ABCMdb

ABCG2 p.Cys438Ser

Predicted by SNAP2:	A: D (75%), D: D (91%), E: D (91%), F: D (91%), G: D (91%), H: D (91%), I: D (75%), K: D (95%), L: D (80%), M: D (80%), N: D (91%), P: D (91%), Q: D (91%), R: D (95%), S: D (66%), T: D (85%), V: D (66%), W: D (91%), Y: D (91%),
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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Publications
[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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Sentences [show]
No. Sentence Comment
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
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
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
127 In the case of BCRP-C438S, which has a mutation in the second transmembrane domain, little expression of BCRP was observed by either western blotting or FACS. Login to comment
128 In addition, the SN-38 sensitivity of PA/C438S cells was equivalent to the parental PA317 cells (Fig. 5c). Login to comment