Name: a "dual-addition" calcium fluorescence assay for the high-throughput screening of recombinant g protein-coupled receptors
Uploaded: 2022-12-02T13:20:00
Description: Watch this Scientific Journal Video about A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors at JoVE.com

This work was supported by the USDA-NIFA-AFRI Animal Health and Well-Being Award (Award number 2022-67015-36336, PVP [Project Director]) and from competitive funds from the Texas A&#38;M AgriLife Research Insect Vector Diseases Grant Program (FY&#39;22-23) to P.V.P. The A.W.E.S.O.M.E. faculty group of the College of Agriculture and Life Sciences, TAMU, is acknowledged for help editing the manuscript. Supplemental Table S2 contains data from an in-house, random, small-molecule library obtained from Dr. James Sacchettini&#39;s laboratory at Texas A&#38;M University and Texas A&#38;M AgriLife Research.

1. Cell maintenance
NOTE: A CHO-K1 cell line that stably expresses the kinin receptor from R. microplus, named BMLK3, was developed by Holmes et al.16. The details of the development of the cell line are presented elsewhere14. All the following steps are performed under sterile conditions in a class II biosafety cabinet.
<ol>
	<li>Grow the recombinant cell line in selective media (F-12K medium containing 10%...

<ol>
	<li>Hanlon, C. D., Andrew, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outside-in+signaling+-+A+brief+review+of+GPCR+signaling+with+a+focus+on+the+Drosophila+GPCR+family.">Outside-in signaling - A brief review of GPCR signaling with a focus on the Drosophila GPCR family.</a> Journal of Cell Science. 128 (19), 3533-3542 (2015).</li><li>Liu, N., Li, T., Wang, Y., Liu, 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+(GPCRs)+in+insects-A+potential+target+for+new+insecticide+development.">G-protein coupled receptors (GPCRs) in insects-A potential target for new insecticide development.</a> Molecules. 26 (10), 2993 (2021).</li><li>Pierce, K. L., Premont, R. T., Lefkowitz, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seven-transmembrane+receptors.">Seven-transmembrane receptors.</a> Nature Reviews Molecular Cell Biology. 3, 639-650 (2002).</li><li>Pietrantonio, P. V., Xiong, C., Nachman, R. J., Shen, Y. <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+in+arthropod+vectors:+Omics+and+pharmacological+approaches+to+elucidate+ligand-receptor+interactions+and+novel+organismal+functions.">G protein-coupled receptors in arthropod vectors: Omics and pharmacological approaches to elucidate ligand-receptor interactions and novel organismal functions.</a> Current Opinion in Insect Science. 29, 12-20 (2018).</li><li>Hilger, D., Masureel, M., Kobilka, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+dynamics+of+GPCR+signaling+complexes.">Structure and dynamics of GPCR signaling complexes.</a> Nature Structural &amp; Molecular Biology. 25 (1), 4-12 (2018).</li><li>Liu, N., Wang, Y., Li, T., Feng, X. <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+(GPCRs):+Signaling+pathways,+characterization,+and+functions+in+insect+physiology+and+toxicology.">G-protein coupled receptors (GPCRs): Signaling pathways, characterization, and functions in insect physiology and toxicology.</a> International Journal of Molecular Sciences. 22 (10), 5260 (2021).</li><li>Hansen, K. B., Br&#228;uner-Osborne, H., Leifert, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FLIPR&#174;+assays+of+intracellular+calcium+in+GPCR+drug+discovery.">FLIPR&#174; assays of intracellular calcium in GPCR drug discovery.</a> G Protein-Coupled Receptors in Drug Discovery. , (2009).</li><li>Bauknecht, P., Jekely, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+combinatorial+deorphanization+of+Platynereis+neuropeptide+GPCRs.">Large-scale combinatorial deorphanization of Platynereis neuropeptide GPCRs.</a> Cell Reports. 12 (4), 684-693 (2015).</li><li>Frooninckx, 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=Neuropeptide+GPCRs+in+C.+elegans.">Neuropeptide GPCRs in C. elegans.</a> Frontiers in Endocrinology. 3, 167 (2012).</li><li>Caers, J., 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=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> Frontiers in Endocrinology. 3, 151 (2012).</li><li>&#352;imo, L., Ko&#269;i, J., Park, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptors+for+the+neuropeptides,+myoinhibitory+peptide+and+SIFamide,+in+control+of+the+salivary+glands+of+the+blacklegged+tick+Ixodes+scapularis.">Receptors for the neuropeptides, myoinhibitory peptide and SIFamide, in control of the salivary glands of the blacklegged tick Ixodes scapularis.</a> Insect Biochemistry and Molecular Biology. 43 (4), 376-387 (2013).</li><li>&#352;imo, L., Park, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropeptidergic+control+of+the+hindgut+in+the+black-legged+tick+Ixodes+scapularis.">Neuropeptidergic control of the hindgut in the black-legged tick Ixodes scapularis.</a> International Journal for Parasitology. 44 (11), 819-826 (2014).</li><li>Liesch, J., Bellani, L. L., Vosshall, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+genetic+characterization+of+neuropeptide+Y-like+receptors+in+Aedes+aegypti.">Functional and genetic characterization of neuropeptide Y-like receptors in Aedes aegypti.</a> PLoS Neglected Tropical Diseases. 7 (10), 2486 (2013).</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> Journal of Visualized Experiments. (50), e2732 (2011).</li><li>Xiong, C., Baker, D., 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=The+cattle+fever+tick,+Rhipicephalus+microplus,+as+a+model+for+forward+pharmacology+to+elucidate+kinin+GPCR+function+in+the+Acari.">The cattle fever tick, Rhipicephalus microplus, as a model for forward pharmacology to elucidate kinin GPCR function in the Acari.</a> Frontiers in Physiology. 10, 1008 (2019).</li><li>Holmes, S. P., Barhoumi, R., Nachman, R. J., 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=Functional+analysis+of+a+G+protein-coupled+receptor+from+the+Southern+cattle+tick+Boophilus+microplus+(Acari:+Ixodidae)+identifies+it+as+the+first+arthropod+myokinin+receptor.">Functional analysis of a G protein-coupled receptor from the Southern cattle tick Boophilus microplus (Acari: Ixodidae) identifies it as the first arthropod myokinin receptor.</a> Insect Molecular Biology. 12 (1), 27-38 (2003).</li><li>Cox, K. J., 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=Cloning,+characterization,+and+expression+of+a+G-protein-coupled+receptor+from+Lymnaea+stagnalis+and+identification+of+a+leucokinin-like+peptide,+PSFHSWSamide,+as+its+endogenous+ligand.">Cloning, characterization, and expression of a G-protein-coupled receptor from Lymnaea stagnalis and identification of a leucokinin-like peptide, PSFHSWSamide, as its endogenous ligand.</a> Journal of Neuroscience. 17 (4), 1197-1205 (1997).</li><li>Dircksen, H., Kastin, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+32+-+Crustacean+bioactive+peptides.">Chapter 32 - Crustacean bioactive peptides.</a> Handbook of Biologically Active Peptides (Second Edition). , 209-221 (2013).</li><li>Halberg, K. A., Terhzaz, S., Cabrero, P., Davies, S. A., Dow, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+the+evolutionary+origins+of+insect+renal+function.">Tracing the evolutionary origins of insect renal function.</a> Nature Communications. 6, 6800 (2015).</li><li>Pietrantonio, P. V., Jagge, C., Taneja-Bageshwar, S., Nachman, R. J., Barhoumi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mosquito+Aedes+aegypti+(L.)+leucokinin+receptor+is+a+multiligand+receptor+for+the+three+Aedes+kinins.">The mosquito Aedes aegypti (L.) leucokinin receptor is a multiligand receptor for the three Aedes kinins.</a> Insect Molecular Biology. 14 (1), 55-67 (2005).</li><li>Radford, J. C., Davies, S. A., Dow, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+G-protein-coupled+receptor+analysis+in+Drosophila+melanogaster+identifies+a+leucokinin+receptor+with+novel+roles.">Systematic G-protein-coupled receptor analysis in Drosophila melanogaster identifies a leucokinin receptor with novel roles.</a> Journal of Biological Chemistry. 277 (41), 38810-38817 (2002).</li><li>Brock, C. M., 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=The+leucokinin-like+peptide+receptor+from+the+cattle+fever+tick,+Rhipicephalus+microplus,+is+localized+in+the+midgut+periphery+and+receptor+silencing+with+validated+double-stranded+RNAs+causes+a+reproductive+fitness+cost.">The leucokinin-like peptide receptor from the cattle fever tick, Rhipicephalus microplus, is localized in the midgut periphery and receptor silencing with validated double-stranded RNAs causes a reproductive fitness cost.</a> International Journal for Parasitology. 49 (3-4), 287-299 (2019).</li><li>N&#228;ssel, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leucokinin+and+associated+neuropeptides+regulate+multiple+aspects+of+physiology+and+behavior+in+Drosophila.">Leucokinin and associated neuropeptides regulate multiple aspects of physiology and behavior in Drosophila.</a> International Journal of Molecular Sciences. 22 (4), 1940 (2021).</li><li>Kim, Y. -. J., 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=Central+peptidergic+ensembles+associated+with+organization+of+an+innate+behavior.">Central peptidergic ensembles associated with organization of an innate behavior.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (38), 14211-14216 (2006).</li><li>Al-Anzi, 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=The+leucokinin+pathway+and+its+neurons+regulate+meal+size+in+Drosophila.">The leucokinin pathway and its neurons regulate meal size in Drosophila.</a> Current Biology. 20 (11), 969-978 (2010).</li><li>Yurgel, M. E., 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+single+pair+of+leucokinin+neurons+are+modulated+by+feeding+state+and+regulate+sleep-metabolism+interactions.">A single pair of leucokinin neurons are modulated by feeding state and regulate sleep-metabolism interactions.</a> PLoS Biology. 17 (2), 2006409 (2019).</li><li>N&#228;ssel, D. R., Zandawala, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+neuropeptide+signaling+in+Drosophila,+from+genes+to+physiology+and+behavior.">Recent advances in neuropeptide signaling in Drosophila, from genes to physiology and behavior.</a> Progress in Neurobiology. 179, 101607 (2019).</li><li>Okusawa, S., Kohsaka, H., Nose, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+and+downstream+leucokinin+neurons+modulate+larval+turning+behavior+in+Drosophila.">Serotonin and downstream leucokinin neurons modulate larval turning behavior in Drosophila.</a> Journal of Neuroscience. 34 (7), 2544-2558 (2014).</li><li>Kersch, C. N., 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=Mosquito+Aedes+aegypti+(L.)+leucokinin+receptor+is+critical+for+in+vivo+fluid+excretion+post+blood+feeding.">Mosquito Aedes aegypti (L.) leucokinin receptor is critical for in vivo fluid excretion post blood feeding.</a> FEBS letters. 585 (22), 3507-3512 (2011).</li><li>Kwon, H., 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=Leucokinin+mimetic+elicits+aversive+behavior+in+mosquito+Aedes+aegypti+(L.)+and+inhibits+the+sugar+taste+neuron.">Leucokinin mimetic elicits aversive behavior in mosquito Aedes aegypti (L.) and inhibits the sugar taste neuron.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (25), 6880-6885 (2016).</li><li>Xiong, C., Baker, D., 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+random+small+molecule+library+screen+identifies+novel+antagonists+of+the+kinin+receptor+from+the+cattle+fever+tick,+Rhipicephalus+microplus+(Acari:+Ixodidae).">A random small molecule library screen identifies novel antagonists of the kinin receptor from the cattle fever tick, Rhipicephalus microplus (Acari: Ixodidae).</a> Pest Management Science. 77 (5), 2238-2251 (2021).</li><li>Torfs, P., 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=The+kinin+peptide+family+in+invertebrates.">The kinin peptide family in invertebrates.</a> Annals of the New York Academy of Sciences. 897 (1), 361-373 (1999).</li><li>Ma, Q., Ye, L., Liu, H., Shi, Y., Zhou, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+Ca2++mobilization+assays+in+GPCR+drug+discovery.">An overview of Ca2+ mobilization assays in GPCR drug discovery.</a> Expert Opinion on Drug Discovery. 12 (5), 511-523 (2017).</li><li>Zhang, J. -. H., Chung, T. D., Oldenburg, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+statistical+parameter+for+use+in+evaluation+and+validation+of+high+throughput+screening+assays.">A simple statistical parameter for use in evaluation and validation of high throughput screening assays.</a> Journal of Biomolecular Screening. 4 (2), 67-73 (1999).</li><li>Zhang, R., Xie, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+for+GPCR+drug+discovery.">Tools for GPCR drug discovery.</a> Acta Pharmacologica Sinica. 33 (3), 372-384 (2012).</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+C.">G&#945;15 and G&#945;16 couple a wide variety of receptors to phospholipase C.</a> Journal of Biological Chemistry. 270 (25), 15175-15180 (1995).</li><li>Murgia, M. V., 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=High-content+phenotypic+screening+identifies+novel+chemistries+that+disrupt+mosquito+activity+and+development.">High-content phenotypic screening identifies novel chemistries that disrupt mosquito activity and development.</a> Pesticide Biochemistry and Physiology. 182, 105037 (2022).</li><li>Lismont, E., 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=Can+BRET-based+biosensors+be+used+to+characterize+G-protein+mediated+signaling+pathways+of+an+insect+GPCR,+the+Schistocerca+gregaria+CRF-related+diuretic+hormone+receptor.">Can BRET-based biosensors be used to characterize G-protein mediated signaling pathways of an insect GPCR, the Schistocerca gregaria CRF-related diuretic hormone receptor.</a> Insect Biochemistry and Molecular Biology. 122, 103392 (2020).</li></ol>

The goal of HTS is to identify hit molecules through screening massive numbers of small molecules. Therefore, the results from this example only represent a small part of a conventional HTS experiment. Furthermore, the hit molecules identified need to be validated in downstream assays such as a dose-dependent assay on the same recombinant cell line and on a CHO-K1 cell line carrying only the empty vector, which can be performed simultaneously to save small molecules. Cytotoxicity assays will help demonstrate that a lack ...

G protein-coupled receptors (GPCRs), which are present from yeast to humans, represent the largest superfamily of receptors in many organisms1. They play critical roles in regulating nearly all biological processes in animals. There are 50-200 GPCRs in the genome of arthropods, meaning they represent the largest membrane receptor superfamily2. They are classified into six major classes, A-F, based on their sequence similarity and functions3. GPCRs transduce various extracellular signals, such as those of hormones, neuropeptides, biogenic amines, glutamate, proton, lipoglycoproteins, and photons4. GPCRs couple to heterotrimer G proteins (G&#945;, G&#946;, and G&#947;) to transmit downstream signals. GPCRs coupled to G&#945;s or G&#945;i/o proteins increase or decrease, respectively, the intracellular 3&#39;, 5&#39;-cyclic adenosine monophosphate (cAMP) levels by activating or inhibiting adenylyl cyclase. GPCRs coupled to G&#945;q/11 induce calcium release from the endoplasmic reticulum calcium stores by activating the phospholipase C (PLC)-inositol-1,4,5-triphosphate (IP3) pathway. GPCRs coupled to G&#945;12/13 activate RhoGTPase nucleotide exchange factors5,6. GPCRs are the target of more than 50% of human drugs and an acaricide, amitraz4. As GPCRs transduce such diverse signals, they are promising targets for developing novel pesticides that disrupt invertebrate-specific physiological functions.
The goal of HTS is to identify hit molecules that can modulate receptor functions. HTS involves assay development, miniaturization, and automation7. Arthropod neuropeptide GPCRs are involved in most physiological functions, such as development, molting and ecdysis, excretion, energy mobilization, and reproduction4. Most of the neuropeptide GPCRs of arthropods and metazoans signal through the calcium signaling cascade2,6,8,9,10, such as in the myoinhibitory peptide and SIFamide receptors of the blacklegged tick Ixodes scapularis; their ligands are antagonistic in hindgut motility assays, with SIF eliciting contraction and MIP inhibiting it11,12. An NPY-like receptor of the yellow fever mosquito, Aedes aegypti, regulates female host seeking13. Compared to other alternative calcium mobilization assays such as the aequorin calcium bioluminescence assay14, the calcium fluorescence assay is easy to perform, does not require the transfection of other recombinant calcium detecting proteins, and is cost-effective. The calcium fluorescence assay produces a prolonged signal compared to the fast kinetic signal obtained in the aequorin calcium bioluminescence assay14,15.
In the example here, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, was recombinantly expressed in the CHO-K1 cell line and used for the calcium fluorescence assay. There is only one kinin receptor gene found in R. microplus; the receptor signals through a Gq protein-dependent signaling pathway and triggers the efflux of Ca2+ from calcium stores into the intracellular space16. This process can be detected and quantified by a fluorophore, which elicits a fluorescence signal when binding calcium ions (Figure 1).
The kinin receptor is an invertebrate-specific GPCR, which belongs to the Class A Rhodopsin-like receptors. Kinin is an ancient signaling neuropeptide that is present in Mollusca, Crustacea, Insecta, and Acari4,17,18. Coleopterans (beetles) lack the kinin signaling system; in the mosquito Aedes aegypti, there is only one kinin receptor that binds three aedeskinins, while Drosophila melanogaster has one kinin receptor with drosokinin as a unique ligand19,20,21. There are no homologous kinins or kinin receptors in vertebrates. Although the exact function of kinin is unknown in ticks, the kinin receptor RNAi-silenced females of R. microplus show significantly reduced reproductive fitness22. Kinins are pleotropic peptides in insects. In Drosophila melanogaster, they are involved in both the central and peripheral nervous regulatory systems23, pre-ecdysis24, feeding25, metabolism26, and sleep activity patterns26,27, as well as larval locomotion28. Kinins regulate hindgut contraction, diuresis, and feeding in the mosquito A. aegypti29,30,31. The kinin peptides have a conserved C-terminal pentapeptide Phe-X1-X2-Trp-Gly-NH2, which is the minimum required sequence for biological activity32. The arthropod specificity, the small size of the endogenous ligand, which makes them amenable to small-molecule interference, and the pleiotropic functions in insects make the kinin receptor a promising target for pest control4.
The &#34;dual-addition&#34; assay (Figure 2) allows the identification of agonists or antagonists in the same HTS assay15. It is adapted from a &#34;dual-addition&#34; assay that is commonly used in the pharmaceutical industry for drug discovery33. In brief, the first addition of drugs into the cell plate allows the identification of potential agonists in the chemical library when a higher fluorescence signal is detected compared to the application of the solvent control. After 5 min of incubation with these small molecules, a known agonist (kinin peptide) is applied to all the wells. Those wells that randomly received an antagonist from the drug plate display a lower fluorescence signal upon agonist addition compared to the control wells that received the solvent in the first addition. This assay then allows the identification of potential agonists and antagonists with the same cells. In a standard HTS project, these hit molecules would be further validated through dose-response assays and by additional biological activity assays, which are not shown here.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64505/64505fig01.jpg" /> 
Figure 1: Illustration of the calcium fluorescence assay mechanism. The Gq protein triggers the intracellular calcium signaling pathway. The kinin receptor (G protein-coupled receptor) was recombinantly expressed in CHO-K1 cells. When the agonist ligand binds to the receptor, the Gq protein associated with the kinin receptor activates PLC, which catalyzes the conversion of a PIP2 molecule into IP3 and DAG. IP3 then binds to the IP3R on the surface of the endoplasmic reticulum, leading to the release of Ca2+ into the cytoplasm, where Ca2+ ions bind to the fluorophores and elicit a fluorescence signal. The fluorescence signal can be obtained by excitation at 490 nm and detected at 514 nm. Abbreviations: GPCR = G protein-coupled receptor; PLC = phospholipase C; PIP2 = phosphatidylinositol 4,5-bisphosphate; IP3 = inositol trisphosphate; DAG = diacylglycerol; IP3R = IP3 receptor. Created with BioRender.com.&#160;<a href="https://www.jove.com/files/ftp_upload/64505/64505fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64505/64505fig02.jpg" /> 
Figure 2: The workflow for the high-throughput screening of small molecules on a G protein-coupled receptor expressed in CHO-K1 cells. (A)&#160;Recombinant CHO-K1 cells stably expressing the kinin receptor were added to the 384-well plate (10,000 cells/well) using a liquid handling system (25 &#181;L/well) and incubated in a humidified CO2 incubator for 12-16 h. (B)&#160;The assay buffer containing the fluorescent dye (25 &#181;L/well) was added into the cell plate using a liquid handling system. The plate was incubated for 30 min at 37 &#176;C for 30 min and equilibrated at RT for another 30 min. (C) The background fluorescence signal of the cells in each well was measured with a plate reader. (D)&#160;Drug solutions from a 384-well library plate and blank solvent (all at 0.5 &#181;L/well) were added into the cellular assay plate using a liquid handling system. (E) Cellular calcium fluorescence responses were measured with the plate reader immediately after the addition of the drug solutions; compound(s) eliciting higher than average fluorescence signals were picked out as agonist hit(s). Antagonist hits that block the GPCR (icon below) were revealed after the addition of the peptide agonist during step G. (F)&#160;In the same assay plate, after 5 min of incubation of the cells with screening compounds, an endogenous agonist peptide Rhimi-K-1 (QFSPWGamide) of the tick kinin receptor was added to each well (1 &#181;M). (G) Cellular fluorescence responses after the agonist peptide addition were measured by the plate reader immediately. Compound(s) inhibiting the fluorescence signal were selected as antagonist hit(s). Abbreviations: GPCR = G protein-coupled receptor; RT = room temperature; RFU = relative fluorescence units. Created with BioRender.com.&#160;<a href="https://www.jove.com/files/ftp_upload/64505/64505fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% trypsin-EDTA</td>
			<td>Gibco Invitrogen</td>
			<td>15050-065</td>
			<td>with phenol red</td>
		</tr>
		<tr>
			<td>0.4% trypan blue</td>
			<td>MilliporeSigma</td>
			<td>T8154</td>
			<td>liquid, sterile</td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>Thermo Fisher</td>
			<td>AM12400</td>
			<td>RNase-free Microfuge Tubes</td>
		</tr>
		<tr>
			<td>5 mL serological pipette</td>
			<td>Corning</td>
			<td>29443-045</td>
			<td>Corning Costar Stripette individually wrapped&#160;</td>
		</tr>
		<tr>
			<td>10 mL serological pipette</td>
			<td>Corning</td>
			<td>29443-047</td>
			<td>Corning Costar Stripette individually wrapped&#160;</td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Falcon</td>
			<td>352196</td>
			<td>sterile</td>
		</tr>
		<tr>
			<td>20 &#181;L filter tips</td>
			<td>USA Scientifc Inc.</td>
			<td>P1121</td>
			<td>sterile, barrier</td>
		</tr>
		<tr>
			<td>25 mL serological pipette</td>
			<td>Corning</td>
			<td>29443-049</td>
			<td>Corning Costar Stripette individually wrapped&#160;</td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Corning</td>
			<td>430828</td>
			<td>graduated, sterile</td>
		</tr>
		<tr>
			<td>150 mL auto-friendly reservior</td>
			<td>Integra Bioscience</td>
			<td>6317</td>
			<td>sterile, individually wrapped for cell seeding in day 1</td>
		</tr>
		<tr>
			<td>150 mL auto-friendly reservior</td>
			<td>Integra Bioscience</td>
			<td>6318</td>
			<td>sterile, stacked, for loading dye in day 2</td>
		</tr>
		<tr>
			<td>384/ 12.5 &#181;L low retention tips</td>
			<td>Integra Bioscience</td>
			<td>6405</td>
			<td>long, sterile filter</td>
		</tr>
		<tr>
			<td>384/ 12.5 &#181;L tips</td>
			<td>Integra Bioscience</td>
			<td>6404</td>
			<td>long, sterile filter</td>
		</tr>
		<tr>
			<td>384-well plate</td>
			<td>Greiner</td>
			<td>781091</td>
			<td>CELLSTAR, clear polystyrene, &#181;Clear, Black/Flat</td>
		</tr>
		<tr>
			<td>Aluminum plate seals</td>
			<td>Axygen Scientific</td>
			<td>PCR-AS-200</td>
			<td>polyester-based</td>
		</tr>
		<tr>
			<td>Aluminum foil wrap</td>
			<td>Walmart</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafty cabinet II</td>
			<td>NuAire</td>
			<td>NU-540-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counter</td>
			<td>Nexcelom</td>
			<td>AutoT4</td>
			<td></td>
		</tr>
		<tr>
			<td>cell counting slides</td>
			<td>Nexcelom</td>
			<td>SD-100</td>
			<td>20 &#181;L chamber</td>
		</tr>
		<tr>
			<td>CO2 humidified incubator</td>
			<td>Thermo Fisher</td>
			<td>Forma Series II</td>
			<td></td>
		</tr>
		<tr>
			<td>Desk Lamp</td>
			<td>SunvaleeyTEK</td>
			<td>RS1000B</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>MilliporeSigma</td>
			<td>276855</td>
			<td>anhydous, &#62;99.9%</td>
		</tr>
		<tr>
			<td>Drug plate</td>
			<td>Corning</td>
			<td>3680</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td>DPBS, 1x without calcium amd magnesium</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Koptec</td>
			<td>2000</td>
			<td></td>
		</tr>
		<tr>
			<td>F-12K Nutrient Mixture&#160;</td>
			<td>Corning</td>
			<td>45000-354</td>
			<td>(Kaighn&#39;s Mod.) with L-glutamine</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Equitech-Bio</td>
			<td>SFBU30</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent calcium assay kit</td>
			<td>ENZO Lifescience</td>
			<td>ENZ-51017</td>
			<td>10x96 tests</td>
		</tr>
		<tr>
			<td>G418 sulfate</td>
			<td>Gibco Invitrogen</td>
			<td>10131-027</td>
			<td>Geneticin selective antibiotic 50 mg/mL</td>
		</tr>
		<tr>
			<td>Hank&#39;s buffer</td>
			<td>MilliporeSigma</td>
			<td>55037C</td>
			<td>HBSS modified, with calcium, with magnesium, without phenol read</td>
		</tr>
		<tr>
			<td>HEPES buffer</td>
			<td>Gibco Invitrogen</td>
			<td>15630-080</td>
			<td>1 Molar</td>
		</tr>
		<tr>
			<td>HTS data storage plateform</td>
			<td>CDD vault&#160;</td>
			<td></td>
			<td>https://www.collaborativedrug.com/</td>
		</tr>
		<tr>
			<td>Liquid handling system</td>
			<td>Integra Bioscience</td>
			<td>Viaflo</td>
			<td>384/12.5 &#181;L</td>
		</tr>
		<tr>
			<td>Plate centrifuge</td>
			<td>Thermo Fisher</td>
			<td>Sorvall ST8</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>BMG technology</td>
			<td>Clariostar</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>MilliporeSigma</td>
			<td>P6407</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhimi-K-1 agonist peptide</td>
			<td>Genscript</td>
			<td>custom order</td>
			<td>QFSPWGamide</td>
		</tr>
		<tr>
			<td>T-75 flask</td>
			<td>Falcon</td>
			<td>353136</td>
			<td></td>
		</tr>
	</tbody>
</table>

a "dual-addition" calcium fluorescence assay for the high-throughput screening of recombinant g protein-coupled receptors

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens.
The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells.
The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a &#34;dual-addition&#34; approach can detect functional agonist and antagonist &#34;hit&#34; molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z&#39; factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 &#181;M final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

An in-house drug plate (SAC2-34-6170) composed of 320 random small molecules was used for demonstrating this HTS assay as an example. The HTS had excellent assay quality with a Z&#39; factor of 0.7 (Table 1). This&#160;Z&#39; factor reflects the assay quality independent of the tested compounds34. A Z&#8242;-factor of 0.5 or greater indicates a good assay signal dynamic range between the RFUs of the positive controls and the negative controls. Assays with a Z...

Watch this Scientific Journal Video about A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors at JoVE.com

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay.

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...

Research

JoVE Journal

Biochemistry

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced ...

Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein

Protein prenylation is a key modification that is responsible for targeting proteins to intracellular membranes. KRAS4b, which is mutated in 22% of human ...

Strategic Screening and Characterization of the Visual GPCR-mini-G Protein Signaling Complex for Successful Crystallization

The key to determining crystal structures of membrane protein complexes is the quality of the sample prior to crystallization. In particular, the choice ...

Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral ...