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.
Kage
Kumie
K
Division of Molecular Biotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-10-6 Ariake, Koto-ku, Tokyo 135-8550, Japan.
Fujita
Toshiro
T
Sugimoto
Yoshikazu
Y
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001943:Breast Neoplasms; D003545:Cysteine; D019281:Dimerization; D018432:Drug Resistance, Multiple / Q000235:genetics; D005260:Female; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors; D006801:Humans; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000235:genetics*; D011487:Protein Conformation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D009363:Neoplasm Proteins; D003545:Cysteine