Identification of an amino acid residue in multidrug resistance protein 1 critical for conferring resistance to anthracyclines

Murine multidrug resistance protein 1 (mrp1), unlike human MRP1, does not c onfer resistance to anthracyclines, Previously, we have shown that a human/ murine hybrid protein containing amino acids 959-1187 of MRP1 can confer re sistance to these drugs. We have now examined the functional characteristic s of mutant proteins in which we have converted individual amino acids in t he comparable region of mrp1 to those present at the respective locations i n MRP1. These mutations had no effect on the drug resistance profile confer red by mrp1 with the exception of converting glutamine 1086 to glutamate, a s it is in the corresponding position (1089) in MRP1. This mutation created a protein that conferred resistance to doxorubicin without affecting vincr istine resistance, or the ability of mrp1 to transport leukotriene C, (LTC4 ) and 17 beta -estradiol 17-(beta -D-glucuronide) (E(2)17 betaG). Furthermo re, mutation Q1086D conferred the same phenotype as mutation Q1086E while t he mutation Q1086N did not detectably alter the drug resistance profile of mrp1, suggesting that an anionic side chain was required for anthracycline resistance. To confirm the importance of MRP1 E10S9 for conferring resistan ce to anthracyclines, we mutated this residue to Gin, Asp, Ala, Leu, and Ly s in the human protein. The mutation E1089D showed the same phenotype as MR P1, while the E1089Q substitution markedly decreased resistance to anthracy clines without affecting LTC4 and E(2)17 betaG transport. Conversion of Glu -1089 to Asn, Ala, or Leu had a similar effect on resistance to anthracycli nes, while conversion to a positive amino acid, Lys, completely eliminated resistance to anthracyclines and vincristine without affecting transport of LTC4, E(2)17 betaG, and the GSH-dependent substrate, estrone-3-sulfate. Th ese results demonstrate that an acidic amino acid residue at position 1089 in predicted TM14 of MRP1 is critical for the ability of the protein to con fer drug resistance particularly to the anthracyclines, but is not essentia l for its ability to transport conjugated organic anions such as LTC4 and E (2)17 betaG.