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

Abstract

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.

Figure 1

Schematic diagram of a breast cancer resistance protein (BCRP) molecule indicating the position of its 12 cysteine residues. We constructed 12 mutants of BCRP by replacing each of these cysteines with serine by site‐directed mutagenesis of the cDNA template.

Figure 2

Detection of mutant breast cancer resistance proteins (BCRP), containing serine substitutions, by western blot analysis. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) was carried out (a) under reducing conditions with heating at 70°C for 10 min, or (b) under non‐reducing conditions with heating at 20°C for 5 min. α‐Tubulin expression was analyzed as a loading control. (c) Western blot analysis of three BCRP mutants with a long exposure showing weak band intensities under non‐reducing conditions. Cellular protein (20 µg/lane) was separated by 5–20% SDS‐PAGE and then transferred onto nitrocellulose membranes. BCRP was detected using mouse anti‐BCRP monoclonal antibody (BXP‐21).

Figure 3

Expression analysis of mutant breast cancer resistance proteins (BCRP) on the cell surfaces of PA317 transfectants by FACS. Trypsinized cells were incubated with (bold line) or without (fine line) the biotinylated antihuman BCRP monoclonal antibody 5D3, followed by incubation with R‐phycoerythrin‐conjugated streptavidin.

Figure 4

Semi‐quantitative reverse transcription–polymerase chain reaction of mRNA in wild‐type and mutant breast cancer resistance protein (BCRP) transfectants. First‐strand cDNA was synthesized with 0.3 µg of total RNA and a BCRP cDNA fragment (315 bp) was amplified by PCR using the indicated cycle numbers. Amplification of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) mRNA (551 bp fragment) was carried out as an internal control.

Figure 5

SN‐38 sensitivity assay of mutant breast cancer resistance protein (BCRP)‐transfected PA317 cells. Drug sensitivity to SN‐38 was examined by cell growth inhibition assays as described in Materials and Methods. All data are representative of the mean values ± SD from triplicate determinations. (a) SN‐38 sensitivity of the indicated mutant BCRP transfectants, which expressed similar levels of BCRP protein to PA/WT. (b) SN‐38 sensitivity of four mutant BCRP transfectants, which expressed lower levels of BCRP protein than PA/WT. PA/C43S, PA/C55S, PA/C284S and PA/C603S transfectants acquired similar or somewhat lower degrees of SN‐38 resistance than PA/WT cells. (c) SN‐38 sensitivity of mutants that expressed very small amounts of monomer BCRP. 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.

Figure 6

Western blot analysis of breast cancer resistance protein (BCRP) expression and SN‐38 drug sensitivity in PA/C603X mutants. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was carried out (a) under reducing conditions with heating at 70°C for 10 min and (b) under non‐reducing conditions with heating at 20°C for 5 min. α‐Tubulin expression was analyzed as a loading control. BCRP was detected using the mouse anti‐BCRP monoclonal antibody BXP‐21. Cellular protein (20 µg) was loaded in each lane. (c) SN‐38 sensitivity of parental PA317, PA/WT, PA/C603D, PA/C603Y, PA/C603S and PA/C603A. Drug sensitivity was examined by cell growth inhibition assays as described in Materials and Methods. All data are representative of the mean values ± SD from triplicate determinations.