Name: parallel interrogation of &#946;-arrestin2 recruitment for ligand screening on a gpcr-wide scale using presto-tango assay
Uploaded: 2020-03-10T14:30:00
Description: Watch this Scientific Journal Video about Parallel Interrogation of Î²-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay at JoVE.com

This work was supported by the Canadian Institutes of Health Research (CIHR grant #MOP142219).

1. Primary screening: cell culture and plate seeding
<ol>
	<li>To prepare poly-L-lysine (PLL)-coated plates, dispense 20 &#956;L/well of a 25 &#956;g/mL stock solution of PLL in white or black 384-well optical bottom plates using an electronic multichannel pipette or a reagent dispenser. Incubate the plates at room temperature for 0.5&#8211;2 h. 
	NOTE: If using the black 384-well plates, expect the background signal to be lower compared to the white plates. Black plates are recommended to reduce bleed-through of ...

<ol>
	<li>Liapakis, 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=The+G-protein+coupled+receptor+family:+actors+with+many+faces.">The G-protein coupled receptor family: actors with many faces.</a> Current pharmaceutical design. 18 (2), 175-185 (2012).</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 (9), 639-650 (2002).</li><li>Kroeze, W. K., Sheffler, D. J., Roth, B. L. <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+at+a+glance.">G-protein-coupled receptors at a glance.</a> Journal of cell science. 116, 4867-4869 (2003).</li><li>Rask-Andersen, M., Alm&#233;n, M. S., 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=Trends+in+the+exploitation+of+novel+drug+targets.">Trends in the exploitation of novel drug targets.</a> Nature Reviews Drug Discovery. 10 (8), 579-590 (2011).</li><li>Ngo, T., 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=Identifying+ligands+at+orphan+GPCRs:+current+status+using+structure-based+approaches.">Identifying ligands at orphan GPCRs: current status using structure-based approaches.</a> British journal of pharmacology. 173 (20), 2934-2951 (2016).</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>Kimple, A. J., Bosch, D. E., Giguere, P. M., Siderovski, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulators+of+G-Protein+Signaling+and+Their+G+Substrates:+Promises+and+Challenges+in+Their+Use+as+Drug+Discovery+Targets.">Regulators of G-Protein Signaling and Their G Substrates: Promises and Challenges in Their Use as Drug Discovery Targets.</a> Pharmacological Reviews. 63 (3), 728-749 (2011).</li><li>Wettschureck, N., Offermanns, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+G+Proteins+and+Their+Cell+Type+Specific+Functions.">Mammalian G Proteins and Their Cell Type Specific Functions.</a> Physiological Reviews. 85 (4), 1159-1204 (2005).</li><li>Denis, C., Sauli&#232;re, A., Galandrin, S., S&#233;nard, J. -. M., Gal&#233;s, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+heterotrimeric+G+protein+activation:+applications+to+biased+ligands.">Probing heterotrimeric G protein activation: applications to biased ligands.</a> Current Pharmaceutical Design. 18 (2), 128-144 (2012).</li><li>Yin, 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=Lipid+G+protein-coupled+receptor+ligand+identification+using+beta-arrestin+PathHunter+assay.">Lipid G protein-coupled receptor ligand identification using beta-arrestin PathHunter assay.</a> The Journal of Biological Chemistry. 284 (18), 12328-12338 (2009).</li><li>Cheng, Z., 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=Luciferase+Reporter+Assay+System+for+Deciphering+GPCR+Pathways.">Luciferase Reporter Assay System for Deciphering GPCR Pathways.</a> Current Chemical Genomics. 4, 84-91 (2010).</li><li>Roth, B. L., Kroeze, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+Approaches+for+Genome-wide+Interrogation+of+the+Druggable+Non-olfactory+G+Protein-coupled+Receptor+Superfamily.">Integrated Approaches for Genome-wide Interrogation of the Druggable Non-olfactory G Protein-coupled Receptor Superfamily.</a> The Journal of Biological Chemistry. 290 (32), 19471-19477 (2015).</li><li>Jean-Charles, P. -. Y., Kaur, S., Shenoy, S. K. <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+Signaling+Through+&#946;-Arrestin-Dependent+Mechanisms.">G Protein-Coupled Receptor Signaling Through &#946;-Arrestin-Dependent Mechanisms.</a> Journal of Cardiovascular Pharmacology. 70 (3), 142-158 (2017).</li><li>Smith, J. S., Rajagopal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+&#946;-Arrestins:+Multifunctional+Regulators+of+G+Protein-coupled+Receptors.">The &#946;-Arrestins: Multifunctional Regulators of G Protein-coupled Receptors.</a> The Journal of Biological Chemistry. 291 (17), 8969-8977 (2016).</li><li>B&#246;hme, I., Beck-Sickinger, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illuminating+the+life+of+GPCRs.">Illuminating the life of GPCRs.</a> Cell Communication and Signaling. 7 (1), 16 (2009).</li><li>Fang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-Free+Receptor+Assays.">Label-Free Receptor Assays.</a> Drug discovery today. Technologies. 7 (1), 5-11 (2011).</li><li>Wang, T., 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=Measurement+of+&#946;-Arrestin+Recruitment+for+GPCR+Targets.+Assay+Guidance+Manual.">Measurement of &#946;-Arrestin Recruitment for GPCR Targets. Assay Guidance Manual.</a> Eli Lilly &amp; Company and the National Center for Advancing Translational Sciences. , (2004).</li><li>Barnea, 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=The+genetic+design+of+signaling+cascades+to+record+receptor+activation.">The genetic design of signaling cascades to record receptor activation.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (1), 64-69 (2008).</li><li>Kroeze, W. K., 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=PRESTO-Tango+as+an+open-source+resource+for+interrogation+of+the+druggable+human+GPCRome.">PRESTO-Tango as an open-source resource for interrogation of the druggable human GPCRome.</a> Nature Structural &amp; Molecular Biology. 22 (5), 362-369 (2015).</li><li>Jordan, M., Schallhorn, A., Wurm, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfecting+Mammalian+Cells:+Optimization+of+Critical+Parameters+Affecting+Calcium-Phosphate+Precipitate+Formation.">Transfecting Mammalian Cells: Optimization of Critical Parameters Affecting Calcium-Phosphate Precipitate Formation.</a> Nucleic Acids Research. 24 (4), 596-601 (1996).</li><li>Baker, J. M., Boyce, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+functional+screening+using+a+homemade+dual-glow+luciferase+assay.">High-throughput functional screening using a homemade dual-glow luciferase assay.</a> Journal of Visualized Experiments. (88), 50282 (2014).</li><li>Li, H., Eishingdrelo, A., Kongsamut, S., Eishingdrelo, H. <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+mediate+14-3-3+signal+transduction.">G-protein-coupled receptors mediate 14-3-3 signal transduction.</a> Signal Transduction and Targeted Therapy. 1 (1), 16018 (2016).</li><li>Walther, C., Ferguson, S. S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minireview:+Role+of+intracellular+scaffolding+proteins+in+the+regulation+of+endocrine+G+protein-coupled+receptor+signaling.">Minireview: Role of intracellular scaffolding proteins in the regulation of endocrine G protein-coupled receptor signaling.</a> Molecular Endocrinology. 29 (6), 814-830 (2015).</li><li>Gurevich, E. V., Benovic, J. L., Gurevich, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arrestin2+expression+selectively+increases+during+neural+differentiation.">Arrestin2 expression selectively increases during neural differentiation.</a> Journal of Neurochemistry. 91 (6), 1404-1416 (2004).</li><li>Shaikh, S., Nicholson, L. F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+the+Tet-On+system+for+inducible+expression+of+RAGE.">Optimization of the Tet-On system for inducible expression of RAGE.</a> Journal of Biomolecular Techniques JBT. 17 (4), 283-292 (2006).</li><li>Blau, H. M., Rossi, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tet+B+or+not+tet+B:+advances+in+tetracycline-inducible+gene+expression.">Tet B or not tet B: advances in tetracycline-inducible gene expression.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (3), 797-799 (1999).</li><li>Roche, O., 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=Development+of+a+Virtual+Screening+Method+for+Identification+of+"Frequent+Hitters"+in+Compound+Libraries.">Development of a Virtual Screening Method for Identification of "Frequent Hitters" in Compound Libraries.</a> Journal of Medicinal Chemistry. 45 (1), 137-142 (2002).</li></ol>

The conformationally dynamic GPCRs are powerhouses of signal transduction. The physiochemical properties of the binding pockets of these heptahelical receptors, as well as their physiological relevance underscore the need for GPCR ligand screening tools. As presented above, the PRESTO-Tango assay is rapid, sensitive and user-friendly, lending itself to drug development. Not only does this assay measure agonist-induced activation, but it can also be used to quantify the activity of antagonists and allosteric modulators<su...

G-protein-coupled receptors (GPCRs) constitute the largest and most diverse family of transmembrane proteins, operating as communication interfaces between a cell and its environment1. The versatility of GPCRs is highlighted by their ability to detect a diverse array of ligands&#8211;from neurotransmitters to nucleotides, peptides to photons, and many more&#8211;as well as their ability to regulate numerous downstream signaling cascades involved in cellular growth, migration, differentiation, apoptosis, cell firing, etc.2,3. Considering their ubiquity and involvement in a multitude of physiological processes, this receptor family is of utmost therapeutic importance, showcased by the fact that more than a third of currently available prescribed medications target GPCRs4. However, these existing therapeutics only target a small subset of the superfamily (an estimated 10%), and the pharmacology of many GPCRs remains unelucidated. Moreover, more than 100 GPCRs exist as orphan receptors, as they have not been matched with an endogenous ligand5. Thus, GPCR ligand screening is critical in deorphanization and drug development, as it paves the path towards lead discovery and optimization, and possibly to the clinical trial phase.
Methods for GPCR ligand screening have traditionally fallen in one of two categories, G-protein dependent or G-protein independent functional assays6. GPCR signaling is regulated by heterotrimeric G-proteins (G&#945;&#946;&#947;), which are activated by the exchange of GTP for GDP bound on the G&#945; subunit7. Signals from the activated receptor are transduced by G-proteins via secondary messengers, such as cAMP, Calcium, DAG, and IP3, to mediate downstream signaling at downstream effectors8. The nature of the functional consequences of G-protein signaling has been exploited to create cell-based assays that reflect receptor activation. These methods, which measure proximal (direct) or distal (indirect) events in G-protein signaling, are most frequently used for GPCR ligand screening and have been principally employed in deorphanization studies6. Examples of assays that directly measure GPCR-mediated G-protein activation include the [35S]GTP&#947;S binding assay, which measures binding of a radiolabeled and non-hydrolyzable GTP analog to the G&#945; subunit, and F&#246;rster/bioluminescence resonance energy transfer (FRET/BRET, respectively) probes to monitor GPCR-G&#945; and G&#945;/G&#947; interactions, which have been gaining more traction over the years9,10. Assays that monitor distal events are the most commonly used tools for GPCR profiling; for example, cAMP and IP1/3 assays measure intracellular accumulation of G-protein dependent secondary messengers, whereas [Ca2+] flux and reporter assays involving specific response elements implicated in G-protein activation (CRE, NFAT-RE, SRE, SRF-RE) examine events further downstream the signaling cascade11. While most of the aforementioned assays can be performed at a high-throughput level, are fairly sensitive, and boast certain assay-specific advantages (e.g., discrimination between full/partial agonists, neutral antagonists and inverse agonists in the case of GTP&#947;S binding, or assay functionality on live cells such as [Ca2+] and IP1/3)6, there are unfortunately no existing G-protein dependent methods befitting the interrogation of the entire druggable GPCR-ome. This is largely due to the native coupling of multiple G-protein subfamilies to GPCRs, resulting in signaling at several cascades and the unknown G-protein coupling at orphan GPCRs. To mitigate this issue, assays have been developed to force promiscuous G-protein coupling through a single common signaling read-out, such as cAMP, and Ca2+, albeit most of them are low-throughput12.
An important aspect of the GPCR lifecycle is the termination of G-protein-dependent signaling, which occurs in large part through the recruitment of &#946;-arrestins which induces dissociation of the G-protein, and ultimately desensitizing the receptor, which is targeted for clathrin-coated internalization13. The most ubiquitously expressed isoforms of &#946;-arrestin are the non-visual &#946;-arrestin1 and &#946;-arrestin2, also denoted as arrestin-2 and arrestin-3, respectively14. Enter G-protein independent cell-based assays, which add a new dimension to GPCR ligand screening; receptor trafficking, label-free whole cell, and &#946;-arrestin recruitment assays are all notable examples. GPCR trafficking assays employ fluorophore-labeled ligands or co-internalized antibodies targeting the receptor15, whereas label-free whole cell assays use biosensors which translate cellular changes induced by ligand binding into quantifiable outputs, such as electrical or optical signals16. Notably, quintessential GPCR- &#946;-arrestin interactions fashion the &#946;-arrestin recruitment assay as an attractive tool in the repertoire of functional assays17. The Tango system, first developed by Barnea et al. only a decade ago, involves the introduction of three exogenous genetic elements: a protein fusion consisting of &#946;-arrestin2 with a tobacco etch virus protease (TEVp), a tetracycline transactivator (tTA) that is tethered to a GPCR via a tobacco etch virus protease cleavage site (TEVcs) and is preceded by a sequence from the C-terminus of the V2 vasopressin receptor (V2 tail) to promote arrestin recruitment, and a reporter luciferase gene whose transcription is triggered by the tTA transcription factor translocation to the nucleus, which is freed from the membrane anchoring following &#946;-arrestin2 recruitment (Figure 1)18. Quantitative readings of GPCR activation and &#946;-arrestin2 recruitment can be subsequently determined by reading for luminescence. A notable distinction is that while receptor trafficking and label-free whole cell methods are relatively low-throughput, the Tango has several advantages, including selective read-out that is specific to the target receptor and sensitivity due to signal integration, which make it a suitable candidate for ligand screening on a larger scale18.
In view of these strategic features, Kroeze et al. developed PRESTO-Tango (Parallel Receptor-ome Expression and Screening via Transcriptional Output-Tango), a high-throughput open-source platform that uses the Tango approach to profile the druggable GPCR-ome in a parallel and simultaneous manner19. Exploiting the &#34;promiscuous&#34; recruitment of &#946;-arrestin2 to nearly all GPCRs, PRESTO-Tango is the first-of-its-kind in terms of cell-based functional assays, enabling rapid &#34;first-round&#34; screening of small molecule compounds at almost all non-olfactory GPCRs, including orphans, independent of the G-protein subfamily coupling.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>384 Well Optical Bottom Plates, Polystyrene Polymer Base, Cell Culture Treated, BLACK, with lid, Sterile</td>
			<td>NUNC</td>
			<td>12-566</td>
			<td></td>
		</tr>
		<tr>
			<td>384 Well Optical Bottom Plates, Polystyrene Polymer Base, Cell Culture Treated, White, with lid, Sterile</td>
			<td>NUNC</td>
			<td>12-566-1</td>
			<td></td>
		</tr>
		<tr>
			<td>384 Well Round Bottom, Polypropylene, Non-Treated, Blue, non-sterile, without lid</td>
			<td>ThermoFisher</td>
			<td>12-565-390</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Wisent</td>
			<td>450-115-EL</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Luciferin, sodium salt</td>
			<td>GoldBio</td>
			<td>LUCNA</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM with L-Glutamine, 4.5g/L Glucose and Sodium Pyruvate</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Xplorer, 12-channel, variable, 15&#8211;300 &#181;L</td>
			<td>Eppendorf</td>
			<td>4861000155</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Xplorer, 12-channel, variable, 5&#8211;100 &#181;L</td>
			<td>Eppendorf</td>
			<td>4861000139</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrix Platemate 2x3</td>
			<td>ThermoFisher</td>
			<td>801-10001</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroBeta 1450 Wallac</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin-Streptomycin</td>
			<td>Wisent</td>
			<td>450-201-EL</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine hydrobromide</td>
			<td>Millipore-Sigma</td>
			<td>P2636-500MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Roth Lab PRESTO-Tango GPCR Kit</td>
			<td>Addgene</td>
			<td>Kit #1000000068</td>
			<td></td>
		</tr>
	</tbody>
</table>

parallel interrogation of &#946;-arrestin2 recruitment for ligand screening on a gpcr-wide scale using presto-tango assay

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent one of the most promising targets for the pharmaceutical industry. Ergo, the design, implementation, and optimization of GPCR ligand screening assays is crucial, as they represent remote-control tools for drug discovery and for manipulating GPCR pharmacology and outcomes. In the past, G-protein dependent assays typified this area of research, detecting ligand-induced events and quantifying the generation of secondary messengers. However, since the advent of functional selectivity, as well as an increased awareness of several other G protein-independent pathways and the limitations associated with G-protein dependent assays, there is a greater push towards the creation of alternative GPCR ligand screening assays. Towards this endeavor, we describe the application of one such resource, the PRESTO-Tango platform, a luciferase reporter-based system that enables the parallel and simultaneous interrogation of the human GPCR-ome, a feat which was previously considered technically and economically unfeasible. Based on a G-protein independent &#946;-arrestin2 recruitment assay, the universality of &#946;-arrestin2-mediated trafficking and signaling at GPCRs makes PRESTO-TANGO an apt tool for studying approximately 300 non-olfactory human GPCRs, including approximately 100 orphan receptors. PRESTO-Tango&#39;s sensitivity and robustness make it suitable for primary high-throughput screens using compound libraries, employed to uncover new GPCR targets for known drugs or to discover new ligands for orphan receptors.

Using the PRESTO-Tango protocol presented herein, a chromaffin granule (CG) extract was screened against 168 non-olfactory GPCR targets, with the majority being orphan receptors. Profiling of said extract was performed by examining &#946;-arrestin2 mobilization at the chosen receptors, based on the principle designed by Barnea et al.18 (Figure 1). Plasmid cDNA of the GPCRs of interest was taken from the PRESTO-Tango GPCR Kit and assembled in two 96 well-plates in the ...

Watch this Scientific Journal Video about Parallel Interrogation of Î²-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay at JoVE.com

Parallel Interrogation of &#946;-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay

Given that GPCRs are attractive druggable targets, GPCR ligand screening is thus indispensable for the identification of lead compounds and for deorphanization studies. Towards these efforts, we describe PRESTO-Tango, an open-source resource platform used for simultaneous profiling of transient &#946;-arrestin2 recruitment at approximately 300 GPCRs using a TEV-based reporter assay.

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent ...

The Presto-Tango platform was devised to execute the parallel and simultaneous interrogation of all non-olfactory GPCRs in the human genome, including that orphan targets to profile ligand-induced beta-arrestin 2 recruitment. The Presto-Tango platform is the only open source resource for profiling the entire GPCR genome in a parallel approach, using a G protein independent beta-arrestin recruitment assay. Demonstrating the processor would be Genevieve Laroche a Research Associate in my lab, and Manel Zighal, a PhD candidate in my lab.Before beginning the procedure add 20 microliters of a 25 microgram per milliliter poly-L-lysine solution to each well of the appropriate experimental number of 384 optical bottom plates and incubate the plates at room temperature for 30 minutes to two hours. At the end of the incubation, flick the plates over the sink to remove the excess poly-L-lysine and add 40 microliters of antibiotic antimycotic solution to each well. Then incubate the plates needed for cell seeding at 37 degrees Celsius and place the rest of the plates at four degrees Celsius for long term storage.To seed HTLA cells for primary screening, gently rinse confluent 150 millimeters cell cultures with 10 milliliters of PBS before treating the cultures with six milliliters of 0.05%trypsin EDTA per dish. When the cells have detached pool the cell suspensions in a 50 milliliter conical tube containing an equal volume of DMEM. And collect the cells by centrifugation.Re-suspend the pellet at a 2.2 times 10 to the fifth cells per milliliter of DMEM concentration and flick the plates over the sink to remove the antibiotic antimycotic solution. Tap the plates to dry and seed 45 microliters of cells into each well. Then place the plates at 37 degrees Celsius and 45%carbon dioxide overnight.To prepare the 384 well DNA source plate for transfection distribute 50 nanograms per milliliter of each plasmid complimentary DNA encoding the GPRC Tango construct of interest in 0.1X Tris-EDTA per well into each well of a 96 well plate. Use a multi-channel pipette to manually transfer 10 microliters of DNA solution from each well of the 96 well plate to individual wells of one 384 well DNA source plate in quadruplicate for each condition as illustrated. Next, transfer 40 microliters of a 0.313 molar calcium chloride solution to each well of the DNA source plate and gently mix the contents of each well.Add 50 microliters of 2x HEPES buffer to each well of the 384 well DNA source plate with gentle mixing. After one minute transfer 10 microliters of the DNA transfection mixture from the 384 well DNA source plate to each well of seeded HTLA cells and incubate the cells in the cell culture incubator overnight. The next day decant the transfected cell medium and slowly add 40 microliters of starving medium to each well taking care not to touch the cells directly.Then add 20 microliters of the vehicle buffer for the alternating rows without compound in 20 microliters of the compound of interest at a three times concentration into the alternating rows, before returning the plate to the cell culture incubator. 16 to 24 hours following the stimulation decant the transfected cell medium and add 20 microliters of freshly prepared glow reagent to each well for a five to 20 minute incubation at room temperature protected from light. Then read the plates on a micro-plate luminescence counter with an integration time of one second per well.For a secondary screening, subculture the HTLA cells in 100 millimeter dishes at a five times 10 to the six cells total cell density in 11 millimeters of complete medium per dish for 24 hours at 37 degrees Celsius. The next day, mixed 10 micrograms of GPCR complimentary DNA with 500 microliters of Tris-EDTA calcium chloride solution with vortexing followed by the addition of 500 microliters of HEPES buffer. After vigorous shaking, incubate the solution for one minute at room temperature and immediately add one milliliter of the solution drop wise onto the cells.Gently rock to evenly distribute the precipitate and place the plate at 37 degrees Celsius for 24 hours. The next day, check the transfection efficiency under a fluorescent cell imager. Transactions greater than 50%coverage are ideal.Gently rinse the transfected cells with versene solution before detaching the cells with three milliliters of 0.05%trypsin EDTA. Collect the dissociated cells by centrifugation and re-suspend the pellet at a four times 10 to the fifth cells per milliliter of starving medium concentration. Then seed 45 microliters of cells into each well of a poly-L-lysine coded 384 well plate and place the plate in the cell culture incubator for at least four hours.To prepare a half log dose-curve response plate add 270 microliters of HBSS supplemented with HEPES and antibiotic antimycotic to all but the last row of a 96 well plate and add 30 microliters of each high and low drug solution to the wells of row H.Transfer the solution from each well of row H into the corresponding well of row G with mixing and continue to serially dilute the drug compounds until the last row of the plate is reached. Using the schematic as a reference, mix 20 microliters of the low column dilutions from rows A through G of the 96 well plate and 20 microliters of the high column dilutions to wells B through H of the 96 well plate to the previously seated 384 well plate. Then incubate the plate at 37 degrees Celsius for a minimum of 16 hours.16 to 24 hours following the stimulation decant the transfected cell medium and add 20 microliters of glow reagent to each well for a five to 20 minute incubation before reading the plates on a micro plate luminescence counter with an integration time of one second per well. In this representative experiment, out of the 168 GPCRs that were interrogated in the primary screening, only dopamine receptor D3 and opsin 5 were contenders as potential active targets. Dopamine receptor D3 produced a significant log2 fold change of 4.7 whereas opsin 5 produced a slightly lower response of 2.39.By comparison, dopamine receptor D2, the positive control for the primary screen produced a log2 fold change of 4.58. Those response curves from the secondary screening demonstrated that chromatin granule extract produce similar signal windows in half maximal effective concentration values to quinpirole confirming its validity as an active hit at dopamine receptor D3.A flat dose-curve similar to the negative control was produced for opsin 5 however ruling out this receptor as a possible target for the chromatin granule extract. Presto-Tango requires special care as variabilities in cell distribution, transfection efficiency and serial drug dilutions can result in compounded error, which can affect the accuracy of the data.Orthogonal methods such as Radioligand-binding approaches, Bioluminescence Resonance Energy Transfer and Cyclic AMP calcium and internalization functional assays can be used to further confirm and characterize ligand receptor interactions.

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 ...

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in ...

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 ...

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts ...

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

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 ...

Screening for Phytoestrogens using a Cell-based Estrogen Receptor  &#946; Reporter Assay

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in ...

NMR-Based Fragment Screening in a Minimum Sample but Maximum Automation Mode

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest ...