Figure 1.
The Lucy tag aids in surface expression of some ORs in the absence of accessory proteins or the Rho tag.
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 Figure 1 and the results for all ORs tested are summarized in Table 1.
Figure 2.
The Lucy tag works synergistically with accessory proteins and tags to promote surface expression of ORs.
Figure 3.
The Lucy tag is a cleavable signal peptide.
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.
Figure 4.
The Lucy tag increases OR protein levels.
(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).
Figure 5.
The Lucy tag does not alter OR signaling.
(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.