PMID: 16528971Mar 15, 2006Paper

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

Journal of Experimental Therapeutics & Oncology
Kanako WakabayashiToshihisa Ishikawa

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-I...Continue Reading

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