title: Identification of cysteine residues critically involved in homodimer formation and protein expression of human ATP-binding cassette transporter ABCG2: a new approach using the flp recombinase system.
abstract: Since ABCG2 is a half-transporter in the ATP-binding cassette (ABC) transporter family, it has been suspected that ABCG2 functions as a homodimer. In the present study, we have investigated the molecular mechanism underlying homodimer formation of ABCG2. Based on the amino acid sequence of ABCG2, three cysteine residues (Cys592, Cys603, and Cys608) are expected to exist in the extracellular loop. To identify a cysteine residue(s) required for homodimer formation, we have substituted those cysteine residues to glycine by site-directed mutagenesis and stably expressed the resulting variants in Flp-In-293 cells. Substitution of the amino acid at position 603 from cysteine to glycine (C603G) completely diminished homodimer formation, whereas substitution of both Cys592 and Cys608 to glycine residues (C592G/C608G) had no effect on homodimer formation. These results strongly suggest that Cys603 is prerequisite for homodimer formation of ABCG2 via a disulfide bond. On the other hand, immunohistochemistry experiments revealed that the C592G/C608G variant is mainly located in intracellular compartments. The C592G/C608G variant exhibited lower activity of ATP-dependent methotrexate (MTX) transport, and its expression did not confer Flp-In-293 cells resistance to SN-38 or mitoxantrone. Cys592 and Cys608 appear to be important for intracellular sorting of the de novo synthesized ABCG2 protein to the plasma membrane. Taken together, cysteine residues in the extra-cellular loop are considered to play pivotal roles in homodimer formation and plasma membrane localization of ABCG2.
authors.affiliation: Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, 226-8501, Japan.
authors.forename: Kanako
authors.initials: K
authors.lastname: Wakabayashi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nakagawa
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Adachi
authors.forename: Isao
authors.initials: I
authors.lastname: Kii
authors.forename: Eiry
authors.initials: E
authors.lastname: Kobatake
authors.forename: Akira
authors.initials: A
authors.lastname: Kudo
authors.forename: Toshihisa
authors.initials: T
authors.lastname: Ishikawa
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution; D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D009363:Neoplasm Proteins; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D003545:Cysteine