A bi-functional IL-6-HaloTag® as a tool to measure the cell-surface expression of recombinant odorant receptors and to facilitate their activity quantification

The functional cell surface expression of recombinant odorant receptors typically has been investigated by expressing N-terminally extended, “tagged” receptors in test cell systems, using antibody-based immunocytochemistry or flow cytometry, and by measuring odorant/receptor-induced cAMP signaling, mostly by an odorant/receptor-induced and cAMP signaling-dependent transcriptional activation of a luciferase-based luminescence assay. In the present protocol, we explain a method to measure the cell-surface expression and signaling of recombinant odorant receptors carrying a bi-functional, N-terminal ‘IL-6-HaloTag®’. IL-6, being a secreted cytokine, facilitates functional cell surface expression of recombinant HaloTag®-odorant receptors, and the HaloTag® protein serves as a highly specific acceptor for cell-impermeant or cell-permeant, fluorophore-coupled ligands, which enable the quantification of odorant receptor expression by antibody-independent, chemical live-cell staining and flow cytometry. Here, we describe how to measure the cell surface expression of recombinant IL-6-HaloTag®-odorant receptors in HEK-293 cells or NxG 108CC15 cells, by live-cell staining and flow cytometry, and how to measure an odorant-induced activation of these receptors by the fast, real-time, luminescence-based GloSensor® cAMP assay.


BACKGROUND
Odorant receptors (ORs) are seven-transmembrane G protein-coupled receptors (GPCRs) [1], and are activated by small volatile compounds [2,3]. The activation of ORs by odorants triggers a cAMP signaling cascade [4], which activates cellular function of olfactory sensory neurons (OSNs) of the nose [5], ultimately enabling the perception, recognition and discrimination of a plethora of volatile stimuli [6,7].
To investigate the molecular mechanisms of human olfactory perception at the receptor level, cell-based bioassays are required that can cope with (1) complexity of odorant/receptor interactions [8], and (2) individually different and sub-optimal cell surface expression of ORs [9][10][11]. In the last two decades, different test cell systems have been developed, which facilitated the process of identification of cognate OR/odorant combinations [2,[12][13][14][15]. The most widely used assay in the recent years has been introduced by the Matsunami group in 2008 [15], a static end point assay, where an activation of OR by odorant results in cAMP signaling, which in turn triggers a cAMP/CRE-dependent transcription of a luciferase transgene, enabling cAMP-proportional luminescence measurements. Most recently, we introduced an alternative, luminescence-based, fast online assay, where a genetically modified, cAMP-binding luciferase [16] (GloSensor ® , Promega) detects odorant/ OR-induced changes in intracellular cAMP, enabling a robust luminescence readout within minutes upon stimulation with odorants [12].
The functional cell-surface expression of recombinant ORs in heterologous cells has been, and until now still is, a major challenge in the field. The identification and co-expression of appropriate signal transducing guanine nucleotide-binding protein G(olf) subunit alpha (Gαolf, GNAL) [4,13], as well as co-expression of accessory proteins and chaperones [11,15], such as receptor transport protein (RTP1S), and the use of N-terminal epitope tags, such as the N-terminal part of rhodopsin (Rho-tag) [2], have largely improved the functional cell surface expression of recombinant ORs in test cell systems, and facilitated their de-orphaning. So far, the most widely used combination of
Store culturing medium at 4°C and pre-warm it at 36°C before usage. D-luciferin stock solution: Prepare a 10 mM HEPES solution and adjust to pH 7.5. Dilute 250 mg of D-luciferin (beetle) monosodium salt in 7.5 ml HEPES. Aliquot into 115 µl stock and store them at −80°C until use.
Buffer for luminescence measurements: Prepare 1M solutions in distilled water for NaCl, KCl, CaCl 2 and HEPES. Solutions have to be autoclaved but can be stored at room temperature. Prepare buffer on the day of use, containing 140 mM NaCl, 20 mM HEPES, 5 mM KCl, 1 mM CaCl 2 and 10 mM D-glucose. Adjust to pH 7.5 by using a 1 M NaOH solution.

2.
Resuspend the cells with 10 ml of culturing medium and transfer the suspension to a 10 cm cell-culture dish. Culture NxG cells in a 37°C incubator with 7% CO 2 . Culture HEK-293 cells in a 37°C incubator with 5% CO 2 .

3.
Check cells under a phase contrast microscope to make sure that the cells are healthy.
The cells should be max. 80% confluent (Fig. 2). Use the cells between the second and max. tenth cell-passage.

Transfer cells for transfection 4.
Aspirate all medium from the cell culture dish. Transfer 1 ml of trypsin-EDTA solution onto the cells. Gently agitate the dish to allow the cells detaching from the bottom of the dish. Add 9 ml of culturing medium to the dish.

5.
Transfer the cell suspension to a 50 ml conical tube and centrifuge for 3 min at 250 g at room temperature. POL Scientific Protocol 6.1.2. By using a multichannel pipette, distribute 100 µl of the cell suspension from the reservoir into each well of a 96-well plate.

6.1.3.
Culture 96-well plates in the incubator overnight before proceeding with step 7.

6.2.2.
Transfer 0.8 ml of the cell suspension into each well of a 12-well plate.

6.2.3.
Culture 12-well plates in the incubator overnight before proceeding with step 7.    Which concentrations should be used to establish a concentration-response relation depends on each odorant/OR combination. The concentration range in which a GPCR may be fully activated by its agonist, assuming a single type of non-cooperative binding sites, typically spans about 2.5 orders of magnitude, and has to be adjusted according to, for example, information available from single concentrations that yielded 'hits' in screening experiments. A limiting factor, however, can be the solubility of odorants to be tested: Many odorants are difficult to dissolve in the solvent/physiological buffer used.

Measuring the activation of ORs
(1) Prepare the buffer, stock solutions of odorants and perform the serial dilutions as described above.
(2) Aspirate all medium from each well by using an 8-channel adapter.
(3) Thaw an appropriate amount of pre-made luciferin stocks. Prepare the buffer/luciferin mixture by adding one stock luciferin to 6 ml buffer containing 0.1% DMSO for one 96-well plate. Using a multichannel pipette, distribute 60 µl buffer/luciferin mixture to each well.
(4) Incubate the plate at room temperature in the dark for 50 min.

POL Scientific
Protocol (5) Measure luminescence with the Glomax ® Multi + detection system. As 'basal level' measure three data points.
(6) Using a multichannel pipette, add 30l of the serial dilution, in case of measuring concentration-response relations, or a single dilution of the desired odorant, in case of measuring receptor screening experiments, to each well (Fig. 3).
(7) Incubate the plate at room temperature in the dark for ≥ 4 min.

8.2.8.
Aspirate all solution and re-suspend the cells with 180 µl serum free medium. Transfer the cell resuspension into a 96-well skirted PCR plate and start flow cytometry analysis.

Data analysis 9.
The data analysis for the cAMP-luminescence assay is performed according to step 9.1, and according to step 9.2 for the flow cytometry assay.

9.1.
Data analysis for the cAMP-luminescence assay

Primary data analysis
The raw luminescence data obtained from the Glomax is saved as an Excel worksheet. Three consecutive data point values before ('basal level') and after odorant application are each averaged, and the respective averaged 'basal level' is subtracted from each signal.

Secondary data analysis -OR screening experiments
(1) The duplicates get averaged.

Protocol
(2) To evaluate responding receptors, a 2-sigma (2σ) or 3-sigma (3σ) threshold (mean of all receptor responses + two or three standard deviations) should be calculated.

Secondary data analysis -non-linear regression
For concentration-response relations, it is conventional to have at least three independent transfection experiments, each performed in triplicates. Amplitudes should be normalized to those of a defined OR/odorant pair within each experiment. Alternatively, OR amplitudes may be normalized to an endogenous GPCR-induced cAMP luminescence signal.
(1) Average the normalized data of all three experiments.
(2) EC 50 values and curves are derived from fitting the function.

Primary data analysis
(1) Open the data files with the MACSQuantify Software.
(2) Set the gate for the live cells to exclude dead cells of the further analysis in the 2D plot. The FSC signal (x-axis) is proportional to the surface area of the measured cell whereas the SSC signal (y-axis) depends on the granularity of the cell.
(3) Upon excitation with an argon laser (488 nm), AlexaFluor 488-labelled cells emit green light. Set the gate for the mock-transfected cells at 1% in the 2D plot. The FITC signal of each mock control defines the distinction between negative and positive cells.
(4) TMR-labelled cells show two excitation maxima: one at around 488 nm and one at around 561 nm. The PE molecule is more efficiently excited at 561 nm and has a fluorescence emission peak at 578 nm. The PE signal of each mock-control defines the distinction between negative and positive cells.
(5) Copy both gates for all other investigated samples.

Cell-surface expression of fluorophore-labelled ORs
Live-cell flow cytometry delivers at least two important parameters, the number of recorded cells that emitted a receptor-related fluorescence signal, and the fluorescence intensity emitted by each recorded cell (Fig.  4E). These two parameters, typically measured from thousands of cells, enable a quantification of OR cell-surface expression. It is conventional to have at least three replicates of the experiment. fitting the logistic function to the data. Arrows indicate EC 50 values (see ref [19], Fig. 1E). E. Flow cytometry-derived fluorescence distribution of ~1000 NxG 108CC15 cells expressing IL-6-HaloTag  -OR8D1, and labeled with cell membrane-impermeant HaloTag  Ligand-Alexa488 (see ref [19] Fig. S3E).

Protocol cAMP luminescence assay
ORs are known to have a so-called constitutive or basal activity, which means that to a certain degree they may activate cAMP signaling even in the absence of an adequate odorant stimulus [21]. This basal activity may vary significantly among the different ORs. As a result, in our GloSensor ® cAMP assay, the luciferase activity could be significantly different when using different ORs. Therefore, it is important to determine the luminescence background of each well, and to subtract the average luminescence value of each well before application of odorant from the respective averaged luminescence values in the presence of odorant, to obtain baseline-subtracted Δ luminescence values. Each Δ luminescence value obtained from an odorant-activated OR can then be normalized against the maximum Δ luminescence value of a control receptor, or the highest Δ luminescence obtained for that particular OR in this experiment.
For OR screening experiments, all amplitudes above a calculated 3σ-threshold may be considered as signals ("hits"), but have to be validated by establishing concentration-response-relations in subsequent transfection experiments.
For concentration-response-relations, typically any OR-specific and odorant concentration-dependent effects may be fitted by a non-linear regression algorithm, which will deliver the effective concentration values at half-maximal response (EC 50 ) (Fig. 4D).
It is conventional to have three replica wells and to replicate the experiment three times. The mean relative luminescence unit (RLU), standard deviation and fitted functions can then be graphed with an appropriate software.

TROUBLESHOOTING
Possible problems and their troubleshooting solutions are listed in Table 3.