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 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 (Gαβγ), which are activated by the exchange of GTP for GDP bound on the Gα 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]GTPγS binding assay, which measures binding of a radiolabeled and non-hydrolyzable GTP analog to the Gα subunit, and Förster/bioluminescence resonance energy transfer (FRET/BRET, respectively) probes to monitor GPCR-Gα and Gα/Gγ interactions, which have been gaining more traction over the years^9,10. 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 GTPγS 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 β-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 β-arrestin are the non-visual β-arrestin1 and β-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 β-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- β-arrestin interactions fashion the β-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 β-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 β-arrestin2 recruitment (Figure 1)¹⁸. Quantitative readings of GPCR activation and β-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 "promiscuous" recruitment of β-arrestin2 to nearly all GPCRs, PRESTO-Tango is the first-of-its-kind in terms of cell-based functional assays, enabling rapid "first-round" screening of small molecule compounds at almost all non-olfactory GPCRs, including orphans, independent of the G-protein subfamily coupling.

Protocol

1. Primary screening: cell culture and plate seeding 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 …

Representative 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 …

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<su…

Materials

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 2×3	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