Document Detail


Affinity-based entrapment of the HER2 receptor in the endoplasmic reticulum using an affibody molecule.
MedLine Citation:
PMID:  18671978     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Erik Vernet; Anna Konrad; Emma Lundberg; Per-Ake Nygren; Torbjörn Gräslund
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2008-07-29
Journal Detail:
Title:  Journal of immunological methods     Volume:  338     ISSN:  0022-1759     ISO Abbreviation:  J. Immunol. Methods     Publication Date:  2008 Sep 
Date Detail:
Created Date:  2008-09-12     Completed Date:  2008-10-21     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  1305440     Medline TA:  J Immunol Methods     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  1-6     Citation Subset:  IM    
Affiliation:
Department of Molecular Biotechnology, Royal Institute of Technology, Albanova University Center, Roslagstullsbacken 21, SE-106 91 Stockholm, Sweden.
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MeSH Terms
Descriptor/Qualifier:
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:
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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