A Naturally Occurring Mutation in MRP1 Results in a Selective Decrease in Organic Anion Transport and in Increased Doxorubicin Resistance

Pharmacogenetics. 2002 Jun;12(4):321-30. doi: 10.1097/00008571-200206000-00008.

Abstract

The human 190 kDa multidrug resistance protein, MRP1, is a polytopic membrane glycoprotein that confers resistance to a wide range of chemotherapeutic agents. It also transports structurally diverse conjugated organic anions, as well as certain unconjugated and conjugated compounds, in a reduced glutathione-stimulated manner. In this study, we characterized a low-frequency (<1%) naturally occurring mutation in MRP1 expected to cause the substitution of a conserved arginine with serine at position 433 in a predicted cytoplasmic loop of the protein. Transport experiments with membrane vesicles prepared from transfected human embryonic kidney cells and HeLa cells revealed a two-fold reduction in the ATP-dependent transport of the MRP1 substrates, leukotriene C4 (LTC4) and oestrone sulphate. Kinetic analysis showed that this reduction was due to a decrease in Vmax for both substrates but Km was unchanged. In contrast, 17beta-oestradiol-17beta-(D-glucuronide) transport by the Arg433Ser mutant MRP1 was similar to that by wild-type MRP1. Fluorescence confocal microscopy showed that the mutant MRP1 was routed correctly to the plasma membrane. In contrast to the reduced LTC4 and oestrone sulphate transport, stably transfected HeLa cells expressing Arg433Ser mutant MRP1 were 2.1-fold more resistant to doxorubicin than cells expressing wild-type MRP1, while resistance to VP-16 and vincristine was unchanged. These results provide the first example of a naturally occurring mutation predicted to result in an amino acid substitution in a cytoplasmic region of MRP1 that shows an altered phenotype with respect to both conjugated organic anion transport and drug resistance.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP-Binding Cassette Transporters / genetics
  • ATP-Binding Cassette Transporters / metabolism
  • Adenosine Triphosphate / pharmacology
  • Antineoplastic Agents / pharmacology*
  • Binding Sites
  • Biological Transport
  • Cell Line
  • Cell Membrane
  • DNA / blood
  • DNA / metabolism
  • DNA Primers / chemistry
  • Doxorubicin / pharmacology*
  • Drug Resistance, Multiple*
  • Estrogens, Conjugated (USP) / metabolism
  • Estrone / analogs & derivatives*
  • Estrone / metabolism*
  • Female
  • HeLa Cells / cytology
  • Heterozygote
  • Humans
  • Immunoblotting
  • Kinetics
  • Leukotriene C4 / metabolism*
  • Male
  • Microscopy, Confocal
  • Multidrug Resistance-Associated Proteins / genetics*
  • Multidrug Resistance-Associated Proteins / metabolism
  • Mutagenesis, Site-Directed
  • Mutation / genetics*
  • Polymerase Chain Reaction
  • Transfection

Substances

  • ATP-Binding Cassette Transporters
  • Antineoplastic Agents
  • DNA Primers
  • Estrogens, Conjugated (USP)
  • Multidrug Resistance-Associated Proteins
  • Leukotriene C4
  • Estrone
  • Doxorubicin
  • Adenosine Triphosphate
  • DNA
  • estrone sulfate