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Abstract
Recent studies of vascular adhesion protein-1 (VAP-1) have greatly advanced our understanding of the important role this protein plays in the establishment and progression of inflammatory disease. To facilitate more detailed studies on the function of VAP-1, we developed a GFP-fusion protein that enabled us to monitor the trafficking of the protein in three selected cell types: hepatic sinusoidal endothelial cells, liver myofibroblasts and an hepatic stellate cell line (LX-2). The fusion protein was detected as punctate cytoplasmic GFP staining, but was present only at low levels at the cell surface in all cell types studied. The subcellular distribution of the protein was not altered in a catalytically inactive mutant form of the protein (Tyr471Phe) or in the presence of exogenous VAP-1 substrate (methylamine) or inhibitor (semicarbazide). The GFP-VAP-1 protein was localized to the Golgi apparatus (GM-130), endoplasmic reticulum (GRP94) and early endosomes (EEA-1). Additional staining for VAP-1 revealed that the overexpressed protein was also present in vesicles that were negative for GFP fluorescent signal and did not express EEA-1. We propose that these vesicles are responsible for recycling the fusion protein and that the fluorescence of the GFP moiety is quenched at the low pH within these vesicles. This feature of the protein makes it well suited for live cell imaging studies where we wish to track protein that is being actively trafficked within the cell in preference to that which is being recycled.
Original language | English |
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Pages (from-to) | 951-61 |
Number of pages | 11 |
Journal | Journal of Neural Transmission |
Volume | 120 |
Issue number | 6 |
Early online date | 9 Mar 2013 |
DOIs | |
Publication status | Published - Jun 2013 |
Keywords
- Vascular adhesion protein-1
- Amine oxidase
- Trafficking
- Imaging
- Liver
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Dive into the research topics of 'Cellular localization and trafficking of vascular adhesion protein-1 as revealed by an N-terminal GFP fusion protein'. Together they form a unique fingerprint.Projects
- 1 Finished
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Lymphocyte Ligands for the Endothelial Adhesion Molecule Vascular Adhesion Protein-1
Adams, D. (Principal Investigator) & Wakelam, M. (Co-Investigator)
15/01/04 → 31/03/07
Project: Research Councils