Role of Cys‐603 in dimer/oligomer formation of the breast cancer resistance protein BCRP/ABCG2

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. ( Cancer Sci 2005; 96: 866–872)