Mutational studies of G553 in TM5 of ABCG2

A residue potentially involved in dimerization

Orsolya Polgar, C. Özvegy-Laczka, Robert W. Robey, Kuniaki Morisaki, Masaki Okada, Akina Tamaki, Gabriella Koblos, N. Elkind, Yvona Ward, Michael Dean, B. Sarkadi, Susan E. Bates

Research output: Contribution to journalArticle

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Abstract

ABCG2 is an ATP-binding cassette half-transporter conferring resistance to chemotherapeutic agents 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) are thought to disrupt heterodimerization. We substituted glycine 553 with leucine (G553L) followed by stable transfection in HEK 293 cells. The mutant was not detectable on the cell surface, and markedly reduced protein expression levels were observed by immunoblotting. A deficiency in N-linked glycosylation was 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 ER localization 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-reactive cross-linkers with multiple arm lengths, suggesting that the monomers are in the proximity of each other but are unable to complete normal trafficking. Interestingly, when expressed in Sf9 insect cells, G553L moves to the cell membrane but is unable to hydrolyze ATP or transport the Hoechst dye. Still, when coexpressed, the mutant interferes with the Hoechst transport activity of the wild-type protein. These data show that glycine 553 is important for protein trafficking and are consistent with, but do not yet prove, its involvement in ABCG2 homodimerization.

Original languageEnglish
Pages (from-to)5251-5260
Number of pages10
JournalBiochemistry
Volume45
Issue number16
DOIs
Publication statusPublished - Apr 25 2006

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Dimerization
Glycine
Leucine
irinotecan
alvocidib
Adenosine Triphosphate
HEK293 Cells
Proteins
Glycosylation
Sf9 Cells
Mitoxantrone
ATP-Binding Cassette Transporters
Confocal microscopy
Molecular mass
Protein Transport
Cell membranes
Immunoblotting
Confocal Microscopy
Amines
Transfection

ASJC Scopus subject areas

  • Biochemistry

Cite this

Polgar, O., Özvegy-Laczka, C., Robey, R. W., Morisaki, K., Okada, M., Tamaki, A., ... Bates, S. E. (2006). Mutational studies of G553 in TM5 of ABCG2: A residue potentially involved in dimerization. Biochemistry, 45(16), 5251-5260. https://doi.org/10.1021/bi0521590

Mutational studies of G553 in TM5 of ABCG2 : A residue potentially involved in dimerization. / Polgar, Orsolya; Özvegy-Laczka, C.; Robey, Robert W.; Morisaki, Kuniaki; Okada, Masaki; Tamaki, Akina; Koblos, Gabriella; Elkind, N.; Ward, Yvona; Dean, Michael; Sarkadi, B.; Bates, Susan E.

In: Biochemistry, Vol. 45, No. 16, 25.04.2006, p. 5251-5260.

Research output: Contribution to journalArticle

Polgar, O, Özvegy-Laczka, C, Robey, RW, Morisaki, K, Okada, M, Tamaki, A, Koblos, G, Elkind, N, Ward, Y, Dean, M, Sarkadi, B & Bates, SE 2006, 'Mutational studies of G553 in TM5 of ABCG2: A residue potentially involved in dimerization', Biochemistry, vol. 45, no. 16, pp. 5251-5260. https://doi.org/10.1021/bi0521590
Polgar, Orsolya ; Özvegy-Laczka, C. ; Robey, Robert W. ; Morisaki, Kuniaki ; Okada, Masaki ; Tamaki, Akina ; Koblos, Gabriella ; Elkind, N. ; Ward, Yvona ; Dean, Michael ; Sarkadi, B. ; Bates, Susan E. / Mutational studies of G553 in TM5 of ABCG2 : A residue potentially involved in dimerization. In: Biochemistry. 2006 ; Vol. 45, No. 16. pp. 5251-5260.
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AU - Özvegy-Laczka, C.

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AU - Morisaki, Kuniaki

AU - Okada, Masaki

AU - Tamaki, Akina

AU - Koblos, Gabriella

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AU - Ward, Yvona

AU - Dean, Michael

AU - Sarkadi, B.

AU - Bates, Susan E.

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