BCRP/ABCG2 confers anticancer drug resistance without covalent dimerization

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• The breast cancer resistance protein (BCRP, ABCG2) is a key transporter involved in multidrug resistance.
• Previous studies indicated that BCRP forms S-S homodimers, with Cys-603 suggested as crucial for this dimerization.
• The role of other extracellular cysteine residues in BCRP dimerization and function remained unclear.

Purpose of the Study:

• To investigate the involvement of extracellular cysteine residues (Cys-592, Cys-608) in BCRP dimerization.
• To determine if BCRP requires covalent dimerization for its drug transporter function.
• To analyze the impact of cysteine mutations on BCRP's drug resistance and substrate recognition.

Main Methods:

• Construction and transfection of double and triple cysteine mutant BCRP cell lines (e.g., BCRP-C592S.C603S, BCRP-C592S.C608S, BCRP-C603S.C608S, BCRP-C592S.C603S.C608S).
• SDS-PAGE under non-reducing conditions to analyze BCRP monomer and dimer formation.
• Assays to measure drug accumulation (e.g., mitoxantrone) and drug resistance.
• Evaluation of sensitivity to fumitremorgin C, a BCRP inhibitor.

Main Results:

• BCRP-C603S.C608S and BCRP-C592S.C603S.C608S mutants existed exclusively as monomers, indicating Cys-603 and Cys-608 are critical for dimer formation.
• BCRP-C592S.C603S.C608S cells exhibited significant multidrug resistance and low mitoxantrone accumulation, demonstrating BCRP function without covalent dimerization.
• Mutant BCRP transfectants showed altered substrate recognition, indicated by increased resistance to fumitremorgin C compared to wild-type BCRP.

Conclusions:

• Cysteine-mediated covalent dimerization is not essential for BCRP's function as a drug transporter.
• Cys-603 and Cys-608 play significant roles in BCRP dimer formation.
• Mutations in extracellular cysteines can alter BCRP substrate specificity and drug resistance profiles.