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Perturbation of membrane microdomains in GLC4 multidrug-resistant lung cancer cells--modification of ABCC1 (MRP1)
Carole Marbeuf-Gueye1, Vérene Stierle, Paiwan Sudwan
1Laboratoire Biophysique Moléculaire, Cellulaire et Tissulaire (BioMoCeTi), UMR CNRS 7033, Université Paris 13 et Paris 6, 74 rue Marcel Cachin, Bobigny, France.
Abstract:
The multidrug resistance-associated protein transporter ABCC1 (MRP1) is an integral plasma membrane protein involved in the multidrug resistance phenotype. It actively expels a number of cytotoxic molecules from cells. To gain insight into the modulation of the functional properties of this integral membrane protein by cholesterol, a main component of the lipid bilayer, we used multidrug-resistant GLC4/ADR cells, which overexpress MRP1. Upon altering the plasma membrane cholesterol content of these cells, membrane localization and the activity of MRP1 were analyzed. A detergent-free methodology was used to separate "light" and "heavy" plasma membrane fractions. Our data show that MRP1 was exclusively found in "light" fractions known as L0 phase membrane microdomains, together with 23% of gangliosides GM1 and 40% of caveolin-1. Depletion of the membrane cholesterol level to 40% by treatment with the cholesterol-chelating agent methyl-beta-cyclodextrin did not modify MRP1 activity, as evidenced either by the rate of efflux of pirarubicin or that of glutathione. Further cholesterol depletion below 40% yielded both a partial shift of MRP1 to the high-density fraction and a decrease of its functionality. Taken together, these data suggest that MRP1 functionality depends on its localization in cholesterol-rich membrane microdomains.
Insights
Cholesterol influences the function of the multidrug resistance-associated protein transporter ABCC1 (MRP1). MRP1 activity decreases when cholesterol levels drop below 40%, indicating its dependence on cholesterol-rich membrane microdomains.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Pharmacology
Background:
- The multidrug resistance-associated protein transporter ABCC1 (MRP1) is an integral plasma membrane protein.
- MRP1 actively effluxes cytotoxic molecules, contributing to the multidrug resistance phenotype.
- Cholesterol is a key component of the lipid bilayer and can modulate membrane protein function.
Purpose of the Study:
- To investigate the impact of plasma membrane cholesterol levels on the localization and activity of MRP1.
- To understand how cholesterol modulates the functional properties of the MRP1 transporter.
Main Methods:
- Utilized multidrug-resistant GLC4/ADR cells overexpressing MRP1.
- Employed a detergent-free methodology to isolate "light" and "heavy" plasma membrane fractions.
- Altered cellular cholesterol content using methyl-beta-cyclodextrin and analyzed MRP1 localization and activity.
Main Results:
- MRP1 was exclusively localized in "light" membrane microdomains (L0 phase) containing GM1 and caveolin-1.
- Cholesterol depletion to 40% did not affect MRP1 activity (pirarubicin or glutathione efflux).
- Further cholesterol depletion below 40% caused MRP1 mislocalization and reduced its functionality.
Conclusions:
- MRP1 functionality is dependent on its localization within cholesterol-rich membrane microdomains.
- Specific cholesterol levels are crucial for maintaining optimal MRP1 activity.
- These findings provide insight into the regulation of transporter function by membrane lipid composition.
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