A Functional Steroid-Binding Element in an ATP-Binding Cassette Multidrug Transporter Saroj Velamakanni, Tavan Janvilisri, Sanjay Shahi, and Hendrik W. van Veen Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom Received May 18, 2007; accepted October 5, 2007 ABSTRACT The human breast cancer resistance protein is an ATP-binding cassette (ABC) multidrug transporter that affects the bioavail- ability of chemotherapeutic drugs and can confer drug resis- tance on cancer cells. It is the second member of the ABCG subfamily, other members of which are associated with human steroid disorders such as hypercholesterolemia, sitosterolemia, and atherosclerosis. The molecular bases of protein-steroid interactions in ABC transporters are unknown. Here, we identify a steroid-binding element in the membrane domain of ABCG2 with a similarity to steroid hormone/nuclear receptors. The element facilitates steroid hormone binding and mediates mod- ulation of ABCG2 activity. The identification of this element might provide an opportunity for the development of new ther- apeutic ligands for ABCG2. ABCG proteins are composed of an N-terminal nucleotide- binding domain followed by a membrane domain with six putative transmembrane helices (TMHs). These half-size molecules dimerize to form functionally active, full-size ABC transporters (Krishnamurthy and Schuetz, 2006; Velama- kanni et al., 2008). ABCG2 plays an important role in the disposition and pharmacological activity of a broad range of compounds, including chemotherapeutic drugs used in the treatment of cancer (Hardwick et al., 2007). The protein is expressed on the apical membrane of cells in tissues with excretory functions, such as the apical pole of trophoblast cells in the placenta, the ducts and lobules of the breast, luminal membrane of villous epithelial cells in the small and large intestines, apical membranes of capillary vessels in the blood-brain barrier, and the canalicular membrane of hepa- tocytes (Maliepaard et al., 2001). In addition to its interaction with multiple drugs, ABCG2 can interact with a variety of steroids, including 17-estradiol (ED), progesterone (PG), testosterone, sulfated estrogens, and 17-estradiol-17-D- glucuronide (Chen et al., 2003; Janvilisri et al., 2003, 2005; Suzuki et al., 2003; Cooray et al., 2006). The interaction with steroids has also been observed for other members of the ABCG subfamily. ABCG1 and ABCG4 promote cholesterol efflux from cells to high-density lipo- proteins (Wang et al., 2004). ABCG1 is highly expressed in macrophages and mediates cholesterol efflux from macrophage foam cells, providing a link between high-density lipoprotein levels and atherosclerosis risk. ABCG5 and ABCG8 are the defective proteins in sitosterolemia and form a heterodimeric transporter that is responsible for dietary sitosterol/cholesterol efflux from enterocytes, thus preventing sterol overaccumula- tion in humans (Berge et al., 2000). The observation that many ABCG proteins can interact with steroids raises interesting questions about the nature of protein-steroid interactions in these transporters. In this article, we describe the identification of a functional steroid-binding element in ABCG2 R482G . The original cDNA encoding this ABCG2 protein was derived from S1-M1– 80 cells, a mitoxantrone-resistant human colon carci- noma cell line, which encodes a glycine at amino acid 482 at the cytoplasmic end of TMH 3, instead of the wild-type arginine (Honjo et al., 2001). The R482G replacement does not signifi- cantly affect the interactions of ABCG2 with Hoechst 33342 and steroid hormones (Robey et al., 2003; Janvilisri et al., 2005; Ozvegy-Laczka et al., 2005). ABCG2 R482G was selected for ease of study with cationic dyes such as ethidium. In addition, its wider pharmacological spectrum enables a more exhaustive characterization of drug-protein interactions than ABCG2 R482 (Clark et al., 2006). Materials and Methods Mutagenesis. ABCG2 mutants were generated with the Quik- Change method (Stratagene, La Jolla, CA) using pGEM-BCRP R482G This study was supported by the Medical Research Council and Association for International Cancer Research. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.038299. ABBREVIATIONS: ABC, ATP-binding cassette; a.u., arbitrary unit; DSG, disuccinimidyl glutarate; ED, 17-estradiol; PG, progesterone; TMH, transmembrane helix; GalP, galactose transporter; LBD, ligand binding domain; hPR, human progesterone receptor-; hER, human estrogen receptor-; HEK, human embryonic kidney. 0026-895X/08/7301-12–17$20.00 MOLECULAR PHARMACOLOGY Vol. 73, No. 1 Copyright © 2008 The American Society for Pharmacology and Experimental Therapeutics 38299/3285932 Mol Pharmacol 73:12–17, 2008 Printed in U.S.A. 12 http://molpharm.aspetjournals.org/content/suppl/2008/06/24/73.1.12.DC1.html Supplemental material to this article can be found at: at ASPET Journals on March 26, 2016 molpharm.aspetjournals.org Downloaded from