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 confer resistance to anthracyclines. Previously, we have shown that a human/murine hybrid protein containing amino acids 959-1187 of MRP1 can confer resistance to these drugs. We have now examined the functional characteristics of mutant proteins in which we have converted individual amino acids in the comparable region of mrp1 to those present at the respective locations in MRP1. These mutations had no effect on the drug resistance profile conferred by mrp1 with the exception of converting glutamine 1086 to glutamate, as it is in the corresponding position (1089) in MRP1. This mutation created a protein that conferred resistance to doxorubicin without affecting vincristine resistance, or the ability of mrp1 to transport leukotriene C(4) (LTC(4)) and 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG). Furthermore, mutation Q1086D conferred the same phenotype as mutation Q1086E while the 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 E1089 for conferring resistance to anthracyclines, we mutated this residue to Gln, Asp, Ala, Leu, and Lys in the human protein. The mutation E1089D showed the same phenotype as MRP1, while the E1089Q substitution markedly decreased resistance to anthracyclines without affecting LTC(4) and E(2)17betaG transport. Conversion of Glu-1089 to Asn, Ala, or Leu had a similar effect on resistance to anthracyclines, while conversion to a positive amino acid, Lys, completely eliminated resistance to anthracyclines and vincristine without affecting transport of LTC(4), E(2)17betaG, and the GSH-dependent substrate, estrone-3-sulfate. These 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 confer drug resistance particularly to the anthracyclines, but is not essential for its ability to transport conjugated organic anions such as LTC(4) and E(2)17betaG.
Cancer Research Laboratories and Department of Pathology, Queen's University, Kingston, Ontario K7L 3N6, Canada.
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Zhang
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Cole
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Deeley
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000633:toxicity*; D000970:Antineoplastic Agents / Q000633:toxicity; D001665:Binding Sites; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D004317:Doxorubicin / Q000633:toxicity*; D018432:Drug Resistance, Multiple; D004958:Estradiol / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D004970:Estrone / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D005047:Etoposide / Q000633:toxicity; D006801:Humans; D007700:Kinetics; D017997:Leukotriene C4 / Q000493:pharmacokinetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014750:Vincristine / Q000633:toxicity*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000903:Antibiotics, Antineoplastic; D000970:Antineoplastic Agents; D011994:Recombinant Proteins; C025483:estradiol-17 beta-glucuronide; D017997:Leukotriene C4; D004970:Estrone; D004958:Estradiol; D014750:Vincristine; D005047:Etoposide; D004317:Doxorubicin; C017296:estrone sulfate