The authors acknowledge the Research Foundation Flanders (FWO-Vlaanderen, Belgium, G.0601.11) and the KU Leuven Research Foundation GOA/11/002. IB, TJ and LT benefit from a fellowship from the FWO-Vlaanderen.

Note: All actions in which cells are involved should be carried out in a sterile environment by working in a laminar flow.
1. Maintenance of the HEK293T Cell Line
<ol>
	<li>Grow the HEK293T cells in a T-75 flask at 37 &#176;C in a 5% CO2 humidified incubator.</li>
	<li>Passage the cells when a confluence of 80% is reached. Note: This normally takes 3 to 4 days. Cells in continuous culture allow 20-25 usable passages for screening.
	<ol>
		<li>Place the Dulbecco&#8217;s phosphate buffered saline (PBS) without calcium chloride and magnesium chloride, PBS-trypsin-ethylenediaminetetraacetic acid (EDTA) (500 ml PBS supplemented with 10 ml trypsin-EDTA solution and 4.5 ml 4% EDTA) and growth medium (500 ml Dulbecco&#8217;s modified eagle&#8217;s medium &#8211; high glucose [DMEM] supplemented with 10% fetal bovine serum [FBS] and 1 mM penicillin-streptomycin [P-S]) at room temperature half an hour in advance.</li>
		<li>Remove the old growth medium from the cells. Rinse off dead cells with 3 ml PBS and remove the PBS again.</li>
		<li>Add 3 ml PBS-trypsin-EDTA, incubate for 1 min at room temperature and remove the solution Note: The morphology of the cells will change from star-shaped to sphere-shaped.</li>
		<li>Loosen the cells from the bottom of the flask by gentle tapping and collect the cells by rinsing the bottom several times with 10 ml of fresh growth medium.</li>
		<li>Transfer 1 ml of the cell culture into a new T-75 flask containing 14 ml fresh growth medium, and incubate at 37 &#176;C in a 5% CO2 humidified incubator.</li>
	</ol>
	</li>
</ol>
2. Transient Transfection of HEK293T Cells
<ol>
	<li>Grow the HEK293T cells in a T-75 flask 3 days before the actual calcium mobilization assay takes place. Make sure to use three T-75 flasks, one for transfection with the receptor construct, one for transfection with an empty expression vector, as negative control, and one for the transfection control. Note: The passaging of the cells is performed as described in step 1.2. When multiple 96-well plates have to be screened, T-150 flasks can be used to get a higher yield of cells (to do this, double all listed quantities in steps 1.2.5, 2.3, 3.1.3 and 3.1.4).</li>
	<li>Incubate the flasks at 37 &#176;C in a 5% CO2 humidified incubator for 20-24 hr until the cell cultures approach 50-70% confluence.</li>
	<li>Co-transfect one population of cells with the expression vector encoding the GPCR of interest and the G&#945;16 construct, the second flask with the empty vector and the G&#945;16 construct, and the third with the transfection control and the G&#945;16 construct. Note: In this protocol, the Drosophila sNPF receptor was cloned in a pcDNA3.1 mammalian expression vector13. JetPRIME was used to perform the transfection, but other transfection reagents can be used as well.
	<ol>
		<li>Add a total of 7.8 &#181;g DNA (3.9 &#181;g receptor construct or empty vector, and 3.9 &#181;g G&#945;16 expression construct) to a 1.5 ml microcentrifuge tube and add 500 &#181;l of the JetPRIME buffer. Mix well by vortexing the tube, and spin down shortly.</li>
		<li>Add 37.5 &#181;l of the JetPRIME reagent, vortex and spin down for 1&#160;min&#160;at 14,000 x g. Incubate the transfection mix 10 min at room temperature.</li>
		<li>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 &#176;C in a 5% CO2 humidified incubator for 20-24 hr.</li>
	</ol>
	</li>
</ol>
3. Calcium Mobilization Assay
<ol>
	<li>Collect the transfected cells and seed them in 96-well black-walled, clear-bottom plates.
	<ol>
		<li>Place the PBS, PBS-Trypsin-EDTA and the DMEM transfer medium (500 ml DMEM supplemented with 10% dialyzed FBS and 1% P-S) at room temperature half an hour in advance.</li>
		<li>Coat the 96-well black-walled, clear bottom plates with 60 &#181;l of PBS with fibronectin (0.0025%) per well (5.85 ml PBS and 150 &#181;l fibronectin [0.1%] per plate). Incubate the plates at room temperature for 1 hr with the lid on. Remove the solution from the wells and incubate the plates again for 1 hr without the lid on at room temperature. Alternatively, it is possible to use pre-coated plates for enhanced cell attachment.</li>
		<li>Take off the old growth medium from the cells and rinse dead cells off with 3 ml PBS and remove the PBS.</li>
		<li>Add 3 ml PBS-Trypsin-EDTA, incubate for 1 min, then remove the solution. Note: The morphology of the cells changes from star-shaped to sphere-shaped.</li>
		<li>Loosen the cells from the bottom of the flask by gentle tapping and collect them by rinsing several times with 10 ml of DMEM transfer medium. Transfer the cells into a 50 ml Falcon tube.</li>
		<li>Count the number of cells per ml (performed with a NucleoCounter here, but a Burker&#8217;s chamber is acceptable). Add 100 &#181;l of the cell culture in a 1.5 ml microcentrifuge tube. Add 100 &#181;l lysis buffer and mix by tapping the tube. Add 100 &#181;l stabilizing buffer and mix by tapping.</li>
		<li>Fill a NucleoCassette with the cell suspension and count the cells with the counter. Multiply the number of cells by three, as the cells were diluted by a factor three when adding the lysis and stabilizing buffer in step 3.1.6.</li>
		<li>Dilute the cells to a final concentration of 600,000 cells/ml and seed 150 &#181;l of the cells per well in the coated plates to obtain a cell density of about 90,000 cells per/well. Avoid air bubbles and tap the plate to spread the cells evenly in order to get a contiguous cell layer. Incubate the plates at 37 &#176;C in a 5% CO2 humidified incubator for 16-24 hr.</li>
	</ol>
	</li>
	<li>Load the cells with the fluorescent dye and prepare the compound plate.
	<ol>
		<li>Prepare Hank&#8217;s balanced salt solution (HBSS)/HEPES/Ca2+/bovine serum albumin (BSA) buffer: add 165 &#181;l CaCl2 stock solution (1 M CaCl2 in distilled water [dH2O] &#8211; store at room temperature), 500 &#181;l HEPES stock solution (1 M HEPES in dH2O, pH 7.4 &#8211; store at room temperature), and 0.05 g BSA to 50 ml HBSS. Note that fatty acid free BSA is used for calcium assays, as fatty acids can activate specific receptors, impairing the calcium signal of the receptor of interest.</li>
		<li>Prepare probenecid solution (100x, 250 mM): dissolve 0.71 g probenecid in 5 ml NaOH (1 M) and add 50 &#181;l HEPES stock solution and 5 ml of the HBSS/HEPES/Ca2+/BSA buffer &#8211; always prepare fresh solution. Note: Probenecid inhibits inorganic anion transporters that can dislodge Fluo-4 from the cytoplasm, thereby reducing the fluorescent signal. It is recommended to perform the dye loading in the presence and absence of probenecid to determine whether inorganic anion transporters present a potential problem in the particular cell lines under investigation, as probenecid might even decrease the agonist-mediated signal.</li>
		<li>Prepare wash buffer: add 500 &#181;l probenecid solution to 50 ml HBSS/HEPES/Ca2+/BSA buffer, adjust the pH to 7.4 and filtrate the buffer solution. Always prepare fresh buffer, and 50 ml buffer per plate is required.</li>
		<li>Prepare 10% pluronic acid solution: mix 50 &#181;l pluronic acid (20% w/v in dimethyl-sulfoxide [DMSO]) with 50 &#181;l DMSO &#8211; if crystallization occurs, heat to 37 &#176;C and always prepare fresh solution.</li>
		<li>Prepare Fluo-4 AM solution (1mM): add 44 &#181;l 10% pluronic acid solution to a vial that contains 50 &#181;g Fluo-4 AM and vortex until it is completely dissolved. Avoid exposure to light to prevent bleaching of the fluorophore.</li>
		<li>Prepare loading buffer: add 8.8 ml DMEM supplemented with 10 mM HEPES and 2.5 mM probenecid (pH 7.4) to the Fluo-4 AM solution (44 &#181;l) and vortex. Always use fresh buffer.</li>
		<li>Discard medium from the cells, wash the cells with 200 &#181;l PBS per well, and remove PBS.</li>
		<li>Add 55 &#181;l loading buffer per well and incubate for 1 hr at room temperature. Wrap the plate in aluminum foil to prevent exposure of the plate to light.</li>
		<li>Make sure to dry the compounds before the screen if they are dissolved in solvents that are detrimental (e.g., acetonitrile) for the cellular expression system.</li>
		<li>Solubilize the peptides in wash buffer in siliconized 1.5 ml microcentrifuge tubes. Prepare a dilution series to perform a concentration-response analysis and to determine the EC50 value of a ligand. Add 70 &#181;l of the ligand in the corresponding well of the compound plate (V-shaped 96-well plates) and include positive (endogenous agonist) and negative controls (wash buffer) on each plate. Note: If a compound is not soluble in the wash buffer, one can try other solvents that are not harmful for the cells under investigation, such as water or solutions that emulsify and solubilize oils and other water-insoluble substances (e.g., Kolliphor EL).</li>
		<li>Measure all samples in triplicate. Keep in mind that 50 &#181;l of a ligand from the compound plate will be transferred into a well with 100 &#181;l wash buffer of the cell plate during the assay, so prepare the compounds at 3x their desired final concentration.</li>
		<li>Discard the loading buffer from the cell plate and add 100 &#181;l wash buffer to each well. Incubate the plate 15 min at room temperature and prevent exposure to light.</li>
		<li>Discard the wash buffer from the cell plate and add 100 &#181;l new wash buffer to each well.</li>
		<li>Incubate the cell plate, the compound plate, and a tip rack (96-well, station device pipette tips) for 15 min at 37 &#176;C.</li>
	</ol>
	</li>
</ol>
4. Assay on the Station Device
<ol>
	<li>Create and load the desired protocol file with the software and activate the temperature control unit for the reading chamber. Here, measure calcium responses at 37 &#176;C for 2 min one row at a time with 1.52 sec interval between successive readings (measuring a complete 96-well plate takes approximately 25 min) at 525 nm. Set the excitation of Fluo-4 to 488 nm and transfer a total volume of 50 &#181;l of compound to the cell plate, 18 sec after the reading start, with the dispense speed set at 26 &#181;l/sec and a pipette height of 135 &#181;l.</li>
	<li>Position the compound and cell plate, and the tip rack in the appropriate drawers of the station device.</li>
	<li>Perform a single read of the cell plate at the same excitation and emission wavelength in the ENDPOINT mode, before starting the actual screen in FLEX mode. Note: This measurement gives relative fluorescence unit (RFU) values and allows detecting variability between wells of the plate. Values between 20,000 and 40,000 are considered as acceptable.</li>
	<li>Start the assay and analyze the data with the software.</li>
</ol>

<ol>
	<li>Bockaert, J., Pin, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+tinkering+of+G+protein-coupled+receptors+an+evolutionary+success.">Molecular tinkering of G protein-coupled receptors an evolutionary success.</a> EMBO J. 18 (7), 1723-1729 (1999).</li><li>Gether, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uncovering+molecular+mechanisms+involved+in+activation+of+G+protein-coupled+receptors.">Uncovering molecular mechanisms involved in activation of G protein-coupled receptors.</a> Endocr Rev. 21 (1), 90-113 (2000).</li><li>Drews, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+discovery+a+historical+perspective.">Drug discovery a historical perspective.</a> Science. 287 (5460), 1960-1964 (2000).</li><li>Marinissen, M. J., Gutkind, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G-protein-coupled+receptors+and+signaling+networks+emerging+paradigms.">G-protein-coupled receptors and signaling networks emerging paradigms.</a> Trends Pharmacol Sci. 22 (7), 368-376 (2001).</li><li>Bendena, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropeptide+physiology+in+insects.">Neuropeptide physiology in insects.</a> Adv Exp Med Biol. 692, 166-191 (2010).</li><li>Van Hiel, M. . B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropeptide+receptors+as+possible+targets+for+development+of+insect+pest+control+agents.">Neuropeptide receptors as possible targets for development of insect pest control agents.</a> Adv Exp Med Biol. 692, 211-226 (2010).</li><li>Tang, X. . L., Wang, Y., Li, D. . L., Luo, J., Liu, M. . Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orphan+G+protein-coupled+receptors+(GPCRs):+biological+functions+and+potential+drug+targets.">Orphan G protein-coupled receptors (GPCRs): biological functions and potential drug targets.</a> Acta Pharmacol Sin. 33 (3), 363-371 (2012).</li><li>Civelli, O., Reinscheid, R. K., Zhang, Y., Wang, Z., Fredriksson, R., Schi&#246;th, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G+protein-coupled+receptor+deorphanizations.">G protein-coupled receptor deorphanizations.</a> Annu Rev Pharmacol Toxicol. 53, 127-146 (2013).</li><li>Mertens, I., Vandingenen, A., Meeusen, T., De Loof, A., Schoofs, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postgenomic+characterization+of+G-protein-coupled+receptors.">Postgenomic characterization of G-protein-coupled receptors.</a> Pharmacogenomics. 5 (6), 657-672 (2004).</li><li>Brough, S. J., Shah, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+aequorin+for+G+protein-coupled+receptor+hit+identification+and+compound+profiling.">Use of aequorin for G protein-coupled receptor hit identification and compound profiling.</a> Methods Mol Biol. 552, 181-198 (2009).</li><li>Gee, K. R., Brown, K. A., Chen, W. N. U., Bishop-Stewart, J., Gray, D., Johnson, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+and+physiological+characterization+of+fluo-4+Ca2+-indicator+dyes.">Chemical and physiological characterization of fluo-4 Ca2+-indicator dyes.</a> Cell Calcium. 27 (2), 97-106 (2000).</li><li>Beets, I., Lindemans, M., Janssen, T., Verleyen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deorphanizing+G+protein-coupled+receptors+by+a+calcium+mobilization+assay.">Deorphanizing G protein-coupled receptors by a calcium mobilization assay.</a> Methods Mol Biol. 789, 377-391 (2011).</li><li>Mertens, I., Meeusen, T., Huybrechts, R., De Loof, A., Schoofs, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+short+neuropeptide+F+receptor+from+Drosophila+melanogaster.">Characterization of the short neuropeptide F receptor from Drosophila melanogaster.</a> Biochem Biophys Res Commun. 297 (5), 1140-1148 (2002).</li><li>Lu, H. -. L., Kersch, C. N., Taneja-Bageshwar, S., Pietrantonio, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+calcium+bioluminescence+assay+for+functional+analysis+of+mosquito+(Aedes+aegypti)+and+tick+(Rhipicephalus+microplus)+G+protein-coupled+receptors.">A calcium bioluminescence assay for functional analysis of mosquito (Aedes aegypti) and tick (Rhipicephalus microplus) G protein-coupled receptors.</a> J. Vis. Exp. (50), e2732 (2011).</li><li>Feng, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterization+of+a+neuropeptide+F-like+receptor+from+Drosophila+melanogaster.">Functional characterization of a neuropeptide F-like receptor from Drosophila melanogaster.</a> Eur. J. Neurosci. 18 (2), 227-238 (2003).</li><li>N&#228;ssel, D. R., Wegener, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+review+of+short+and+long+neuropeptide+F+signaling+in+invertebrates+any+similarities+to+vertebrate+neuropeptide+Y+signaling.">A comparative review of short and long neuropeptide F signaling in invertebrates any similarities to vertebrate neuropeptide Y signaling.</a> Peptides. 32 (6), 1335-1355 (2011).</li><li>Grimmelikhuijzen, C. J. P., Hauser, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mini-review+The+evolution+of+neuropeptide+signaling.">Mini-review The evolution of neuropeptide signaling.</a> Regul Pept. 177, S6-S9 (2012).</li><li>Caers, J., Verlinden, H., Zels, S., Vandersmissen, H. P., Vuerinckx, K., Schoofs, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=More+than+two+decades+of+research+on+insect+neuropeptide+GPCRs+an+overview.">More than two decades of research on insect neuropeptide GPCRs an overview.</a> Front Endocrinol (Lausanne. 3 (151), 1-30 (2012).</li><li>Kozak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analysis+of+5&#8217;-noncoding+sequences+from+699+vertebrate+messenger+RNAs).">An analysis of 5&#8217;-noncoding sequences from 699 vertebrate messenger RNAs).</a> Nucleic Acids Res. 15 (20), 8125-8148 (1987).</li><li>Offermanns, S., Simon, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G&#945;15+and+G&#945;16+couple+a+wide+variety+of+receptors+to+phospholipase.">G&#945;15 and G&#945;16 couple a wide variety of receptors to phospholipase.</a> CJ Biol Chem. 270 (25), 15175-15180 (1995).</li><li>Ral Conklin, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carboxyl-terminal+mutations+of+Gq&#945;+and+Gs&#945;+that+alter+the+fidelity+of+receptor+activation.">Carboxyl-terminal mutations of Gq&#945; and Gs&#945; that alter the fidelity of receptor activation.</a> Mol Pharmacol. 50 (4), 885-890 (1996).</li><li>Kostenis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+G&#945;16+the+optimal+tool+for+fishing+ligands+of+orphan+G-protein-coupled+receptors.">Is G&#945;16 the optimal tool for fishing ligands of orphan G-protein-coupled receptors.</a> Trends Pharmacol Sci. 22 (11), 560-564 (2001).</li><li>Robas, N. M., Fidock, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+orphan+G+protein-coupled+receptor+ligands+using+FLIPR+assays.">Identification of orphan G protein-coupled receptor ligands using FLIPR assays.</a> Methods Mol Biol. 306, 17-26 (2005).</li><li>Caers, J., Peeters, L., Janssen, T., De Haes, W., G&#228;de, G., Schoofs, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-activity+studies+of+Drosophila+adipokinetic+hormone+(AKH)+by+a+cellular+expression+system+of+dipteran+AKH+receptors.">Structure-activity studies of Drosophila adipokinetic hormone (AKH) by a cellular expression system of dipteran AKH receptors.</a> Gen Comp Endocrinol. 177 (3), 332-337 (2012).</li><li>Peeters, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pharmacological+study+of+NLP-12+neuropeptide+signaling+in+free-living+and+parasitic+nematodes.">A pharmacological study of NLP-12 neuropeptide signaling in free-living and parasitic nematodes.</a> Peptides. 34 (1), 82-87 (2012).</li></ol>

The authors have nothing to disclose.

The fluorescence-based calcium mobilization assay was successfully applied to confirm the functional characterization of the Drosophila sNPF peptidergic signaling system, which was already performed by Mertens et al. with a bioluminescence assay and by Feng et al. with an electrophysiological assay13,15. The EC50 values obtained with the fluorescence assay in HEK293T cells are about 10-fold less than those obtained with the bioluminescence assay performed in CHO cells (Drome-sNPF-1: fluo = 2.04 nM, lumi = 51 nM; Drome-sNPF-2: fluo = 5.89 nM, lumi = 42 nM; Drome-sNPF-3: fluo = 5.55 nM, lumi = 31 nM; Drome-sNPF-4: fluo = 0.50 nM, lumi = 75 nM). These variations can be explained by several factors, including the fact that one of the used expression systems may be better suited for functional expression of a given receptor, or the folding of some receptors can be less efficient in certain cell types. The EC50 values of all four Drome-sNPF peptides are in the nanomolar range when tested on their receptor with both the fluorescence and the bioluminescence assay, generally supporting the physiological relevance of their peptide-receptor interaction in vivo.
Note that no transfection control with a receptor for which the activating ligand is known was included in the screen presented here, because the Drosophila sNPF signaling system is normally the transfection control in experimental setups. The positive control with an endogenous ligand of the HEK293T cells (PAR1) and the negative control (wash buffer) were included in the screen. The results of PAR1 showed that the cells were in a good condition. The negative control (wash buffer) did not elicit a fluorescent signal, which indicates that the medium in which the peptides are dissolved was free of any contaminants that could influence the results.
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 &#181;M). 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 EC50 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 G&#945;16 subunit. When a calcium response is measured, it means that the receptor couples with an endogenous G&#945;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&#8217;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 alanine-substitution series on the receptor allows to determine the importance of each of the amino acids for receptor activation24,25.
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 G&#945;16 subunit has the advantage that it binds to most GPCRs, but may also have a dominant-negative effect on receptors that endogenously couple via G&#945;q22. 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 G&#945;q-coupled receptors in the absence or presence of G&#945;16. Alternative assays that are independent of the interacting G protein to detect receptor activation can also be performed, such as the translocation of GFP-labeled 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.

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 transduction1. Their natural activating ligands comprise a wide diversity of external stimuli including peptides, biogenic amines, odorants, glycoproteins, and photons2. 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 GPCRs3,4. Besides their great importance in the pharmaceutical industry, GPCRs are also in the spotlight for the development of a new generation of species-specific insecticides5,6 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 applications7.

Since the genomic era, the reverse pharmacology strategy is widely applied for the deorphanization of GPCRs8. The approach implies that an orphan receptor is used as a &#8216;hook&#8217; to &#8216;fish out&#8217; 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 molecules9. 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 plate12.

Here, the fluorescence-based calcium mobilization assay is thoroughly described and illustrated by the deorphanization process of the Drosophila melanogaster short neuropeptide F (sNPF) receptor. This neuropeptidergic signaling system was originally characterized by Mertens et al. in 200213 with a calcium bioluminescence assay performed in Chinese hamster ovary (CHO) cells14 and by Feng et al. in 2003 with an electrophysiological assay using Xenopus oocytes15. The presence of the sNPF signaling system seems to be limited to the phylum of Arthropoda, where it is implicated in a wide range of processes including the regulation of feeding, growth, stress reactions, locomotion, and circadian rhythms16.

Research on neuropeptidergic signaling systems in insects may not only lead to new targets for the development of insecticides, but the knowledge of their functioning may also be extrapolated towards other organisms as many signaling systems have been generally well conserved throughout evolution17. In the last decade, great progress has been made in the deorphanization process of insect neuropeptide GPCRs. Despite these efforts, only a small number of receptors have been matched to their cognate ligand, and loads of sequence information for new orphan GPCRs has become available due to the booming of genomics18. The availability of medium/high-throughput screening approaches, like the fluorescence-based calcium mobilization assay that has proven to be a widely applied technique9,18, 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 sequence19 is added to the 5&#8217; 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 Gq family (e.g., murine G&#945;15 or human G&#945;16 [used here]) or chimeric G proteins (e.g., G&#945;qi5) that interact with most GPCRs and induce the release of calcium can be co-expressed20,21,22. 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 G&#945;16 subunit, will be replaced by a guanosine triphosphate (GTP) molecule. This provokes the dissociation of the heterotrimeric G protein in a G&#945;16 and G&#946;&#947; subunit. The G&#945;16 subunit activates phospholipase C&#946; (PLC&#946;), which in turn hydrolyzes the membrane-bound phosphatidylinositol bisphosphate (PIP2) resulting in diacylglycerol (DAG) and inositol trisphosphate (IP3). IP3 will spread throughout the cytoplasm and activates IP3-dependent 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 Kd(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 nm11. 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 EC50 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 (PAR1), which acts as a PAR1 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&#8217;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 fluorescence-based calcium mobilization assay is depicted in Figure 1.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51516/51516fig1highres.jpg" src="/files/ftp_upload/51516/51516fig1.jpg" /> 
Figure 1. Overall scheme of the fluorescence-based calcium mobilization assay. Automated liquid handling and simultaneous fluorescence measurements are performed with the station device microplate reader, driven by the software. The station device contains three drawers: one for the cell plate, compound plate and tip rack. The build-in pipettor transfers compounds from one column of the compound plate to the corresponding column of the cell plate (step 1). Each well of the cell plate contains a monolayer of HEK293T cells that have been co-transfected with the GPCR of interest and the promiscuous G&#945;16 subunit. When a compound activates the receptor, the G&#945;16-bound GDP is replaced by GTP. The G&#945;16 subunit subsequently dissociates from the G&#946;&#947; complex and activates phospholipase C&#946; (PLC&#946;), which in turn hydrolyzes phosphatidylinositol bisphosphate (PIP2) resulting in diacylglycerol (DAG) and inositol trisphosphate (IP3). IP3 activates IP3-dependent calcium channels present in the membrane of the endoplasmic reticulum, inducing the release of calcium into the cytoplasm. The interaction of calcium with Fluo-4 (with which the cells are loaded prior to compound addition) results in a fluorescent signal (step 2). The software presents the results as relative fluorescent unit (RFU) values in function of time, and peak heights correlate with the ligand concentration in a concentration-dependent manner. These data can then be converted into a concentration-response curve to determine the EC50 value of a ligand-receptor pair (step 3).

<table border="0" cellpadding="0" cellspacing="0" width="610">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">HEK293T cells</td>
			<td style="width:109px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Dulbecco&#39;s Phosphate Buffered Saline (PBS)</td>
			<td style="width:109px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Trypsin-EDTA solution (0.25%)</td>
			<td style="width:109px;">Sigma-Aldrich</td>
			<td style="width:119px;">T4049</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA)</td>
			<td style="width:109px;">MP Biomedicals</td>
			<td style="width:119px;">195173</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Dulbecco&#39;s Modified Eagle&#39;s Medium - high glucose&#160; (DMEM)</td>
			<td style="width:109px;">Sigma-Aldrich</td>
			<td style="width:119px;">D5796</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:109px;">Sigma-Aldrich</td>
			<td style="width:119px;">F7524</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Penicillin-Streptomycin (P-S)</td>
			<td style="width:109px;">Sigma-Aldrich</td>
			<td style="width:119px;">P4333</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:215px;">jetPRIME</td>
			<td style="width:109px;">Polyplus transfection</td>
			<td style="width:119px;">114-01</td>
			<td style="width:167px;">FuGENE HD Transfection Reagent (Promega); Lipofectamine LTX &#38; Plus Reagent (Life technologies)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Dialyzed Fetal Bovine Serum (FBS)</td>
			<td style="width:109px;">Sigma-Aldrich</td>
			<td style="width:119px;">F0392</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Fibronectin from human plasma</td>
			<td style="width:109px;">Sigma-Aldrich</td>
			<td style="width:119px;">F0895</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Reagent A100, Lysis buffer</td>
			<td style="width:109px;">Chemometec</td>
			<td style="width:119px;">910-0003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Reagent B, Stabilizing buffer</td>
			<td style="width:109px;">Chemometec</td>
			<td style="width:119px;">910-0002</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:215px;">CaCl2</td>
			<td style="width:109px;">Sigma-Aldrich</td>
			<td style="width:119px;">C3881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">HEPES</td>
			<td style="width:109px;">Sigma-Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:109px;">Sigma-Aldrich</td>
			<td>A4503</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">HBSS buffer: Hank&#39;s Balanced Salt Solution</td>
			<td style="width:109px;">Sigma-Aldrich</td>
			<td>H8264</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Probenecid</td>
			<td style="width:109px;">Sigma-Aldrich</td>
			<td style="width:119px;">P8761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">NaOH (1 M)</td>
			<td>Vel</td>
			<td>2781</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Pluronic acid</td>
			<td>Invitrogen</td>
			<td>P-3000MP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Dimethyl-sulfoxide (DMSO)</td>
			<td style="width:109px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8418</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Fluo-4 AM</td>
			<td>Invitrogen</td>
			<td>F14201</td>
			<td style="width:167px;">Fluo-3, Rhod-2, Fluo-5, Calcium Green-1,.. (Invitorgen)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">TPP tissue culture flasks (T-75 and T-150)</td>
			<td style="width:109px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z707503 and Z707554</td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:215px;">FlexStation device</td>
			<td style="width:109px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">NOVOstar (BMG Labtechnologies); FLIPR (Fluorometric Imaging Plate Reader) (Molecular Devices)</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:215px;">Black-walled polystyrene plates (96 wells) with clear bottom</td>
			<td style="width:109px;">Greiner Bio-One</td>
			<td style="width:119px;">655090</td>
			<td style="width:167px;">Corning 96 well flat clear bottom black polystyrene poly-D-lysine coated microplates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">NucleoCassette</td>
			<td style="width:109px;">Chemometec</td>
			<td style="width:119px;">941-0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">NucleoCounter NC-100</td>
			<td style="width:109px;">Chemometec</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Microcentrifuge tubes, siliconized</td>
			<td style="width:109px;">BioCision</td>
			<td style="width:119px;">BCS-2470</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Polystyrene V-shaped 96-well plates</td>
			<td style="width:109px;">Greiner Bio-One</td>
			<td>651101</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">96-Well, FlexStation Pipet Tips</td>
			<td style="width:109px;">Molecular Devices</td>
			<td>9000-0912</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Soft Max Pro software</td>
			<td style="width:109px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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 G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 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 G&#945;16, 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.

Concentration series ranging from 50 &#181;M 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 G&#945;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 (PAR1 &#8211; 1 &#181;M) elicited strong activation of the endogenous protease-activated receptor-1, leading to a high fluorescent signal (&#177; 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 EC50 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&#160;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 &#177; 1.48 nM [95% confidence interval]), Drome-sNPF-2 (5.89 &#177; 3.74 nM), Drome-sNPF-3 (5.55 &#177; 3.95 nM) and Drome-sNPF-4 (0.50 &#177; 1.01 nM) are similar, indicating that they are equally potent to activate the receptor.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51516/51516fig2highres.jpg" src="/files/ftp_upload/51516/51516fig2.jpg" /> 
Figure 2. Graphical output of the software for a receptor-mediated fluorescence response of a concentration series. Twelve final concentrations (ranging from 50 &#181;M to 0.001 nM) of the Drome-sNPF-1 peptide were tested on the Drome-sNPF receptor. Activation is expressed in relative fluorescent unit (RFU) values. The upper graph gives the results of the six highest concentrations and the lower graph of the six lowest concentrations. The positive control (+) is PAR1 (1 &#181;M) and the negative control (-) is wash buffer.
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51516/51516fig3highres.jpg" src="/files/ftp_upload/51516/51516fig3.jpg" /> 
Figure 3. Preliminary concentration-response curves of Drosophila sNPF peptides determined by a single fluorescence-based calcium mobilization assay. The concentration-response curves of the Drosophila sNPF receptor transiently expressed in HEK293T cells for the four Drosophila sNPF peptides are shown in blue. For the negative controls (shown in red), peptides were tested on HEK293T cells transfected with an empty pcDNA3.1 vector. The fluorescent responses are shown as relative (%) to the highest value (100% activation). The curves are the result of one experiment in which each concentration series was measured in triplicate. The vertical bars represent standard errors of the mean (SEM), which sometimes are smaller than the used symbols (in that case, only the symbols are depicted).
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/51516/51516fig4highres.jpg" src="/files/ftp_upload/51516/51516fig4.jpg" /> 
Figure 4. Concentration-response curves and corresponding EC50 values of Drosophila sNPF peptides determined by three independent fluorescence-based calcium mobilization assays. The concentration response curves of the Drosopihla sNPF receptor transiently expressed in HEK293T cells for the four Drosophila sNPF peptides are the result of three independent measurements each performed in triplicate (n &#8805; 9). The fluorescent responses are shown as relative (%) to the highest value (100% activation). Asterisks indicate concentrations for which n &#8804; 9. Error bars indicate SEM, which sometimes are smaller than the used symbols (in that case, only the symbols are depicted). EC50 values are shown with their 95% confidence intervals.

Watch this Scientific Journal Video about Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay at JoVE.com

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

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

characterization-g-protein-coupled-receptors-fluorescence-based

Education

Research

JoVE Core

JoVE Journal

Cell Biology

Molecular Biology

Biology

Unit 2

Unit 1

Cell Signaling Pathways

Signaling Networks of G Protein-coupled Receptors

SCIENTISTS IN ACTION

39.3K Views.  KU Leuven. The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

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

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

Quantifying Agonist Activity at G Protein-coupled Receptors

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors.
Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (Kb) is much greater than that for the inactive state (Ka). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (Kobs), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (&#949;) (Figure 2).
Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the Kobs and relative efficacy of an agonist 1,2. In this report, we show how to modify this analysis to estimate the agonist Kb value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate Kb in absolute units of M-1.
Our method of analyzing agonist concentration-response curves 3,4 consists of global nonlinear regression using the operational model 5. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of Kobs and a parameter proportional to efficacy (&tau;). The estimate of &tau;Kobs of one agonist, divided by that of another, is a relative measure of Kb (RAi) 6. For any receptor exhibiting constitutive activity, it is possible to estimate a parameter proportional to the efficacy of the free receptor complex (&tau;sys). In this case, the Kb value of an agonist is equivalent to &tau;Kobs/&tau;sys 3.
Our method is useful for determining the selectivity of an agonist for receptor subtypes and for quantifying agonist-receptor signaling through different G proteins.

A method for estimating the affinity constant of an agonist for the active state (Kb) of a G protein-coupled receptor is described. The analysis provides absolute or relative measures of Kb depending on whether constitutive receptor activation is measurable. Our method applies to various responses downstream from receptor activation.

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. It is likely to play a role in preventing accumulation of recombinogenic intermediates that would otherwise accumulate at transiently stalled replication forks, consistent with a hyper-recombinant phenotype of cells lacking WRN. In humans, the exonuclease domain comprises an N-terminal portion of a much larger protein that also possesses helicase activity, together with additional sites important for DNA and protein interaction. By contrast, in Drosophila, the exonuclease activity of WRN (DmWRNexo) is encoded by a distinct genetic locus from the presumptive helicase, allowing biochemical (and genetic) dissection of the role of the exonuclease activity in genome stability mechanisms. Here, we demonstrate a fluorescent method to determine WRN exonuclease activity using purified recombinant DmWRNexo and end-labeled fluorescent oligonucleotides. This system allows greater reproducibility than radioactive assays as the substrate oligonucleotides remain stable for months, and provides a safer and relatively rapid method for detailed analysis of nuclease activity, permitting determination of nuclease polarity, processivity, and substrate preferences.

Exonucleases play critical roles in ensuring genome stability. Loss of WRN exonuclease function results in premature aging. Studying substrates and other requirements of the nuclease in vitro can help elucidate its role in vivo. Here we demonstrate a rapid and reproducible fluorescence-based assay to measure its nuclease activity.

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...

G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group developed an intra-molecular FRET sensor to detect the interaction between G&#945; subunits and GPCRs in live cells following agonist stimulation. Here, we detail the protocol for detecting changes in FRET between the &#946;2-adrenergic receptor and the G&#945;s C-terminus peptide upon treatment with 100 &#181;M isoproterenol hydrochloride as previously characterized1. Our FRET sensor is a single polypeptide consisting serially of a full-length GPCR, a FRET acceptor fluorophore (mCitrine), an ER/K SPASM (systematic protein affinity strength modulation) linker, a FRET donor fluorophore (mCerulean), and a G&#945; C-terminal peptide. This protocol will detail cell preparation, transfection conditions, equipment setup, assay execution, and data analysis. This experimental design detects small changes in FRET indicative of protein-protein interactions, and can also be used to compare the strength of interaction across ligands and GPCR-G protein pairings. To enhance the signal-to-noise in our measurements, this protocol requires heightened precision in all steps, and is presented here to enable reproducible execution.

Simple methods to detect the selective activation of G proteins by G protein-coupled receptors remain an outstanding challenge in cell signaling. Here, F&#1255;rster resonance energy transfer (FRET) biosensors have been developed by pairwise tethering a GPCR to G protein peptides to probe conformational changes at controlled concentrations in live cells.

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group ...

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli. Here, we describe an experimental method to detect the subcellular localization and internalization of GPCR in HEK293 cells with confocal microscopy. In addition, this experiment provides some details about cell culture and transfection. This protocol is compatible with a variety of widely available fluorescent markers and is applicable to the visualization of the subcellular localization of a majority of proteins, as well as of the internalization of GPCR. This technique should enable researchers to efficiently manipulate GPCR gene expression in mammalian cell lines and should facilitate studies on GPCR subcellular localization and internalization.

This protocol describes confocal microscopy detection of G protein-coupled receptor (GPCR) internalization in mammalian cells. It includes the basic cell culture, transfection, and confocal microscopy procedure and provides an efficient and easily interpretable method to detect the subcellular localization and internalization of fusion-expressed GPCR.

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent ...