As central orchestrators of cellular and physiological processes, G protein-coupled receptors (GPCRs) mediate the transduction of extracellular stimuli into conformationally-driven intracellular signals. Comprised of more than 800 members in the human genome, the diversity of this superfamily of membrane proteins is shaped by both the multiplicity of ligands they respond to, as well as the diverse array of signaling pathways they coordinate. Moreover, GPCRs function in conjunction with protein interactors, whose identities and abundances vary by virtue of tissue- and/or cellular-specific expression.
The dynamism of GPCR signaling events is due to the receptors’ conformational and locational changes throughout their life cycle, including activation, desensitization, internalization and resensitization. Although there is great diversity of ligands among them, GPCRs share a common fundamental mechanism of receptor activation. GPCRs in their inactive conformation are coupled to a heterotrimeric G-proteins complex, formed of a Gα subunit bound to GDP and Gβγ dimer stabilizing the inactive conformation of the heterotrimer. Activation of the GPCR results in conformational changes which enable the exchange of bound GDP by Gα for GTP, resulting in the dissociation of Gα-GTP and Gβγ-subunits from the receptor, which transduce different downstream signaling cascades depending on the nature of the GPCR and the subclasses of the G-protein subunits, composing the basis of G-protein dependent signaling. This classical paradigm posits that activation can be induced not only by agonist binding, but also by virtue of GPCRs’ ability to spontaneously adopt active conformations in the absence of agonist, termed constitutive activity. Although it is now widely recognized that all GPCRs exhibit spontaneous activation, albeit at varying degrees, a large-scale quantification of constitutive activity across the GPCRome, including druggable and orphan receptors, has yet to be conducted.
To prevent overstimulation, active GPCRs can be desensitized, wherein kinases such as GRKs phosphorylate the receptor at specific serine/threonine residues, typically C-terminal or intracellular loop 3 (IL3) sites. Phosphorylation in turn leads to the recruitment of arrestins, the most well-known and characterized scaffold proteins comprising four isoforms, the two visual arrestins, arrestin-1 and arrestin-4, that are confined to retinal cones and rods, and the ubiquitously expressed nonvisual arrestins, β-arrestin-1 and −2. While nonvisual arrestins have been shown to bind to hundreds of different GPCR members, the vast majority of demonstrations have been conducted with β-arrestin-2, with few studies addressing the contributions of the relevant but often forgotten β-arrestin-1 isoform. Besides inducing receptor desensitization through steric hindrance of the G-protein binding site, arrestins also redirect GPCR signaling to alternative G-protein independent pathways such as MAPKs, JNKs, and Src. Additionally, the engagement of arrestin initiates receptor internalization via dynamin- and clathrin-dependent endocytosis. Besides canonical arrestin-mediated endocytosis, increasing evidence has emerged describing GPCRs internalizing independently of arrestins.
Herein, we describe a comprehensive screening and interrogation platform evolved from the PRESTO-Tango, capable of the simultaneous interrogation of β-arrestin-2 recruitment at ~300 non-olfactory druggable GPCRs. The reconstruction of this system was sought in part to increase the dynamic range and sensitivity of the original system, specifically improving the TRE promoter and TEV protease elements, and to expand its versatility beyond monitoring β-arrestin-2. Indeed, our platform, named Tango-Trio, includes monoclonal cell lines expressing trackers of β-arrestin-2, β-arrestin-1, and FYVE domain for internalization, all sharing the common luciferase reporter lineage. Moreover, their cumate-inducible nature enables the study of the various GPCR state-dependent and independent activities. Hereafter, we refer to the following states: the manifest agonist-induced active state; the constitutive active state, which represents ligand-independent activated receptor; steady-state, which refers to state-independent interaction level; and the basal level, which includes the steady-state plus constitutively active receptor pool, which cannot be discriminated in most cases. We are revealing divergent basal versus agonist-dependent β-arrestin-1/2 couplings, selectivities, and receptor endocytosis signatures across the GPCRome. We report the basal sigmoidal-fitted activities of more than 200 class A GPCRs, including ~50 orphans. Our findings represent a step towards uncovering the differences behind the mechanisms of constitutive versus agonist-induced activation, as well as state-independent activity. Moreover, we believe the Tango-Trio platform could facilitate the development of new GPCR-acting drugs and deorphanization efforts.
The PRESTO-Tango has a number of advantages, including selective read-out as the response is specific to the target receptor, sensitivity due to signal integration to produce a read-out, and the ability to study a multitude of GPCRs as the assay is independent of the G protein family the receptor signals through. As such, we exploited these strategic features to undergird the development of the Tango-Trio platform, while addressing its original limitations, chiefly the tetracycline-response element (TRE) promoter and tobacco etch virus (TEV) protease.
To stringently control gene expression, tTA binding to tetO7 permits transcriptional activation of the luciferase reporter. However, the main limitation to the Tet system is the leakiness due to the strong positional effects on the tetO7 minimal promoter, resulting in relatively high background transcription. In turn, this would lead to basal expression that would not be dependent on the tTA, which is intended to be cleaved from the GPCR by the β-arrestin2-TEV fusion protein. The second-generation promoter called TRE-Tight (Clonetech), redesigned tetO7 to remove potential bindings sites of endogenous transcription factors within the operon such as ISRE and GATA, renders this promoter virtually silent in the absence of induction. As expected, lower RLU counts were obtained with TRE-Tight, but the induction factor remained higher for the TRE-Tight promoter (4.5 fold) compared to TRE (2.7 fold) (Fig. 1a); the dopamine D2 receptor (DRD2) Tango receptor was used as it is a strong β-arrestin2 recruiter. Thus, the minimal basal leakage and increased fold window suggest TRE-Tight to be an improved promoter for Tango-Trio and reduce potential arrestin-independent modulation of the reporter activity.
Fig. 1: Optimization of the dynamic range, sensitivity, and specificity of the Tango-Trio platform.
a Comparison of TRE and TRE-Tight. Promoters were cloned upstream luc2, and expression vectors were transfected in HEK293T cells along with the β-arrestin2-TEV fusion protein and DRD2. Transfected cells were stimulated with the DRD2 specific agonist quinpirole. b Selection and pharmacological characterization of the monoclonal reporter cell line HTTL (HEK293T-TRE-Tight-Luc2) compared to the original HTL (HEK293T-TRE-Luc) cell line. c Comparison of TEV and TEV219 proteases. β-arrestin2 was cloned to both proteases, and transfected in HTL cells with DRD2. Transfected cells were stimulated with the DRD2 specific agonist quinpirole. HTTL-B2 and HTLA were transfected with HTR2A (e), HTR2B (f), HTR1B (g), and F2R (h) and stimulated, along with untransfected cells (d), with dose-response curve of PMA and in presence/absence of 10 µM JAK inhibitor I. HTTL-B2 and HTLA dose-response curves at various targets: DRD2 to quinpirole (i), HTR5A to serotonin (j), CHRM4 to carbachol (k), OPRM1 to DAMGO (l), ADRB3 to isoproterenol (m), and PTGDR to prostaglandin D2 (n). o–r Comparison of the specificity of HTTL-B2 and HTLA readouts. Cell lines were transfected with GPCRs that activate the Jak/STAT Pathway and stimulated with serial dilutions of untreated FBS (o), heat-inactivated (p), dialyzed (q), and Tet-System Approved (r) sera. HTTL-B2 was maintained in cumate-containing media throughout. Dose- response curves were built using XY analysis for non-linear regression curve and the 3-parameters dose-response stimulation function. Data are presented as mean values, with error bars representing SD. Data are representative of 2 biological replicates, with 3 technical replicates each. Generic receptor codes refer to the GPCR-Tango constructs.
Based on these observations, we generated a monoclonal TRE-Tight Luciferase reporter cell line for the Tango-Trio platform as an improvement over the HTL (HEK293T cells stably expressing TRE-Luc) cells of the original Tango assay (Fig. 1b). Although it is unknown whether Luc or Luc+ was used in creating the HTL cell line, we opted to clone TRE-Tight upstream the Luc2 gene (Promega), a markedly improved variant over its predecessors with significantly lower levels of cryptic transcription from the coding region and codon optimized expression. Henceforth referred to as HTTL (HEK293T TRE-Tight-Luc), our reporter cell line had a comparable level of maximal expression to HTL but possessed a much lower baseline compared to its counterpart, resulting in a larger induction factor.
The Tango system involves a protein fusion consisting of β-arrestin-2 with TEV, which cleaves the engineered GPCR following β-arrestin-2 recruitment to the receptor to release the tTA. However, one limitation of the WT TEV is that it undergoes self-cleavage, generating a trun
