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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.
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.
Insights
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.