ABCMdb

ABCG2 p.Cys608Ser

Predicted by SNAP2:	A: D (71%), D: D (85%), E: D (85%), F: D (85%), G: D (85%), H: D (85%), I: D (71%), K: D (85%), L: D (85%), M: D (80%), N: D (85%), P: D (85%), Q: D (85%), R: D (85%), S: D (75%), T: D (80%), V: D (66%), W: D (91%), Y: D (80%),
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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62 Because BCRP protein expression levels in PA/C438S, PA/ C592S and PA/C608S cells were found to be remarkably decreased under non-reducing conditions (Fig. 2b), they were further examined by overexposing the immunoblot (Fig. 2c). Login to comment
69 For the PA /C592S and PA/C608S species, which may have mutations in the epitope for the 5D3 antibody located on a extracellular domain of BCRP, BCRP expression on the cell surface was undetectable by FACS, but protein could be detected at low levels by western blotting. 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
110 PA/C592S and PA/C608S showed decreased drug resistance and the sensitivity of PA/C438S cells was similar to that of the parental PA317 cells. 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

[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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254 They found that C592S and C608S had impaired 5D3 binding; however, these two mutants showed very low expression levels in this study (9). 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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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
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
78 The BCRP expression levels in PA /C608S-mix, PA /C603SÆC608S-mix, and PA /C592SÆC603SÆC608S-mix cells were too low to evaluate dimer /monomer formation (Fig. 2b). 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
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
110 PA /C592S transfectants exhibited intermediate levels of resistance to SN-38 whereas PA /C608S transfectants showed only marginal levels of resistance to this drug. 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
163 (16) PA /C608S cells also expressed a BCRP monomer whereas PA/ C592S cells expressed a BCRP dimer. Login to comment
175 In PA /C608S cells, Cys-603 can also construct an intramolecular disulfide bridge with Cys-592, and therefore a single mutation on Cys-608 may weaken dimerization of BCRP. Login to comment
228 Significant expression of a 75-kDa BCRP was also found in PA /C592S, PA /C592SÆC608S, and PA/ C592SÆC603SÆC608S-cl.7 cells, whereas the PA/C608S cells mainly expressed a 70-kDa BCRP PA317 PA/WT-mix PA/C592S·C603S ·C608S-cl.7 BCRP Nuclei Merge 20 µm 20 µm 20 µm 20 µm 20 µm 20 µm 20 µm 20 µm 20 µm Fig. 8. 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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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