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Absence of N-linked glycosylation does not affect plasma membrane localization of breast cancer resistance protein (BCRP/ABCG2)

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Abstract

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.

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Abbreviations

ABC:

ATP-binding cassette

BCRP:

Breast cancer resistance protein

CHO:

Chinese hamster ovary

ERM:

Ezrin-Radixin-Moesin

MDCK:

Madin-Darby canine kidney

MDR:

Multidrug resistance

MRP:

Multidrug resistance-associated protein

PCR:

Polymerase chain reaction

PDZ:

PSD-95-Dlh-ZO1

PKC:

Protein kinase C

TAP:

Transporter associated with antigen processing

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Acknowledgements

This work was supported by the Dutch Cancer Society (grants NKI 1999-2060 and NKI 2000-2143)

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Authors and Affiliations

  1. Division of Experimental Therapy, Netherlands Cancer Institute, Amsterdam, 1066 CX, The Netherlands

    Karin Mohrmann, Maria A. J. van Eijndhoven, Alfred H. Schinkel & Jan H. M. Schellens

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  1. Karin Mohrmann
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  2. Maria A. J. van Eijndhoven
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  3. Alfred H. Schinkel
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  4. Jan H. M. Schellens
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Correspondence to Jan H. M. Schellens.

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Mohrmann, K., van Eijndhoven, M.A.J., Schinkel, A.H. et al. Absence of N-linked glycosylation does not affect plasma membrane localization of breast cancer resistance protein (BCRP/ABCG2). Cancer Chemother Pharmacol 56, 344–350 (2005). https://doi.org/10.1007/s00280-005-1004-5

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