Localization of the Human Breast Cancer Resistance Protein (BCRP/ABCG2) in Lipid Rafts/Caveolae and Modulation of Its Activity by Cholesterol in Vitro

Abstract

Breast cancer resistance protein (BCRP/ABCG2) is an active efflux pump that belongs to the ATP-binding cassette (ABC) transporter family. It is located in various tissues involved in drug absorption, distribution, and elimination and plays an important role in multidrug resistance. For P-glycoprotein, another member of the ABC transporter family, it is well established that it is at least partly located in cholesterol and sphingolipid-enriched domains of the plasma membrane called "lipid rafts" and that the composition of the membrane lipids may modulate its efflux activity. This study addressed the compartmentalization of BCRP in the plasma membrane and the influence of membrane cholesterol on the efflux activity of BCRP. As a cell model, we used the canine kidney epithelial cell line MDCKII-BCRP transfected with the cDNA encoding human BCRP and the corresponding parental cell line MDCKII. Cholesterol depletion with methyl-␤-cyclodextrin (M␤CD) provoked a 40% decrease in BCRP activity (p Ͻ 0.01) assessed with flow cytometry (pheophorbide A efflux assay). Cholesterol repletion with M␤CD/cholesterol-inclusion complexes restored BCRP function, and cholesterol saturation of native cells did not further enhance BCRP activity. Coimmunoprecipitation experiments indicated a physical interaction between BCRP and caveolin-1, and Western blot analysis after density gradient ultracentrifugation demonstrated that BCRP is located in detergent-resistant membranes that also contain caveolin-1. In conclusion, our results demonstrate for the first time that BCRP is located in membrane rafts and that cholesterol has impact on its efflux activity.

Lipid Regulation of the ABCB1 and ABCG2 Multidrug Transporters

“…ABCG2 was found in Triton X-100-insoluble lipid rafts in close interaction with cholesterol and caveolin (Storch et al, 2007). As discussed below, in the case of ABCG2, cholesterol is an essential modulator of the transport activity; thus, raft localization may be advantageous for its proper functioning.…”

“…Both ABCB1 and ABCG2 have been reported to reside in detergentinsoluble rafts (Ismair et al, 2009;Orlowski, Martin, & Escargueil, 2006;Radeva, Perabo, & Sharom, 2005;Storch, Ehehalt, Haefeli, & Weiss, 2007) and in close interaction with cholesterol and caveolin. In the canalicular membranes of hepatocytes, several ABC transporters, including ABCB4 (MDR3), ABCB11 (BSEP, S-P-gp), ABCC2 (MRP2), and ABCG5/G8, have also been shown to reside in raft domains (Ismair et al, 2009).…”

“…Cholesterol depletion of the cell membranes has been reported to decrease both ABCB1 and ABCG2 function (Storch et al, 2007;Troost, Lindenmaier, Haefeli, & Weiss, 2004). For assessing a direct modulation of these transporters by membrane cholesterol, various functional assays should be combined, e.g., measurement of vanadate-sensitive, basal, or drug-stimulated ATPase activity, direct drug transport in isolated membranes, or combined ATPase and transport activity by isolated and reconstituted proteins (see Section 1).…”

Methyl-β-cyclodextrin Suppresses Hyaluronan Synthesis by Down-regulation of Hyaluronan Synthase 2 through Inhibition of Akt

“…Cholesterol and Hyaluronan Synthesis-Cholesterol is an important component of the plasma membrane, enriched in the heterogenous and dynamic lipid raft microdomains that have been suggested to regulate the function of multiple plasma membrane proteins (51,52). We have previously shown that overexpressed GFP-HAS3 is partly colocalized in lipid rafts and that M␤CD removes the hyaluronan synthesis-induced microvilli, a site for active hyaluronan synthesis.…”

The Caveolin-1 Connection to Cell Death and Survival

“…Additionally, Caveolin-1 function in this context has been linked to another ABC transporter, the BCRP protein, since it was shown that Caveolin-1 co-distributes with BCRP in detergent-resistant membrane fractions and interacts there with BCRP [142,148]. Interestingly, in Caveolin-1 knock-down cells, BCRP activity was reduced and the cells became more sensitive to mitoxantrone-induced reduction in cell proliferation [142].…”