Absence of N-linked glycosylation does not affect plasma membrane localization of breast cancer resistance protein (BCRP/ABCG2)
| Autor: | Karin Mohrmann, Maria A.J. van Eijndhoven, Jan H.M. Schellens, Alfred H. Schinkel |
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| Rok vydání: | 2005 |
| Předmět: |
Cancer Research
Glycosylation Abcg2 Mutant ATP-binding cassette transporter CHO Cells Transfection Toxicology chemistry.chemical_compound Cricetulus Dogs N-linked glycosylation Cricetinae ATP Binding Cassette Transporter Subfamily G Member 2 Animals Humans Pharmacology (medical) Asparagine Phosphorylation Pharmacology biology Multidrug resistance-associated protein 2 Cell Membrane Subcellular localization Molecular biology Neoplasm Proteins Oncology chemistry biology.protein ATP-Binding Cassette Transporters |
| Zdroj: | Cancer Chemotherapy and Pharmacology. 56:344-350 |
| ISSN: | 1432-0843 0344-5704 |
| Popis: | Breast cancer resistance protein (BCRP/ABCG2) is an ATP-binding cassette (ABC) multidrug transporter that confers resistance to various anticancer drugs like topotecan and mitoxantrone. To obtain more insight in its cellular functioning, we investigated phosphorylation and N-linked glycosylation of BCRP. In the epithelial Madin-Darby canine kidney (MDCK) cell line, we did not detect phosphorylation of BCRP, in contrast to MRP2, which was phosphorylated. In the ovarian carcinoma cell line T8 also no phosphorylated BCRP was detected. As BCRP in both lines effectively transports drugs, it appears that phosphorylation of BCRP (if it occurs at all) is not needed for drug transport. We further mutated the asparagine residues 418, 557 and 596 in three putative N-linked glycosylation motifs of BCRP to alanines. Mutant proteins were expressed in CHO9 and MDCKII cells by transient transfection and characterized by Western blot and immunofluorescence analysis. We found that only BCRP-N596A and a mutant with all three asparagines mutated (triple mutant) were not glycosylated anymore, indicating that only asparagine 596 is normally glycosylated. The mutation of asparagine 596 (or 418) had little effect on the subcellular localization of BCRP, indicating that N-linked glycosylation is not essential for routing to the plasma membrane. However, BCRP-N557A and the triple mutant were mainly localized intracellularly, probably in the endoplasmic reticulum, suggesting that this mutation disrupted proper routing of BCRP. |
| Databáze: | OpenAIRE |
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