title: Membrane topology of the human breast cancer resistance protein (BCRP/ABCG2) determined by epitope insertion and immunofluorescence.
abstract: The human breast cancer resistance protein (BCRP/ABCG2) mediates efflux of drugs and organic anions across the plasma membrane. Hydropathy analysis suggests that BCRP consists of a nucleotide-binding domain (residues approximately 1-395) and a membrane-spanning domain (MSD) (residues approximately 396-655); however, its exact topology structure remains unknown. In this study, we determined the topology structure of BCRP by inserting hemagglutinin (HA) tags in its predicted hydrophilic regions of the MSD. HA-tagged BCRP mutants were expressed in HEK cells and tested for their ability to efflux mitoxantrone and BODIPY-prazosin. Polarity of the inserted tags with respect to the plasma membrane was determined by immunofluorescence. All of the mutants were expressed at levels comparable to wild-type BCRP as revealed by immunoblotting with specific antibodies against BCRP and the HA tag. Insertions at residues 423, 454, 462, 499, 529, 532, and 651 produced functional mutants, whereas insertions at residues 560, 594, and 623 resulted in mutants with significantly reduced activity and insertions at residues 387, 420, 474, and 502 completely abrogated the activity. HA tags inserted at residues 387, 474, 529, 532, 560, and 651 were localized intracellularly, whereas those inserted at residues 420, 423, 454, 499, 502, 594, and 623 revealed an extracellular location. Residue 462 was localized in a transmembrane (TM) segment. These results provide the first direct experimental evidence in support of a 6-TM model for BCRP with the amino and carboxyl termini of the MSD located intracellularly. These data may have important implications for understanding the transport mechanism of BCRP.
authors.affiliation: Department of Pharmaceutics, School of Pharmacy, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Honggang
authors.initials: H
authors.lastname: Wang
authors.forename: Eun-Woo
authors.initials: EW
authors.lastname: Lee
authors.forename: Xiaokun
authors.initials: X
authors.lastname: Cai
authors.forename: Zhanglin
authors.initials: Z
authors.lastname: Ni
authors.forename: Lin
authors.initials: L
authors.lastname: Zhou
authors.forename: Qingcheng
authors.initials: Q
authors.lastname: Mao
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D001943:Breast Neoplasms / Q000737:chemistry* / Q000276:immunology; D002462:Cell Membrane / Q000378:metabolism; D019008:Drug Resistance, Neoplasm; D018604:Epitope Mapping / Q000379:methods*; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D006801:Humans; D008565:Membrane Proteins; D008942:Mitoxantrone / Q000493:pharmacokinetics; D050505:Mutant Proteins; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D011224:Prazosin / Q000493:pharmacokinetics; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D050505:Mutant Proteins; D009363:Neoplasm Proteins; D008942:Mitoxantrone; D011224:Prazosin