Parallel Interrogation of β-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay

Summary

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 β-arrestin2 recruitment at approximately 300 GPCRs using a TEV-based reporter assay.

Abstract

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 β-arrestin2 recruitment assay, the universality of β-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'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.

Introduction

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–from neurotransmitters to nucleotides, peptides to photons, and many more–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 p....

Protocol

1. Primary screening: cell culture and plate seeding

1. To prepare poly-L-lysine (PLL)-coated plates, dispense 20 μL/well of a 25 μg/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 0.5–2 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 luminescence between adjacent wells.
2. To preserve the coated plates and wash off the excess PLL, remove the PLL by flicking it over the sink, tap dry over a pa....

Results

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 β-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 desir.......

Discussion

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

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
384 Well Optical Bottom Plates, Polystyrene Polymer Base, Cell Culture Treated, BLACK, with lid, Sterile	NUNC	12-566
384 Well Optical Bottom Plates, Polystyrene Polymer Base, Cell Culture Treated, White, with lid, Sterile	NUNC	12-566-1
384 Well Round Bottom, Polypropylene, Non-Treated, Blue, non-sterile, without lid	ThermoFisher	12-565-390
Antibiotic-Antimycotic	Wisent	450-115-EL
D-Luciferin, sodium salt	GoldBio	LUCNA
DMEM with L-Glutamine, 4.5g/L Glucose and Sodium Pyruvate	Corning	10-013-CV
Eppendorf Xplorer, 12-channel, variable, 15–300 µL	Eppendorf	4861000155
Eppendorf Xplorer, 12-channel, variable, 5–100 µL	Eppendorf	4861000139
Matrix Platemate 2x3	ThermoFisher	801-10001
MicroBeta 1450 Wallac	Perkin Elmer
Penicilin-Streptomycin	Wisent	450-201-EL
Poly-L-Lysine hydrobromide	Millipore-Sigma	P2636-500MG
Roth Lab PRESTO-Tango GPCR Kit	Addgene	Kit #1000000068