A cleavable N-terminal signal peptide promotes widespread olfactory receptor surface expression in HEK293T cells.

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

Identification and characterization of novel renal sensory receptors.

Recent studies have highlighted the important roles that "sensory" receptors (olfactory receptors, taste receptors, and orphan "GPR" receptors) play in a variety of tissues, including the kidney. Although several studies have identified important roles that individual sensory receptors play in the kidney, there has not been a systematic analysis of the renal repertoire of sensory receptors. In this study, we identify novel renal sensory receptors belonging to the GPR (n = 76), olfactory receptor (n = 6), and taste receptor (n = 11) gene families. A variety of reverse transcriptase (RT)-PCR screening strategies were used to identify novel renal sensory receptors, which were subsequently confirmed using gene-specific primers. The tissue-specific distribution of these receptors was determined, and the novel renal ORs were cloned from whole mouse kidney. Renal ORs that trafficked properly in vitro were screened for potential ligands using a dual-luciferase ligand screen, and novel ligands were identified for Olfr691. These studies demonstrate that multiple sensory receptors are expressed in the kidney beyond those previously identified. These results greatly expand the known repertoire of renal sensory receptors. Importantly, the mRNA of many of the receptors identified in this study are expressed highly in the kidney (comparable to well-known and extensively studied renal GPCRs), and in future studies it will be important to elucidate the roles that these novel renal receptors play in renal physiology

RT-PCR with mouse whole kidney cDNA as template to identify novel renal olfactory receptors.

<p>Olfr99 (A), Olfr545 (B), Olfr691 (C), Olfr693 (D), Olfr31(E) and Olfr1426(F) expression is detectible in mouse whole kidney cDNA by PCR and sequencing confirms the identity of amplified products. Mock RT template controls are negative for OR GSP sets and β-actin (not shown). The white arrow indicates the band of the expected size for each olfactory receptor.</p

The Lucy tag is a cleavable signal peptide.

<p>HA-Flag-Rho-Olfr691 (A and B) or HA-Lucy-Flag-Rho-Olfr691 (C and D) constructs were expressed in HEK293T cells along with RTP1S. Cells were fixed and stained with both an HA and Flag antibody (A and C) to detect total tagged OR or surface labeled with the HA and Flag antibodies (B and D) to detect surface-associated OR. HA surface stain is observed only in the absence of the Lucy tag, indicating a functional Lucy cleavage site.</p

Dose response curves for the novel Olfr691 ligands.

<p>Dose response curves show that Olfr691 has the highest affinity for valproate when co-expressed with RTP1S in HEK293T cells, with an EC₅₀ value of 0.4778 mM; however 4-pentenoate induced the strongest cAMP responses at all doses when compared to isobutyrate, valerate and valproate. NT represents measurements obtained from non-treated cells (with no ligand) transfected with Olfr691 and RTP1S.</p

The Lucy tag aids in surface expression of some ORs in the absence of accessory proteins or the Rho tag.

<p>ORs were cloned and expressed in HEK293T cells without Rho or Lucy tags (OR), with a Rho tag (Rho-OR), with the Lucy tag (Lucy-OR) or with both the Lucy and Rho tags (Lucy-Rho-OR). The cells were then surface labeled with a Flag antibody to detect membrane-associated OR. Images were taken for each OR at equal exposure for all conditions. To assess OR surface expression, the entirety of each coverslip was systematically scanned and scored based on detectable surface immunofluorescence. A ‘+’ was scored for those ORs whose surface expression was detectable in >90% of all fields of view while ORs received an ‘*’ if surface expression was found in <50% of all fields of view. A complete lack of detectable surface expression was scored as a ‘-‘. Results for 6 representative ORs are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068758#pone-0068758-g001" target="_blank">Figure 1</a> and the results for all ORs tested are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068758#tab1" target="_blank">Table 1</a>.</p

The Lucy tag increases OR protein levels.

<p>(A) Rho-tagged and Lucy-Rho-tagged ORs (L-OR) were immunoprecipated from HEK293T cells using monoclonal M2 Flag beads. Bound (B) lysates were immunoblotted with the Flag antibody to detect total OR levels. The arrow indicates the mature OR product at 39 kb. The input was also immunoblotted with the Flag antibody and then stripped and reprobed for β-actin to ensure equal loading. (B) An ELISA was performed for HEK293T cells expressing either Rho-tagged ORs or Lucy-Rho-tagged ORs to detect total OR levels using a monoclonal Flag antibody. Total protein levels are graphed as absorbance in arbitrary units. The dashed line indicates the background as measured by a non-transfected (NT) control. All measurements were performed in quadruplicate and the error bars indicate the SEM. An * represents significance as measured by the student T-test (Rho- OR vs. Lucy-OR) with P ≤ 0.005 and a + represents significance with a P ≤ 0.05. The Lucy tag increased total OR expression of ORs, as shown in both (A) and (B).</p

Relative response values at 0.5 mM for Olfr691 ligands.

<p>The structures of the ligands are shown in the figure for reference.</p

The Lucy tag does not alter OR signaling.

<p>(A) A luciferase reporter assay was performed for both Rho-Olfr691(R-691) and Lucy-Rho-Olfr691 (L-R-691) with and without RTP1S. Cells expressing the Olfr691 constructs were grown in a 96-well plate and exposed to the known Olfr691 ligand, isovaleric acid (0-5 mM). Error bars represent the SEM. By ANOVA and Student-Newman-Keuls, no concentrations of isovaleric acid activated R-691 (n/s = no significance). For R-691 + RTP1S, L-691, and L-691 + RTP1S, P ≤ 0.05 for 0 mM vs. all doses of isovaleric acid (marked by an *). In addition, for R-691 + RTP1S, P ≤ 0.05 for 5 vs 0.5, 0.25 and 0.1, 1 vs 0.25 and 0.1, 0.5 vs 0.1. For L-691, P ≤ 0.05 for 5 vs 0.1, 0.25 and 0.5. For L-691 + RTP1S, P ≤ 0.05 for 5 vs 0.5, 0.25 and 0.1, 1 vs 0.5, 0.25 and 0.1, 0.5 vs 0.1. (B) A luciferase reporter assay was performed for mOREG (EG), Rho-mOREG (R-EG), Lucy-mOREG (L-EG) and Lucy-Rho-mOREG (L-R-EG), all in the absence of RTP1S. Cells expressing the mOREG constructs were grown in a 96-well plate and exposed to the known mOREG ligand, eugenol (100-300 µM). Error bars represent the SEM. By ANOVA and Student-Newman-Keuls, all concentrations of eugenol significantly activated (P ≤ 0.05) eg, R-EG, L-EG and L-R-EG as compared to 0 µM (marked by an *). In addition, 100 and 300 µM eugenol were significant from each other (P ≤ 0.05) for both L-EG and L-R-EG. In both A and B, the Firefly: Renilla ratio was measured and compared to the non-treated control. An increase in the ratio indicates OR activation. Both Lucy-Rho-691 and Lucy-tagged mOREG constructs were activated with their ligands indicating that the Lucy tag does not alter OR signaling.</p

The Lucy tag works synergistically with accessory proteins and tags to promote surface expression of ORs.

<p>The Lucy tag works synergistically with accessory proteins and tags to promote surface expression of ORs.</p