Affinity-based entrapment of the HER2 receptor in the endoplasmic reticulum using an affibody molecule.
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.
Vernet
Erik
E
Department of Molecular Biotechnology, Royal Institute of Technology, Albanova University Center, Roslagstullsbacken 21, SE-106 91 Stockholm, Sweden.
Konrad
Anna
A
Lundberg
Emma
E
Nygren
Per-Ake
PA
Gräslund
Torbjörn
T
D000595:Amino Acid Sequence; D049109:Cell Proliferation; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D008969:Molecular Sequence Data; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; C508053:ERBB2 protein, human