title: Role of basic residues within or near the predicted transmembrane helix 2 of the human breast cancer resistance protein in drug transport.
abstract: The human breast cancer resistance protein (BCRP/ABCG2) mediates efflux of drugs and xenobiotics out of cells. In this study, we investigated the role of five basic residues within or near transmembrane (TM) 2 of BCRP in transport activity. Lys(452), Lys(453), His(457), Arg(465), and Lys(473) were replaced with Ala or Asp. K452A, K453D, H457A, R465A, and K473A were stably expressed in human embryonic kidney (HEK) cells, and their plasma membrane expression and transport activities were examined. All of the mutants were expressed predominantly on the plasma membrane of HEK cells. After normalization to BCRP levels, the activities of K452A and H457A in effluxing mitoxantrone, boron-dipyrromethene-prazosin, and Hoechst33342 were increased approximately 2- to 6-fold compared with those of wild-type BCRP, whereas the activities of K453D and R465A were decreased by 40 to 60%. Likewise, K452A and H457A conferred increased resistance to mitoxantrone and 7-ethyl-10-hydroxy-camptothecin (SN-38), and K453D and R465A exhibited lower resistance. The transport activities and drug-resistance profiles of K473A were not changed. These mutations also differentially affected BCRP ATPase activities with a 2- to 4-fold increase in V(max)/K(m) for K452A and H457A and a 40 to 70% decrease for K453D and R465A. These mutations may induce conformational changes as manifested by the altered binding of the 5D3 antibody to BCRP in the presence of prazosin and altered trypsin digestion. Molecular modeling and docking calculations indicated that His(457) and Arg(465) might be directly involved in substrate binding. In conclusion, we have identified several basic residues within or near TM2 that may be important for interaction of substrates with BCRP.
authors.affiliation: Department of Pharmaceutics, School of Pharmacy, University of Washington, Seattle, Washington 98185, USA.
authors.forename: Xiaokun
authors.initials: X
authors.lastname: Cai
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Bikadi
authors.forename: Zhanglin
authors.initials: Z
authors.lastname: Ni
authors.forename: Eun-Woo
authors.initials: EW
authors.lastname: Lee
authors.forename: Honggang
authors.initials: H
authors.lastname: Wang
authors.forename: Mark F
authors.initials: MF
authors.lastname: Rosenberg
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 / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000317:Adrenergic alpha-Antagonists / Q000494:pharmacology; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001693:Biological Transport, Active; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D018613:Microscopy, Confocal; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D011224:Prazosin / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D014162:Transfection; D015262:Xenobiotics / Q000378:metabolism
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000317:Adrenergic alpha-Antagonists; D009363:Neoplasm Proteins; D004364:Pharmaceutical Preparations; D015262:Xenobiotics; D000251:Adenosine Triphosphatases; D011224:Prazosin