Functional analysis of human olfactory receptors with a high basal activity using LNCaP cell line

Abstract

Humans use a family of more than 400 olfactory receptors (ORs) to detect odorants. However, deorphanization of ORs is a critical issue because the functional properties of more than 80% of ORs remain unknown, thus, hampering our understanding of the relationship between receptor function and perception. HEK293 cells are the most commonly used heterologous expression system to determine the function of a given OR; however, they cannot functionally express a majority of ORs probably due to a lack of factor(s) required in cells in which ORs function endogenously. Interestingly, ORs have been known to be expressed in a variety of cells outside the nose and play critical physiological roles. These findings prompted us to test the capacity of cells to functionally express a specific repertoire of ORs. In this study, we selected three cell lines that endogenously express functional ORs. We demonstrated that human prostate carcinoma (LNCaP) cell lines successfully identified novel ligands for ORs that were not recognized when expressed in HEK293 cells. Further experiments suggested that the LNCaP cell line was effective for functional expression of ORs, especially with a high basal activity, which impeded the sensitive detection of ligand-mediated activity of ORs. This report provides an efficient functional assay system for a specific repertoire of ORs that cannot be characterized in current cell systems.

Fig 1. HEK293 cell-based screening of human ORs for six perfumery raw materials.

A. Primary screening of human ORs using 384-well plates. 378 ORs were expressed in HEK293 cells, and they were stimulated with a single concentration of each odorant. HEK293 cells were co-transfected with each OR, CRE/luc2PpGL4.29, pRL-CMV, and RTP1S. Odorant solutions were diluted in the growth medium without FBS. X-axis lists each of the 378 ORs, and color bars represent OR families. Y-axis indicates the response of cells expressing each OR as fold increase where a signal from stimulated cells was divided by that from non-stimulated cells expressing the same OR (mean values from two screening replicates). B. Verification of dose-dependent responsiveness of cell-expressing candidate ORs using 96-well plates. Each OR was co-transfected with CRE/luc2PpGL4.29, pRL-CMV, and RTP1S. 32 pairs of ORs and their agonists that meet our statistical criteria for dose-dependent responses are shown. The grey-shaded ORs do not fit a sigmoidal curve because their responses were not saturated in the range of concentrations tested due to less sensitivity. Y-axis indicates the fold increase value, and data are shown as mean ± SE from three independent experiments.

Fig 2. Capacity of three cell lines as a functional expression cell system for ORs.

A total of 412 ORs were expressed in different cell lines. Each OR was co-transfected with CRE/luc2PpGL4.29, pRL-CMV, and RTP1S in HEK293 cells whereas Gαolf was also co-transfected in the other three cell lines. They were stimulated with a mixture of six odorants in the growth medium without FBS. The X-axis lists each of the 412 ORs, and color bars represent OR families. Y-axis indicates the response of cells expressing each OR as a fold increase. Data are presented as the mean values from two screening replicates. The dashed line denotes mean + 2SD of all the examined ORs except for those validated in HEK293 cells. The name of the receptor is described if the response value in the follow-up dose-response analysis meets our criteria (S3–S6 Figs).

Fig 3. Activations of specific ORs were selectively monitored in LNCaP cells.

Dose-response curves of OR1A1 (top, black), OR51T1 (middle, magenta), and TAAR1 (bottom, blue) expressed in HEK293 (left) or LNCaP cells (right) against increasing concentrations of the odorant mixture. Each OR was co-transfected with CRE/luc2PpGL4.29, pRL-CMV, RTP1S and Gαolf. X-axis indicates final concentrations of each of the six components of the mixture. Ringer’s solution was used to dissolve the odorant mixture. Assays were also conducted on mock-transfected cells transfected with empty vector, CRE/luc2PpGL4.29, pRL-CMV, RTP1S, and Gαolf. (white). Y-axis indicates the fold increase in response. Data are shown as mean ± SE of three independent experiments.

Fig 4. Identification of novel pairs of ligands and ORs in LNCaP cells.

Dose-response curves of OR51T1 (A, magenta) and TAAR1 (B, blue) expressed in HEK293 (left) or LNCaP cells (right) against increasing concentrations of each odorant. Each OR in both cell lines was co-transfected with CRE/luc2PpGL4.29, pRL-CMV, RTP1S and Gαolf. Ringer’s solution was used to dissolve the odorant mixture. Y-axis indicates the fold increase in response. Data are shown as mean ± SE of three independent experiments.

Fig 5. Capacity of HEK293 and LNCaP cells for membrane trafficking of nascent OR proteins and for controlling background intracellular signal from non-stimulated ORs.

A. Immunohistochemical analysis (live-cell staining) of HEK293 and LNCaP cells expressing OR51T1 or TAAR1. M3AChR was tested as a positive control of GPCRs for efficient cell-surface expression. All three tested receptors were expressed as fused proteins with an N-terminal FLAG-tag, and they were detected using an anti-FLAG antibody. Scale, 100 μm. B, C. Difference in background signal levels from constitutive activity of ORs in HEK293 (green) and LNCaP (purple) cells. Each type of cell was transfected with CRE/luc2PpGL4.29, pRL-CMV, RTP1S and Gαolf. X-axis indicates transfected ORs. After 24 h of transfection, cells were stimulated with growth medium without odorants, and luciferase activity was measured. Data are presented as CRE-Luc ratio (luminescence intensity of firefly luciferase divided by the luminescence intensity of Renilla luciferase) (B) or %forskolin (C) where CRE-Luc ratio in each transfection condition is presented as normalized values and CRE-Luc ratio in response to 10 μM forskolin was set to 100%. D. Dose-response of HEK293 cells and LNCaP cells to forskolin. Each type of cell was transfected with an empty vector, CRE/luc2PpGL4.29, and pRL-CMV. E. Background signal levels of 412 ORs expressed in HEK293 cells are shown as CRE-Luc ratios. HEK293 cells were transfected with CRE/luc2PpGL4.29, pRL-CMV, and RTP1S. Color bars represent OR families. F. IBMX (0.1 mM) treatment increased OR51T1-mediated basal signal. G. IBMX (0.1 mM) treatment enhanced 10 μM forskolin-mediated responses more robustly in mock-transfected LNCaP cell than in HEK293 cells. H, I. Correlation analysis of response amplitudes of 40 ORs to the odorant mixture in HEK293 cells or LNCaP cells. The 40 ORs were those selected to have response amplitudes above mean + 2SD from all examined ORs in each cell line of the primary screening (Fig 2). X- and Y-axis indicates log2 of response amplitude (fold increase) in each cell line. The Pearson’s correlation coefficient (r) and its p-value are displayed on top of each panel. J. Co-expression of PDE1C (20 ng/well) decreases OR51T1-derived basal signal in HEK293 cells. K. Co-expression of PDE1C decreases 10 μM forskolin-mediated responses drastically in mock-transfected HEK293 cells than in LNCaP cells. L. Co-transfection of different amounts of plasmid coding PDE1C failed to elicit an l-carvone-mediated response in OR51T1-expressing cells as compared to that in mock-transfected control. Data are shown as mean ± SE of three to four independent experiments (B-G, and J-L).