title: Effect of cysteine mutagenesis on the function and disulfide bond formation of human ABCG2.
abstract: ABCG2 is a member of the ATP-binding cassette (ABC) transporter superfamily. Its overexpression causes multidrug resistance in cancer chemotherapy. Based on its apparent half size in sequence when compared with other traditional ABC transporters, ABCG2 has been thought to exist and function as a homodimer linked by intermolecular disulfide bonds. However, recent evidence suggests that ABCG2 may exist as a higher form of oligomers due to noncovalent interactions. In this study, we attempted to create a cysless mutant ABCG2 as a tool for further characterization of this molecule. However, we found that the cysless mutant ABCG2 is well expressed but not functional. Mapping of the cysteine residues showed that three cysteine residues (Cys284, Cys374, and Cys438) are required concurrently for the function of ABCG2 and potentially for intramolecular disulfide bond formation. We also found that the cysteine residues (Cys592, Cys603, and Cys608) in the third extracellular loop are involved in forming intermolecular disulfide bonds and that mutation of these residues does not affect the expression or drug transport activity of human ABCG2. Thus, we conclude that Cys284, Cys374, and Cys438, which may be involved in intramolecular disulfide bond formation, are concurrently required for ABCG2 function, whereas Cys592, Cys603, and Cys608, potentially involved in intermolecular disulfide bond formation, are not required.
authors.affiliation: Department of Pharmacology and Toxicology, IU Simon Cancer Center, Walther Oncology Center/Walther Cancer Institute, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
authors.forename: Yang
authors.initials: Y
authors.lastname: Liu
authors.forename: Youyun
authors.initials: Y
authors.lastname: Yang
authors.forename: Jing
authors.initials: J
authors.lastname: Qi
authors.forename: Hui
authors.initials: H
authors.lastname: Peng
authors.forename: Jian-Ting
authors.initials: JT
authors.lastname: Zhang
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D000818:Animals; D002460:Cell Line; D003545:Cysteine / Q000235:genetics*; D004220:Disulfides / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D011485:Protein Binding / Q000235:genetics; D018411:Spodoptera
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D004220:Disulfides; D009363:Neoplasm Proteins; D003545:Cysteine