title: A cleavable N-terminal signal peptide promotes widespread olfactory receptor surface expression in HEK293T cells.
abstract: 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.
authors.affiliation: Department of Physiology, Johns Hopkins University School of Medicine, Baltimore MD, USA.
authors.forename: Blythe D
authors.initials: BD
authors.lastname: Shepard
authors.forename: Niranjana
authors.initials: N
authors.lastname: Natarajan
authors.forename: Ryan J
authors.initials: RJ
authors.lastname: Protzko
authors.forename: Omar W
authors.initials: OW
authors.lastname: Acres
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Pluznick
mesh_terms: D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism*; D003001:Cloning, Molecular; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals; D021381:Protein Transport; D018035:Receptors, Odorant / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012243:Rhodopsin / Q000235:genetics
chemical_list: D021382:Protein Sorting Signals; D018035:Receptors, Odorant; D012243:Rhodopsin