ABCMdb

ABCG2 p.Asn557Ala

Predicted by SNAP2:	A: D (91%), C: D (91%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (95%), I: D (91%), K: D (95%), L: D (91%), M: D (91%), P: D (95%), Q: D (95%), R: D (95%), S: D (95%), T: D (91%), V: D (91%), W: D (95%), Y: D (91%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: N, I: D, K: D, L: D, M: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: N,

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Publications
[hide] Absence of N-linked glycosylation does not affect ... Cancer Chemother Pharmacol. 2005 Oct;56(4):344-50. Epub 2005 May 5. Mohrmann K, van Eijndhoven MA, Schinkel AH, Schellens JH
Absence of N-linked glycosylation does not affect plasma membrane localization of breast cancer resistance protein (BCRP/ABCG2).
Cancer Chemother Pharmacol. 2005 Oct;56(4):344-50. Epub 2005 May 5., [PMID:15875186]

Abstract [show]
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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No. Sentence Comment
8 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. Login to comment
45 BCRP-N418A, BCRP-N557A and BCRP-N596A were made using pcDNA3/BCRP wild type (GenBank accession number AY017168; generously provided by Susan Bates) as template. Login to comment
46 BCRP-N418A/ N557A was made using overlap extension PCR with pGEM-T Easy/BCRP-N418A as template and BCRP-N418A/N557A/N596A was made with pGEM-T Easy/ BCRP-N418A/N557A as template again using overlap extension PCR. Login to comment
106 In BCRP-N557A transfectants, only the lower band was detected. As this band was PNGase F sensitive, BCRP-N557A was still glycosylated. Login to comment
130 These results suggest that both BCRP-N557A and the triple mutant were not properly targeted to the plasma membrane as a consequence of the N557A mutation. Login to comment
156 Empty vector pcDNA3 or pcDNA3 with BCRP or mutant BCRP, BCRP-N418A, BCRP-N557A and BCRP-N596A, was transiently transfected into CHO9 cells or MDCKII cells using FuGENE6. Login to comment
159 N non-glycosylated C core-glycosylated G fully glycosylated In our experiments, BCRP-N557A was apparently core-glycosylated and localized in the ER. Login to comment
169 We cannot exclude that in our study also a small fraction of BCRP-N557A reaches the plasma membrane. Login to comment
187 BCRP-N557A and a triple mutant is localized intracellularly. Login to comment
188 Empty vector pcDNA3 or pcDNA3 with BCRP or mutant BCRP, BCRP-N418A, BCRP-N557A and BCRP-N596A, was transiently transfected into CHO9 cells or MDCKII cells using FuGENE6. Login to comment

[hide] Intracellular trafficking of MDR transporters and ... Curr Top Med Chem. 2009;9(2):197-208. Porcelli L, Lemos C, Peters GJ, Paradiso A, Azzariti A
Intracellular trafficking of MDR transporters and relevance of SNPs.
Curr Top Med Chem. 2009;9(2):197-208., [PMID:19200005]

Abstract [show]
Multi-drug resistance (MDR) frequently contributes to the failure of chemotherapeutic treatments in cancer patients. Mechanisms underlying the development of MDR have been extensively studied and are considered multifactorial. Among them, the ATP-Binding Cassette (ABC) family of proteins plays a pivotal role. Processes of cellular distribution and subcellular localization of MDR-ABC proteins are not yet well explored and to enlighten these topics could be crucial to understand cellular drug uptake and retention. In this review, we analysed literature data concerning i) intracellular trafficking of MDR-ABC proteins (BCRP, P-gp and MRP1) and ii) mechanisms altering their cellular localization and trafficking. Moreover, we describe single nucleotide polymorphisms (SNP) that have been reported for some multidrug resistance (MDR) transporters, such as BCRP and P-gp, emphasizing their ability to affect the expression, function and localization of the transporters, with implications on drug resistance phenotypes.

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54 Diop & Hrycyna [30] indicated that glycosylation is not required for transporter function or localization of ABCG2 in the plasma membrane, conversely, Mohrmann suggested that the intracellular localization of BCRP-N557A could disrupt proper routing of the transporter [28]. Login to comment