Mutational Studies of G553 in TM5 of ABCG2: A Residue Potentially Involved in Dimerization
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- 作者：Orsolya Polgar ; Csilla Ozvegy-Laczka ; Robert W. Robey ; Kuniaki Morisaki ; Masaki Okada ; Akina Tamaki ; Gabriella Koblos ; N. Barry Elkind ; Yvona Ward ; Michael Dean ; Balazs Sarkadi ; and Susan E. Bates
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：April 25, 2006
- 年：2006
- 卷：45
- 期：16
- 页码：5251 - 5260
- 全文大小：919K
- 年卷期：v.45,no.16(April 25, 2006)
- ISSN：1520-4995
文摘
ABCG2 is an ATP-binding cassette half-transporter conferring resistance to chemotherapeuticagents such as mitoxantrone, irinotecan, and flavopiridol. With its one transmembrane and one ATP-binding domain, ABCG2 is thought to homodimerize for function. One conserved region potentially involved in dimerization is a three-amino acid sequence in transmembrane segment 5 (residues 552-554).Mutations in the corresponding residues in the Drosophila white protein (an orthologue of ABCG2) arethought to disrupt heterodimerization. We substituted glycine 553 with leucine (G553L) followed bystable transfection in HEK 293 cells. The mutant was not detectable on the cell surface, and markedlyreduced protein expression levels were observed by immunoblotting. A deficiency in N-linked glycosylationwas suggested by a reduction in molecular mass compared to that of the 72 kDa wild-type ABCG2.Similar results were observed with the G553E mutant. Confocal microscopy demonstrated mostly ERlocalization of the G553L mutant in HEK 293 cells, even when coexpressed with the wild-type protein.Despite its altered localization, the G553L and G553E mutants were cross-linked using amine-reactivecross-linkers with multiple arm lengths, suggesting that the monomers are in the proximity of each otherbut are unable to complete normal trafficking. Interestingly, when expressed in Sf9 insect cells, G553Lmoves to the cell membrane but is unable to hydrolyze ATP or transport the Hoechst dye. Still, whencoexpressed, the mutant interferes with the Hoechst transport activity of the wild-type protein. These datashow that glycine 553 is important for protein trafficking and are consistent with, but do not yet prove,its involvement in ABCG2 homodimerization.