Functional analysis of the nucleotide binding domains of the multidrug resistance protein (MRP).
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.