A cleavable N-terminal signal peptide promotes widespread olfactory receptor surface expression in HEK293T cells
| Autor: | Jennifer L. Pluznick, Niranjana Natarajan, Blythe D. Shepard, Ryan J. Protzko, Omar W. Acres |
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| Jazyk: | angličtina |
| Rok vydání: | 2013 |
| Předmět: |
Signal peptide
Rhodopsin Molecular Sequence Data Gene Expression lcsh:Medicine Protein Sorting Signals Biology Receptors Odorant 03 medical and health sciences 0302 clinical medicine FLAG-tag medicine Humans Amino Acid Sequence Cloning Molecular Receptor lcsh:Science Peptide sequence 030304 developmental biology G protein-coupled receptor 0303 health sciences Multidisciplinary Olfactory receptor Cell Membrane HEK 293 cells lcsh:R Protein Transport HEK293 Cells medicine.anatomical_structure Biochemistry lcsh:Q Olfactory epithelium 030217 neurology & neurosurgery Research Article |
| Zdroj: | PLoS ONE, Vol 8, Iss 7, p e68758 (2013) PLoS ONE |
| ISSN: | 1932-6203 |
| Popis: | Olfactory receptors (ORs) are G protein-coupled receptors that detect odorants in the olfactory epithelium, and comprise the largest gene family in the genome. Identification of OR ligands typically requires OR surface expression in heterologous cells; however, ORs rarely traffic to the cell surface when exogenously expressed. Therefore, most ORs are orphan receptors with no known ligands. To date, studies have utilized non-cleavable rhodopsin (Rho) tags and/or chaperones (i.e. Receptor Transporting Protein, RTP1S, Ric8b and G(αolf)) to improve surface expression. However, even with these tools, many ORs still fail to reach the cell surface. We used a test set of fifteen ORs to examine the effect of a cleavable leucine-rich signal peptide sequence (Lucy tag) on OR surface expression in HEK293T cells. We report here that the addition of the Lucy tag to the N-terminus increases the number of ORs reaching the cell surface to 7 of the 15 ORs (as compared to 3/15 without Rho or Lucy tags). Moreover, when ORs tagged with both Lucy and Rho were co-expressed with previously reported chaperones (RTP1S, Ric8b and G(αolf)), we observed surface expression for all 15 receptors examined. In fact, two-thirds of Lucy-tagged ORs are able to reach the cell surface synergistically with chaperones even when the Rho tag is removed (10/15 ORs), allowing for the potential assessment of OR function with only an 8-amino acid Flag tag on the mature protein. As expected for a signal peptide, the Lucy tag was cleaved from the mature protein and did not alter OR-ligand binding and signaling. Our studies demonstrate that widespread surface expression of ORs can be achieved in HEK293T cells, providing promise for future large-scale deorphanization studies. |
| Databáze: | OpenAIRE |
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