| Affinity-based entrapment of the HER2 receptor in the endoplasmic reticulum using an affibody molecule. | |
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MedLine Citation:
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PMID: 18671978 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Interference with the export of cell surface receptors can be performed through co-expression of specific affinity molecules designed for entrapment in the endoplasmic reticulum during the export process. We describe the investigation of a small (6 kDa) non-immunoglobulin-based HER2 receptor binding affibody molecule (Z(HER2:00477)), for use in affinity mediated entrapment of the HER2 receptor in the ER. Constructs encoding Z(HER2:00477) or a control affibody protein, with or without ER-retention peptide extensions (KDEL), were expressed in the HER2 over-expressing cell line SKOV-3. Intracellular expression of the full-length affibody constructs could be confirmed by probing cell extracts by Western blotting. Confocal immunofluorescence microscopy experiments showed extensive co-localization of the HER2 receptor and Z(HER2:00477)-KDEL in the ER, whereas the use of a KDEL-extended control affibody molecule resulted in distinct and separate signals from cell surface-localized HER2 receptor and ER-localized affibody protein. This indicated a capability of the Z(HER2:00477)-KDEL fusion protein to functionally interfere with the export process of HER2 receptor in a specific manner. Using flow cytometry and cell proliferation analyses, it could be shown that expression of the Z(HER2:00477)-KDEL fusion construct in the SKOV-3 cell line resulted both in a marked reduction in cell surface level of HER2 receptors and that the cell population doubling time was significantly increased. Expression of the Z(HER2:00477)-KDEL fusion protein in additional cell lines of different origin and with different expression levels of endogenous HER2 receptor compared to SKOV-3, also resulted in depletion of the cell surface levels of HER2 receptor. This indicated upon a general ability of the Z(HER2:00477)-KDEL fusion protein to functionally interfere with the export process of HER2. |
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Authors:
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Erik Vernet; Anna Konrad; Emma Lundberg; Per-Ake Nygren; Torbjörn Gräslund |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2008-07-29 |
Journal Detail:
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Title: Journal of immunological methods Volume: 338 ISSN: 0022-1759 ISO Abbreviation: J. Immunol. Methods Publication Date: 2008 Sep |
Date Detail:
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Created Date: 2008-09-12 Completed Date: 2008-10-21 Revised Date: 2009-11-19 |
Medline Journal Info:
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Nlm Unique ID: 1305440 Medline TA: J Immunol Methods Country: Netherlands |
Other Details:
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Languages: eng Pagination: 1-6 Citation Subset: IM |
Affiliation:
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Department of Molecular Biotechnology, Royal Institute of Technology, Albanova University Center, Roslagstullsbacken 21, SE-106 91 Stockholm, Sweden. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Amino Acid Sequence Cell Proliferation Cells, Cultured Endoplasmic Reticulum / metabolism* Flow Cytometry Humans Molecular Sequence Data Receptor, erbB-2 / analysis, metabolism* Recombinant Fusion Proteins / metabolism |
| Chemical | |
Reg. No./Substance:
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0/Recombinant Fusion Proteins; EC 2.7.10.1/ERBB2 protein, human; EC 2.7.10.1/Receptor, erbB-2 |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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