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 wildtype 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 are known to be involved in multidrug resistance. Breast cancer resistance protein (BCRP/ABCG2/MXR) is a new member of the ABC transporter family, (6-8) with a C-terminal transmembrane domain and an N-terminal ATP-binding domain, (9) consisting of 655 amino acids. BCRP has been studied as a molecular target for anticancer drug resistance because of its ability to confer resistance to mitoxantrone, 7-ethyl-10-hydroxycamptothecin (SN-38) and topotecan in cells by pumping out these structurally unrelated drugs. (10 -16) We previously showed that BCRP might form a homodimer, bridged by disulfide bonds, and that BCRP function was impaired by dominant-negative mutants through S-S-dependent homodimerization.(17) In our present study we removed each of the possible disulfide bonds that may be involved in BCRP dimerization by site-directed mutagenesis of the cysteine residues in this protein. As shown in Fig. 1, BCRP has six cysteines in the cytoplasm, three of which are intramembranous and three that are extracellular. We constructed 12 BCRP mutants for these analyses, each with a serine substitution in place of a cysteine, using site-directed mutagenesis of BCRP cDNA, and examined the resulting effects on both protein structure and function. We show from our data that Cys-603 is likely to be an important residue for the stable oligomerization of BCRP. Materials and MethodsCell culture and drug sensitivity assay PA317 amphotropic retrovirus packaging cells were grown in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum, at 37°C in a humidified incubator with 5% CO 2 . The sensitivity of the mutant BCRP-transfected cells to SN-38 was evaluated by the inhibition of cell growth after incubation at 37°C in the presence of various...

Gout-causing Q141K mutation in ABCG2 leads to instability of the nucleotide-binding domain and can be corrected with small molecules

“…This possibility that protein-protein interactions are key for ABCG2 dimerization is a relatively new idea. Early work (24,42) showed that the simple withdrawal of reducing agent from the Laemmli buffer allowed the visualization of the ABCG2 dimer on a Western blot and proposed therefore that disulfide bonds between cysteines, specifically the C603 residue, was the critical residue in ABCG2 dimerization (43)(44)(45). However, more recent work has showed that the cysteines were not necessary for dimerization and that in vitro ABCG2 dimerized and trafficked without the need for disulfide bonds (43,(46)(47)(48), suggesting the importance of protein-protein interactions in ABCG2 dimerization.…”

Effect of Cysteine Mutagenesis on the Function and Disulfide Bond Formation of Human ABCG2

“…However, Bhatia et al (2005) observed no effect of similar mutations on the trafficking or function of the mutant form of human ABCG2. Furthermore, Kage et al (2005) found that mutation of Cys603 had no effect on ABCG2 function. Thus, it is not yet clear whether formation of intermolecular disulfide bonds is responsible for ABCG2 dimerization and whether cysteine residues are important for its membrane targeting and function.…”

ABCG2 Transports and Transfers Heme to Albumin through Its Large Extracellular Loop*

“…Approximately 70 -80 residues long, ECL3 is large enough to constitute a full domain, although no specific role has been assigned to it. It contains three cysteine residues reported to be involved in both intermolecular (Cys 603 -Cys 603 ) and intramolecular (Cys 592 -Cys 608 ) disulfide bridges (31)(32)(33)(34). ECL3 also contains a conformational epitope recognized by the 5D3 monoclonal antibody (24), the interaction of which is prevented upon reduction of the intramolecular disulfide bridge Cys 603 -Cys 603 (35), making 5D3 a powerful conformation-sensing tool.…”