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Mutational studies of G553 in TM5 of ABCG2: a residue potentially involved in dimerization
Orsolya Polgar1, Csilla Ozvegy-Laczka, Robert W Robey
1Medical Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Building 10, Room 13N240, Bethesda, Maryland 20892, USA.
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.
Insights
Glycine 553 in ABCG2 transporter is crucial for proper protein trafficking and cell surface expression. Mutations here impair function and suggest involvement in homodimerization for drug resistance.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- ABCG2 (ATP-binding cassette subfamily G member 2) is a transporter protein involved in multidrug resistance.
- It is known to function as a homodimer, with potential dimerization interfaces in transmembrane segments.
- A conserved region in transmembrane segment 5 (residues 552-554) is hypothesized to be involved in ABCG2 homodimerization.
Purpose of the Study:
- To investigate the role of glycine 553 in transmembrane segment 5 of ABCG2 in protein trafficking and function.
- To determine if mutations at this residue affect ABCG2 homodimerization and its ability to transport substrates.
Main Methods:
- Site-directed mutagenesis was used to create glycine 553 to leucine (G553L) and glycine 553 to glutamate (G553E) mutants of ABCG2.
- Mutant proteins were expressed in HEK 293 cells and Sf9 insect cells.
- Techniques included immunoblotting, N-linked glycosylation analysis, confocal microscopy, amine-reactive cross-linking, and Hoechst dye transport assays.
Main Results:
- The G553L and G553E mutants showed significantly reduced protein expression and were not detected on the cell surface in HEK 293 cells.
- Mutants exhibited reduced molecular mass, suggesting impaired N-linked glycosylation, and were primarily localized in the endoplasmic reticulum.
- Cross-linking experiments indicated that mutant monomers are in proximity but fail to traffic correctly.
- In Sf9 cells, G553L localized to the membrane but lost ATP hydrolysis and Hoechst transport activity, and interfered with wild-type ABCG2 function when coexpressed.
Conclusions:
- Glycine 553 is essential for the proper trafficking and cell surface localization of ABCG2.
- While not definitively proven, the data strongly suggest that glycine 553 plays a role in ABCG2 homodimerization and functional transport.
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