3.7 ± 0.1 1.3 ± 0.1 2.8 52 PA / C603S-cl.3 106 ± 7.3 7.9 ± 0.3 13.4 57 ± 5.9 15 ± 0.7 3.8 146 PA / C603S-cl.7 18 ± 2.0 4.1 ± 0.2 4.4 28 ± 3.9 12 ± 1.0 2.3 135 PA / C592SÆC608S-cl.7 8.4 ± 0.5 4.0 ± 0.2 2.1 3.3 ± 0.2 3.3 ± 0.1 1.0 151 PA / C592SÆC603SÆC608S-cl.7 4.1 ± 0.3 4.6 ± 0.3 0.9 7.5 ± 0.2 6.0 ± 0.8 1.3 145 †The cells were incubated for 4 days at 37°C in the presence or absence of various concentrations of anticancer agents in the presence or absence of 1 lM fumitremorgin C (FTC). Login to comment
245 Fumitremorgin C (FTC) strongly reversed the resistance of PA /WT and PA /C592S cells to SN-38 and mitoxantrone, and partially reversed the drug resistance of PA /C603S cells. Login to comment

[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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136 Importantly, Shigeta et al. showed that the triple-mutant C592S/C603S/C608S retained substantial resistance to mitoxantrone, topotecan, and SN-38 [86]. Login to comment
300 The recent study by Shigeta et al. [86] also demonstrated that the triple-mutant C592S/C603S/C608S in PA317 cells retained substantial activity with some alteration in substrate recognition, although its level of expression was significantly decreased and its subcellular distribution was altered. Login to comment

[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
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
230 Cys592 Ser/Cys603 Ser-BCRP and Cys592 Ser/Cys608 Ser-BCRP were detected as dimers, while Cys603 Ser/Cys608 Ser-BCRP and Cys592 Ser/Cys603 Ser/Cys608 Ser-BCRP as monomers, suggesting that no other Cys are involved in dimerization. Login to comment
231 At the same time, cells transfected with a triple mutant, Cys592 Ser/Cys603 Ser/Cys608 Ser-BCRP, showed significant drug resistance and a low accumulation of mitoxantrone which indicated that dimer formation might not be required for BCRP functioning as a transporter. Login to comment