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A naturally occurring mutation in MRP1 results in a selective decrease in organic anion transport and in increased
Silke Conrad1, Hans-Martin Kauffmann, Ken-ichi Ito
1Food Chemistry and Environmental Toxicology, University of Kaiserslautern, Kaiserslautern, Germany.
Pharmacogenetics
|June 4, 2002
Summary
A naturally occurring mutation in the multidrug resistance-associated protein 1 (MRP1) alters its transport of organic anions and impacts doxorubicin resistance. This study details the functional consequences of the Arg433Ser mutation in MRP1.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- The human 190 kDa multidrug resistance-associated protein 1 (MRP1) is a membrane glycoprotein conferring resistance to chemotherapy.
- MRP1 transports various organic anions and compounds, influenced by reduced glutathione.
- Understanding MRP1 mutations is crucial for cancer therapy and drug resistance research.
Purpose of the Study:
- To characterize a rare, naturally occurring Arg433Ser mutation in MRP1.
- To investigate the impact of this mutation on MRP1's substrate transport and drug resistance profiles.
- To determine the functional consequences of amino acid substitution in a predicted cytoplasmic loop of MRP1.
Main Methods:
- Transport assays using membrane vesicles from transfected cells (HEK and HeLa).
- Kinetic analysis (Vmax, Km) of MRP1 substrates like leukotriene C4 (LTC4) and oestrone sulphate.
- Fluorescence confocal microscopy to assess mutant MRP1 localization.
- Drug resistance assays with doxorubicin, VP-16, and vincristine in stably transfected HeLa cells.
Main Results:
- The Arg433Ser MRP1 mutant showed a two-fold reduction in ATP-dependent transport of LTC4 and oestrone sulphate, primarily due to decreased Vmax.
- Transport of 17beta-oestradiol-17beta-(D-glucuronide) was unaffected by the Arg433Ser mutation.
- Cells expressing Arg433Ser MRP1 exhibited 2.1-fold increased resistance to doxorubicin, but unchanged resistance to VP-16 and vincristine.
- Mutant MRP1 was correctly localized to the plasma membrane.
Conclusions:
- The Arg433Ser mutation in MRP1 alters its transport activity for specific conjugated organic anions.
- This mutation confers increased resistance to doxorubicin, suggesting a link between anion transport function and drug resistance phenotype.
- This study provides the first evidence of a naturally occurring MRP1 mutation in a cytoplasmic region affecting both transport and drug resistance.