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Functional analysis of the nucleotide binding domains of the multidrug resistance protein (MRP)
Q Zhu1, H Sun, M S Center
1Division of Biology, Kansas State University, Manhattan 66506, USA.
Abstract:
HeLa cells were transfected with full-length multidrug resistance protein (MRP) cDNA and with MRP cDNAs that had been mutated at certain nucleotide binding domains. Stable transfectants were isolated and those producing equivalent amounts of P190 were tested in cytotoxicity assays using a variety of chemotherapeutic agents. The results demonstrate that deletions in the C-motif of NBD1 or the A-motif of NBD2 have a pronounced effect in reducing resistance levels to adriamycin, vincristine, or etoposide (VP-16). Single-site mutations of lysine in these same motifs reduce IC50 values but less than that observed with the deletion mutants. Additional studies have demonstrated an increase in drug accumulation and reduction in drug efflux in NBD deletion and single-site mutants. The results of this study therefore identify two lysines of the NBD A- and C-motifs that are critical for MRP-mediated multidrug resistance. The results also provide definitive evidence that resistance occurring as a result of MRP overexpression is related to enhanced levels of an ATP-dependent efflux pump.
Insights
Mutations in key areas of the multidrug resistance protein (MRP) significantly reduce cancer drug resistance. Specific lysines in nucleotide-binding domains are critical for MRP function and ATP-dependent drug efflux.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Multidrug resistance (MRP) proteins are crucial in cancer chemotherapy, mediating resistance to various chemotherapeutic agents.
- Understanding the molecular mechanisms of MRP function is vital for developing strategies to overcome drug resistance.
Purpose of the Study:
- To investigate the role of specific nucleotide-binding domains (NBDs) within the MRP protein in conferring multidrug resistance.
- To identify critical amino acid residues and motifs essential for MRP-mediated drug efflux and cellular resistance.
Main Methods:
- HeLa cells were transfected with wild-type and mutated multidrug resistance protein (MRP) cDNAs.
- Stable transfectants producing equivalent P190 levels were subjected to cytotoxicity assays with various chemotherapeutic agents.
- Drug accumulation and efflux assays were performed on selected mutants.
Main Results:
- Deletions in the NBD1 C-motif or NBD2 A-motif significantly reduced resistance to adriamycin, vincristine, and etoposide (VP-16).
- Single-site mutations of critical lysines in these motifs also reduced resistance, but to a lesser extent than deletions.
- Mutants exhibited increased intracellular drug accumulation and decreased drug efflux.
Conclusions:
- Two specific lysines within the NBD A- and C-motifs are critical for MRP-mediated multidrug resistance.
- MRP overexpression confers resistance through an enhanced ATP-dependent efflux pump mechanism, highlighting key functional domains for therapeutic targeting.