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Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous Gα16 construct is co-transfected. Following ligand binding, activation of the Gα16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous Gα16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.


Introduction
G protein-coupled receptors (GPCRs) constitute one of the largest and most diverse families among all cell surface proteins. Their presence in vertebrates, invertebrates, plants, yeast, and slime mold, as well as in Protozoa and the earliest diploblastic Metazoa indicates that GPCRs are among the oldest molecules linked with signal transduction 1 . Their natural activating ligands comprise a wide diversity of external stimuli including peptides, biogenic amines, odorants, glycoproteins, and photons 2 . As such, these receptor-ligand signaling systems are involved in a great variety of physiological processes. The broad functional spectrum makes them ideally suited for the development of therapeutic drugs that cover a wide range of human diseases. About 50-60% of the current drug targets are represented by GPCRs 3,4 . Besides their great importance in the pharmaceutical industry, GPCRs are also in the spotlight for the development of a new generation of species-specific insecticides 5,6 and pesticides in general. Because the natural ligands of many GPCRs are still unidentified, they are classified as orphan GPCRs.
The deorphanization of these receptors will improve the understanding of their physiological roles in organisms and may uncover putative targets for new drug applications 7 .
Since the genomic era, the reverse pharmacology strategy is widely applied for the deorphanization of GPCRs 8 . The approach implies that an orphan receptor is used as a 'hook' to 'fish out' its activating ligand from a biological extract or from a library of synthetic compounds. The GPCR of interest is therefore cloned and subsequently transfected in a cellular expression system. In the most commonly used methods, receptor activation is determined by measuring changes in the concentration of second messenger molecules 9 . The main receptor screening assays rely on calcium-sensitive bioluminescent proteins (e.g., aequorin) 10 or fluorescent calcium indicators (e.g., Fluo-4) 11 . The fluorescence-based assays, in which receptor-expressing cells are loaded with a fluorescent calcium indicator prior to ligand screening, have the advantage that they allow high-throughput screening due to their ease of use, short reading time, and the flexibility of screening multiple orphan receptors on a single plate . The availability of medium/high-throughput screening approaches, like the fluorescence-based calcium mobilization assay that has proven to be a widely applied technique 9,18 , is therefore invaluable.
The fluorescence-based calcium mobilization assay as described here is performed in the human embryonic kidney 293T (HEK293T) cell line and uses a fluorescent probe to determine changes in intracellular calcium concentrations upon receptor activation. To guarantee high expression and translation levels of the receptor, a Kozak consensus sequence 19 is added to the 5' end of the receptor-coding sequence, which is subsequently cloned in an expression vector (e.g., pcDNA vector series for mammalian cell lines). As it is difficult to predict the endogenous G protein-coupling of an orphan GPCR based on sequence information alone, the second messenger molecules (e.g., calcium or cAMP) that are modulated after receptor activation often remain unknown prior to ligand identification. To circumvent this problem, promiscuous G proteins of the G q family (e.g., murine Gα 15 or human Gα 16 [used here]) or chimeric G proteins (e.g., Gα qi5 ) that interact with most GPCRs and induce the release of calcium can be co-expressed 20,21,22 . Upon binding of the ligand to its receptor, the GPCR undergoes a conformational change that leads to the activation of specific intracellular pathways. The guanosine diphosphate (GDP) molecule, bound under resting conditions to the Gα 16 subunit, will be replaced by a guanosine triphosphate (GTP) molecule. This provokes the dissociation of the heterotrimeric G protein in a Gα 16 and Gβγ subunit. The Gα 16 subunit activates phospholipase Cβ (PLCβ), which in turn hydrolyzes the membrane-bound phosphatidylinositol bisphosphate (PIP 2 ) resulting in diacylglycerol (DAG) and inositol trisphosphate (IP 3 ). IP 3 will spread throughout the cytoplasm and activates IP 3dependent calcium channels present in the membrane of the endoplasmic reticulum, which induces the release of calcium into the cytoplasm.
The calcium release upon receptor activation occurs within seconds and can be detected by loading cells before the screening assay with a calcium-sensitive dye, like Fluo-4 acetoxymethyl (AM) 11 . The AM ester group enables the fluorophore to cross the cell membrane and is cleaved off by cytoplasmic esterases once inside the cell. Consequently, the negative charges of the fluorescent dye are unmasked, preventing it from diffusing out of the cell and allowing it to interact with calcium ions. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions only containing calcium concentrations in the nanomolar range. However, when calcium is released upon receptor activation, the signal can increase concentration-dependently to more than a 100-fold, hereby ensuring a large signal-to-noise ratio. Fluo-4 also exhibits a large dynamic range for reporting [calcium] around a K d (calcium) of 345 nM, making it suitable to measure physiologically relevant calcium changes in a wide range of cells. The excitation of Fluo-4 occurs at 488 nm and the emission fluorescence is measured at 525 nm 11 . Fluorimeters like the fluorescence imaging plate reader (FLIPR) 23 , the NOVOstar, or the FlexStation (station device) 12 are medium/high-throughput systems that allow simultaneous compound addition and the detection of the Fluo-4 signal upon receptor activation for every well in an assay plate. The calcium mobilization assay described here relies on the station device 96-well microplate system.
The SoftMax Pro software (software) is used to operate the station device as well as for data analysis. The program immediately displays the results as graphs in 96-well format. Multiple wells can be selected simultaneously to compare the outcome of these wells on the same graph. The relative fluorescent unit (RFU) values of wells in each column are simultaneously measured for a period of two minutes, starting before the addition of compounds to the wells and continuing after measurement of the fluorescent signal following receptor activation. Typically, the trend of an agonist curve aligns with the baseline until an activating compound is added to the cells, resulting in a rapid increase of the fluorescent signal. The peak height is correlated with the final agonist concentration in the well. After the peak, the fluorescent signal slowly drops towards the baseline level. The RFU measurements can be converted into concentration-response curves to determine the EC 50 value (half maximal effective concentration) of a ligand. In general, at least three independent screens, each including three replicas of a concentration series, should be performed to compose a reliable concentration-response curve.
It is recommended to include several positive and negative controls in the experimental design. First of all, a transfection control, i.e. the implementation of a receptor with a known ligand, should be tested. This allows verifying whether the transfection agent was operational. Incorporation of a control experiment with an agonist for an endogenous receptor of the cell line and a negative control (e.g., wash buffer) are also recommended to monitor the health and viability of the cells and to exclude the possibility that the wash buffer was contaminated with a factor that could elicit an auto-fluorescent response. Frequently used agonists are a peptide derived from the protease-activated receptor-1 (PAR 1 ), which acts as a PAR 1 selective agonist, or carbachol, which activates the acetylcholine receptor. Cells transfected with an empty expression vector should also be tested to exclude that active compounds interact with the cell's endogenous receptors. Optimization of several parameters described in the protocol below may be required for different signaling systems. A schematic figure of the complete fluorescencebased calcium mobilization assay is depicted in Figure 1.  3. Co-transfect one population of cells with the expression vector encoding the GPCR of interest and the Gα 16 construct, the second flask with the empty vector and the Gα 16 construct, and the third with the transfection control and the Gα 16 construct. Note: In this protocol, the Drosophila sNPF receptor was cloned in a pcDNA3.1 mammalian expression vector 13 . JetPRIME was used to perform the transfection, but other transfection reagents can be used as well.

Transient Transfection of HEK293T Cells
1. Add a total of 7.8 µg DNA (3.9 µg receptor construct or empty vector, and 3.9 µg Gα 16 expression construct) to a 1.5 ml microcentrifuge tube and add 500 µl of the JetPRIME buffer. Mix well by vortexing the tube, and spin down shortly. 2. Add 37.5 µl of the JetPRIME reagent, vortex and spin down for 1 min at 14,000 x g. Incubate the transfection mix 10 min at room temperature. 3. Add the transfection mix dropwise to the cell culture medium and make sure to pipette directly into the medium avoiding contact with the walls of the culture flask. Incubate the flasks at 37 °C in a 5% CO 2 humidified incubator for 20-24 hr.

Representative Results
Concentration series ranging from 50 µM to 0.001 nM were tested for all four Drosophila sNPF peptides (Drome-sNPF-1: AQRSPSLRLRFamide, Drome-sNPF-2: SPSLRLRFamide, Drome-sNPF-3: PQRLRWamide, Drome-sNPF-4: PMRLRWamide) on HEK293T cells that transiently express the Drosophila sNPF receptor and the promiscuous Gα 16 subunit. The GPCR was activated by all four peptides in final concentrations up to 0.1 nM, and receptor activation was concentration-dependent. Figure 2 depicts the graphs corresponding to one of three replicas of the Drome-sNPF-1 concentration series. The negative control (wash buffer) did not induce a fluorescent signal, while the positive control (PAR 1 -1 µM) elicited strong activation of the endogenous protease-activated receptor-1, leading to a high fluorescent signal (± 30,000 RFU). It should be noted that a deviation of the typical curve may indicate abnormalities. For example, an ongoing rise of the curve without returning to the baseline might be related to non-receptor-mediated signals, such as the presence of calcium ionophores, or a disrupted lipid bilayer causing calcium leaks.
The software allows entering formulas to determine the percentage of activation or the standard errors of the different concentrations among others. The resulting data are used to compose preliminary concentration-response curves to estimate the EC 50 values, as shown for the four Drome-sNPF peptides in Figure 3. The curves of Figure 3 also include the data for the negative control where the concentration series of the Drome-sNPF peptides were tested on HEK293T cells transfected with an empty pcDNA3.1 vector. The results indicate that the addition of the peptides to these cells have no effect on endogenous receptors, but indeed activate the receptor of interest. The fluorescence-based calcium mobilization assay was then repeated three times independently. Based on the preliminary concentration-response curves of the initial screen, the tested concentrations were adapted to cover the dynamic range of the curve (Figure 4). The EC50 values of Drome-sNPF-1 (2.04 ± The previously characterized Drosophila sNPF signaling system was used here to explain the fluorescence-based calcium mobilization assay. For this purpose, concentration series of the activating ligands were tested immediately. However, when an orphan receptor is brought to overexpression in a screening assay to test a library containing hundreds of compounds, it is recommended to first screen with relatively high final concentrations of the ligands (e.g., 10 or 1 µM). Following the detection of an activating compound, a dilution series of that compound can be screened in order to compose a concentration-response curve and to determine the EC 50 value.
Once the activating ligand of a receptor is determined, the intracellular signaling pathway can be further investigated by adapting the protocol. The assay can be performed as described above, but without co-transfecting the Gα 16 subunit. When a calcium response is measured, it means that the receptor couples with an endogenous Gα q subunit of the cellular expression system. When no fluorescent signal is observed, protocols to measure changes in concentrations of other secondary messengers (e.g., cAMP) can be applied.
Structure-activity relationship (SAR) studies can also be carried out to define the peptide's core sequence required for receptor activation. First, truncated sequences are assessed to define the minimal amino acid sequence of the peptide that is still able to activate the receptor.
Next, peptides can be tested in which systematically every amino acid has been substituted for an alanine residue. Testing synthetic alaninesubstitution series on the receptor allows to determine the importance of each of the amino acids for receptor activation 24,25 .
Despite its frequent use and proven efficacy, it has to be emphasized that the assay described here may need some adaptations to gain optimal results for specific receptors of interest. The Gα 16 subunit has the advantage that it binds to most GPCRs, but may also have a dominantnegative effect on receptors that endogenously couple via Gα q 22 . In this case, it may be useful to test different combinations of G proteins during the optimization of a novel calcium assay and to compare the results for Gα q -coupled receptors in the absence or presence of Gα 16 . Alternative assays that are independent of the interacting G protein to detect receptor activation can also be performed, such as the translocation of GFPlabeled arrestin, or the detection of changes in membrane potential (e.g., by the FLIPR Membrane Potential Assay Kit). Besides the Fluo-4 AM used here, a wide array of other calcium-sensitive fluorophores, each with its own spectral and chemical properties, is available. The most appropriate fluorophore can be selected based on the GPCR, cell type and the available plate reader, but experimental verification is necessary. The amounts of transfected DNA and the DNA/transfection reagent ratio need to be determined for each receptor-transfection reagent-cell line combination. Finally, it should be kept in mind that cells in continuous culture only allow 20-25 usable passages to perform the screening assays.

Disclosures
The authors have nothing to disclose.