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 Gαolf) 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 Gαolf), 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.

Defining an olfactory receptor function in airway smooth muscle cells

“…1d, Flag-tagged full-length constructs encoding OR1J1, OR6A2 and OR51E2 were able to traffic to the cell surface under certain conditions; surface expression of OR51E2 was the most pronounced among the three ORs. In fact, OR51E2 incorporated into the plasma membrane without the aid of an N-terminal leucine-rich cleavable signal peptide (Lucy tag, as previously described34), or co-expression of RTP1S and Ric8b chaperones (Fig. 1d).…”

“…ORs identified in ASM by degenerate primer PCR were cloned into the PME18S mammalian expression vector containing N terminal Lucy, Flag and Rho tags as previously described34. These constructs were then transfected into HEK 293T cells with and without the chaperone proteins RTP1S and Ric8b.…”

How does your kidney smell? Emerging roles for olfactory receptors in renal function

“…Olfr78 in the afferent arteriole [64]), these receptors clearly must be signaling through a different G protein. Consistent with this, ORs in vitro can also couple to G s (or, under specific conditions, G q ) [31; 32; 39; 60; 64; 65]. In addition, a handful of ORs have been found to be expressed in the vomeronasal organ, the site of pheromone sensing in the mouse, where they are also thought to couple to other G proteins [66; 67].…”

“…A second possible approach to screening ORs is to use a variety of aids to help the OR achieve surface expression when exogenously expressed in vitro . A number of studies have identified either chaperones or N-terminal tags which aid in OR surface expression [39–53], and when successful this allows for a high-throughput approach focused on a specific OR.…”

“…As mentioned above, functional heterologous expression must be achieved before ligand screening can occur in vitro . To date, with the help of various tags and chaperone proteins, 9 different renal murine ORs have been found to traffic to the cell surface in HEK293T cells [39; 60]. Ligand screens have been performed on these ORs, leading to the identification of several different OR-ligand pairs.…”