Membrane Topology of the Human Breast Cancer Resistance Protein (BCRP/ABCG2) Determined by Epitope Insertion and Immunofluorescence
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- 作者：Honggang Wang ; Eun-Woo Lee ; Xiaokun Cai ; Zhanglin Ni ; Lin Zhou ; Qingcheng Mao
- 刊名：Biochemistry
- 出版年：2008
- 出版时间：December 30, 2008
- 年：2008
- 卷：47
- 期：52
- 页码：13778-13787
- 全文大小：377K
- 年卷期：v.47,no.52(December 30, 2008)
- ISSN：1520-4995
文摘
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 1−395) and a membrane-spanning domain (MSD) (residues 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.