Monitoring G protein-coupled receptor activation using an adenovirus-based β-arrestin bimolecular fluorescence complementation assay

G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors and are involved in a variety of pathological conditions including cancer and cardiovascular, metabolic, neurological, and autoimmune diseases. GPCRs are being intensively investigated as targets for therapeutic intervention, and the β-arrestin recruitment assay has become a popular tool for analyzing GPCR activation. Here, we report a high-throughput method for cloning GPCR cDNAs into adenoviral bimolecular fluorescence complementation (BiFC) vectors and performing the β-arrestin BiFC assay in cells transduced with recombinant adenoviruses. An analysis of the activation of somatostatin receptor 2 (SSTR2) with the adenovirus-based β-arrestin BiFC assay showed that the assay is suitable for quantifying SSTR2 activation in response to specific agonists or antagonists. Furthermore, the adenovirus-based β-arrestin BiFC assay was able to detect the activation of a broad range of GPCRs. Collectively, our data indicate that the adenovirus-based β-arrestin BiFC assay can serve as a simple and universal platform for studying GPCR activation and thus will be useful for high-throughput screening of drugs that target GPCRs.     

Adaptation of aequorin functional assay to high throughput screening

AequoScreen, a cellular aequorin-based functional assay, has been optimized for luminescent high-throughput screening (HTS) of G protein-coupled receptor (GPCRs). AequoScreen is a homogeneous assay in which the cells are loaded with the apoaequorin cofactor coelenterazine, diluted in assay buffer, and injected into plates containing the samples to be tested. A flash of light is emitted following the calcium increase resulting from the activation of the GPCR by the sample. Here we have validated a new plate reader, the Hamamatsu Photonics FDSS6000, for HTS in 96- and 384-well plates with CHO-K1 cells stably coexpressing mitochondrial apoaequorin and different GPCRs (AequoScreen cell lines). The acquisition time, plate type, and cell number per well have been optimized to obtain concentration-response curves with 4000 cells/well in 384-well plates and a high signal:background ratio. The FDSS6000 and AequoScreen cell lines allow reading of twenty 96- or 384-well plates in 1 h with Z' values of 0.71 and 0.78, respectively. These results bring new insights to functional assays, and therefore reinforce the interest in aequorin-based assays in a HTS environment.     

A homogeneous enzyme fragment complementation-based beta-arrestin translocation assay for high-throughput screening of G-protein-coupled receptors

G-protein-coupled receptors (GPCRs) represent one of the largest gene families in the human genome and have long been regarded as valuable targets for small-molecule drugs. The authors describe a new functional assay that directly monitors GPCR activation. It is based on the interaction between beta-arrestin and ligand-activated GPCRs and uses enzyme fragment complementation technology. In this format, a GPCR of interest is fused to a small (approximately 4 kDa), optimized alpha fragment peptide (termed ProLink) derived from beta-galactosidase, and beta-arrestin is fused to an N-terminal deletion mutant of beta-galactosidase (termed the enzyme acceptor [EA]). Upon activation of the receptor, the beta-arrestin-EA fusion protein binds the activated GPCR. This interaction drives enzyme fragment complementation, resulting in an active beta-galactosidase enzyme, and thus GPCR activation can be determined by quantifying beta-galactosidase activity. In this report, the authors demonstrate the utility of this technology to monitor GPCR activation and validate the approach using a Galphai-coupled GPCR, somatostatin receptor 2. Potential application to high-throughput screens in both agonist and antagonist screening modes is exemplified.     

Characterization of serotonin 5-hydroxytryptamine-1A receptor activation using a phospho-extracellular-signal regulated kinase 2 sensor

The activation of G-protein-coupled receptors (GPCRs) can result in the stimulation of numerous signaling networks that extend beyond canonical secondary messenger-dependent pathways. It is well-established that many of these diverse networks converge on the MAPK pathway, resulting in the activation of extracellular-signal regulated kinase 1/2 (ERK). Since the link between GPCRs and ERK can be modulated via both G-protein-dependent and -independent mechanisms, measurement of ERK phosphorylation may serve as an ideal surrogate for GPCR activation. We have combined BacMam-mediated gene delivery of the GFP-ERK2 with a time-resolved Foerster resonance energy transfer (TR-FRET) immunoassay for the measurement of intracellular phospho-ERK2 levels. Together these technologies enable a flexible platform for measuring GPCR and MAPK activation in the cell line of interest. This technology has been applied to the measurement of activation of the serotonin 5-hydroxytryptamine-1A (5-HT_1A) receptor expressed in CHO-K1 cells. In addition to demonstrating the flexibility of this assay platform, we provide the first reported profile for 5-HT_1A receptor-mediated ERK activation using a panel of known Parkinson’s disease drugs. Our results demonstrate the value of using ERK activation as a downstream sensor for GPCR function, providing an attractive complement to upstream endpoints such as ligand occupancy and binding of GTPγS.     

Functional assays for screening GPCR targets

G-protein-coupled receptors (GPCRs) are valuable molecular targets for drug discovery. An important aspect of the early drug discovery process is the design and implementation of high-throughput GPCR functional assays that allow the cost-effective screening of large compound libraries to identify novel drug candidates. Several functional assay kits based on fluorescence and/or chemiluminescence detection are commercially available for convenient screen development, each having advantages and disadvantages. In addition, new GPCR biosensors and high-content imaging technologies have recently been developed that hold promise for the development of functional GPCR screens in living cells.     

ERK5 Activation by Gq-Coupled Muscarinic Receptors Is Independent of Receptor Internalization and β-Arrestin Recruitment

G-protein-coupled receptors (GPCRs) are known to activate both G protein- and β-arrestin-dependent signalling cascades. The initiation of mitogen-activated protein kinase (MAPK) pathways is a key downstream event in the control of cellular functions including proliferation, differentiation, migration and apoptosis. Both G proteins and β-arrestins have been reported to mediate context-specific activation of ERK1/2, p38 and JNK MAPKs. Recently, the activation of ERK5 MAPK by Gq-coupled receptors has been described to involve a direct interaction between Gαq and two novel effectors, PKCζ and MEK5. However, the possible contribution of β-arrestin towards this pathway has not yet been addressed. In the present work we sought to investigate the role of receptor internalization processes and β-arrestin recruitment in the activation of ERK5 by Gq-coupled GPCRs. Our results show that ERK5 activation is independent of M1 or M3 muscarinic receptor internalization. Furthermore, we demonstrate that phosphorylation-deficient muscarinic M1 and M3 receptors are still able to fully activate the ERK5 pathway, despite their reported inability to recruit β-arrestins. Indeed, the overexpression of Gαq, but not that of β-arrestin1 or β-arrestin2, was found to potently enhance ERK5 activation by GPCRs, whereas silencing of β-arrestin2 expression did not affect the activation of this pathway. Finally, we show that a β-arrestin-biased mutant form of angiotensin II (SII; Sar1-Ile4-Ile8 AngII) failed to promote ERK5 phosphorylation in primary cardiac fibroblasts, as compared to the natural ligand. Overall, this study shows that the activation of ERK5 MAPK by model Gq-coupled GPCRs does not depend on receptor internalization, β-arrestin recruitment or receptor phosphorylation but rather is dependent on Gαq-signalling.     

Cell-based high-throughput screening assay system for monitoring G protein-coupled receptor activation using beta-galactosidase enzyme complementation technology

A novel cell-based functional assay to directly monitor G protein-coupled receptor (GPCR) activation in a high-throughput format, based on a common GPCR regulation mechanism, the interaction between beta-arrestin and ligand-activated GPCR, is described. A protein-protein interaction technology, the InteraX trade mark system, uses a pair of inactive beta-galactosidase (beta-gal) deletion mutants as fusion partners to the protein targets of interest. To monitor GPCR activation, stable cell lines expressing both GPCR- and beta-arrestin-beta-gal fusion proteins are generated. Following ligand stimulation, beta-arrestin binds to the activated GPCR, and this interaction drives functional complementation of the beta-gal mutant fragments. GPCR activation is measured directly by quantitating restored beta-gal activity. The authors have validated this assay system with two functionally divergent GPCRs: the beta2-adrenergic amine receptor and the CXCR2 chemokine-binding receptor. Both receptors are activated or blocked with known agonists and antagonists in a dose-dependent manner. The beta2-adrenergic receptor cell line was screened with the LOPAC trade mark compound library to identify both agonists and antagonists, validating this system for high-throughput screening performance in a 96-well microplate format. Hit specificity was confirmed by quantitating the level of cAMP. This assay system has also been performed in a high-density (384-well) microplate format. This system provides a specific, sensitive, and robust methodology for studying and screening GPCR-mediated signaling pathways.     

Transactivation of the epidermal growth factor receptor mediates parathyroid hormone and prostaglandin F2 alpha-stimulated mitogen-activated protein kinase activation in cultured transgenic murine osteoblasts

Recent data suggest that G protein-coupled receptors (GPCRs), including those for PTH and prostaglandins (PGs), contribute to the proliferation and differentiation of osteoblasts in vivo. To understand how these signals are transduced, we studied activation of the ERK1/2 MAPK cascade in cultures of differentiating TMOb murine osteoblasts. In TMOb cells, stimulation of endogenous Gs/Gq-coupled PTH receptors, Gq-coupled PGF2 alpha receptors, and Gi/Gq-coupled lysophosphatidic acid receptors, but not Gs-coupled PGE2 receptors, caused a rapid 5- to 10-fold increase in ERK1/2 phosphorylation. GPCR-stimulated ERK1/2 activation coincided with increased tyrosine phosphorylation of epidermal growth factor (EGF) receptors and was blocked by the EGF receptor inhibitor, tyrphostin AG1478, and the metalloprotease inhibitor, batimastat, suggesting that the response involved transactivation of EGF receptors through the proteolytic release of an EGF receptor ligand. To further examine the mechanism of PTH-stimulated EGF receptor transactivation, we employed COS-7 cells expressing the rat PTH receptor. Here, stimulation with PTH(1-34) caused proteolysis of hemagglutinin epitope-tagged heparin binding-EGF, increased tyrosine autophosphorylation of EGF receptors, and AG1478-sensitive ERK1/2 activation. When PTH receptor-expressing COS-7 cells were placed in a mixed culture with cells lacking the PTH receptor but expressing a green fluorescent protein-tagged ERK2, stimulation with PTH(1-34) induced phosphorylation of green fluorescent protein-ERK2 that was abolished by either batimastat or tyrphostin AG1478. These data suggest that autocrine/paracrine cross-talk between EGF receptors and Gi- or Gq/11-coupled GPCRs represents the predominant mechanism of GPCR-mediated activation of ERK1/2 in cultured TMOb osteoblasts.     

Development of a novel DnaE intein-based assay for quantitative analysis of G-protein-coupled receptor internalization

G-protein-coupled receptor (GPCR) internalization provides a G-protein-subtype-independent method for assaying agonist-stimulated activation of receptors. We have developed a novel assay that allows quantitative analysis of GPCR internalization based on the interaction between activated GPCRs and β-arrestin2 and on Nostoc punctiforme DnaE intein-mediated reconstitution of Renilla luciferase fragments. This assay system was validated using four functionally divergent GPCRs treated with agonists and antagonists. The EC(50) values obtained for the known agonists and antagonists are in close agreement with the results of previous reports, indicating that this assay system is sensitive enough to permit quantification of GPCR internalization. This rapid and quantitative assay, therefore, could be used universally as a functional cell-based assay for GPCR high-throughput screening during drug discovery.     

A reporter assay for G-protein-coupled receptors using a B-cell line suitable for stable episomal expression

We have established a cAMP response element (CRE)-mediated reporter assay system for G-protein-coupled receptors (GPCRs) using an oriP-based estrogen-inducible expression vector and the B-cell line (GBC53 or GBCC71) that expresses EBNA-1 and is adapted to serum-free culture. GBC53 harbors a GAL4-ER expression unit and a CRE-luciferase gene in the genome, and GBCC71 also harbors expression units for two chimeric Gαs proteins (Gs/q and Gs/i). Introduction of a GPCR expression plasmid into GBC53 or GBCC71 creates polyclonal stable transformants in 2 weeks, and these are easily expanded and used for assays after induction of the GPCR expression. Using GBC53, we detected ligand-dependent signals of Gs-coupled GPCRs such as glucagon-like peptide 1 receptor (GLP1R) and β2 adrenergic receptor (β2AR) with high sensitivity. Interestingly, we also detected constitutive activity of β2AR. Using GBCC71, we detected ligand-dependent signals of Gq- or Gi-coupled GPCRs such as H1 histamine receptor and CXCR1 chemokine receptor in addition to Gs-coupled GPCRs. An agonist, antagonist, or inverse agonist was successfully evaluated in this system. We succeeded in constructing a 384-well high-throughput screening (HTS) system for GLP1R. This system enabled us to easily and rapidly make a large number of efficient GPCR assay systems suitable for HTS as well as ligand hunting of orphan GPCRs.     

A fluorescent reporter assay for the detection of ligands acting through Gi protein-coupled receptors

Accompanying the advances in basic biology of G protein-coupled receptors (GPCRs) is the practical need among biopharmaceutical companies for sensitive assays to assess GPCR function, particularly formats that are compatible with high-throughput drug screening. Here we describe a novel cell-based assay format for the high-throughput detection of ligands for Gi protein-coupled receptors. Two Gi-GPCRs, mu-opioid receptor (mu-OPR) and 5-hydroxytryptamine receptor la (5HT1aR) are employed as model receptor targets. The key feature of this assay system is the isolation of stable, clonal Chinese hamster ovary (CHO) cell lines that carry three separate expression plasmids: (1) a chimeric Gq/i5 protein (which re-directs a negative Gi-type signal to a positive Gq-type response), (2) a given Gi-GPCR, and (3) a beta-lactamase (beta1a) reporter gene responsive to Gi-GPCR signaling. Cell-based assays built using this format show appropriate rank order of potency among a reference set of receptor agonist and antagonist compounds. Such assays are also robust, reliable, and can be used for industrial-scale applications such as high-throughput screening for drug leads.     

Protein kinase C (PKC)ζ-mediated Gαq stimulation of ERK5 protein pathway in cardiomyocytes and cardiac fibroblasts

Gq-coupled G protein-coupled receptors (GPCRs) mediate the actions of a variety of messengers that are key regulators of cardiovascular function. Enhanced Gα(q)-mediated signaling plays an important role in cardiac hypertrophy and in the transition to heart failure. We have recently described that Gα(q) acts as an adaptor protein that facilitates PKCζ-mediated activation of ERK5 in epithelial cells. Because the ERK5 cascade is known to be involved in cardiac hypertrophy, we have investigated the potential relevance of this pathway in cardiovascular Gq-dependent signaling using both cultured cardiac cell types and chronic administration of angiotensin II in mice. We find that PKCζ is required for the activation of the ERK5 pathway by Gq-coupled GPCR in neonatal and adult murine cardiomyocyte cultures and in cardiac fibroblasts. Stimulation of ERK5 by angiotensin II is blocked upon pharmacological inhibition or siRNA-mediated silencing of PKCζ in primary cultures of cardiac cells and in neonatal cardiomyocytes isolated from PKCζ-deficient mice. Moreover, upon chronic challenge with angiotensin II, these mice fail to promote the changes in the ERK5 pathway, in gene expression patterns, and in hypertrophic markers observed in wild-type animals. Taken together, our results show that PKCζ is essential for Gq-dependent ERK5 activation in cardiomyocytes and cardiac fibroblasts and indicate a key cardiac physiological role for the Gα(q)/PKCζ/ERK5 signaling axis.     

Comparative analysis of functional assays for characterization of agonist ligands at G protein-coupled receptors

A variety of functional assays are available for agonist or antagonist screening of G protein-coupled receptors (GPCRs), but it is a priori not predictable which assay is the most suitable to identify agonists or antagonists of GPCRs with therapeutic value in humans. More specifically, it is not known how a given set of GPCR agonists compares in different functional assays with respect to potency and efficacy and whether the level of the signaling cascade that is analyzed has any impact on the detection of agonistic responses. To address this question, the authors used the recently cloned human S1P(5) receptor as a model and compared a set of 3 lipid ligands (sphingosine 1-phosphate [S1P], dihydro sphingosine 1-phosphate [dhS1P], and sphingosine) in 5 different functional assays: GTPgammaS binding, inhibition of adenylyl cyclase activity, mobilization of intracellular Ca(2+) via the FLIPR and aequorin technology, and MAP kinase (ERK1/2) activation. S1P induced agonistic responses in all except the ERK1/2 assays with EC(50) values varying by a factor of 10. Whereas dhS1P was identified as a partial agonist in the GTPgammaS assay, it behaved as a full agonist in all other settings. Sphingosine displayed partial agonistic activity exclusively in GTPgammaS binding assays. The findings suggest that assays in a given cellular background may vary significantly with respect to suitability for agonist finding and that ligands producing a response may not readily be detectable in all agonist assays.     

G-protein-coupled receptors in drug discovery: nanosizing using cell-free technologies and molecular biology approaches

Signal transduction by G-protein-coupled receptors (GPCRs) underpins a multitude of physiological processes. Ligand recognition by the receptor leads to activation of a generic molecular switch involving heterotrimeric G-proteins and guanine nucleotides. Signal transduction has been studied extensively with both cell-based systems and assays comprising isolated signaling components. Interest and commercial investment in GPCRs in areas such as drug targets, orphan receptors, high throughput screening, biosensors, and so on will focus greater attention on assay development to allow for miniaturization, ultra-high throughput and, eventually, microarray/biochip assay formats. Although cell-based assays are adequate for many GPCRs, it is likely that these formats will limit the development of higher density GPCR assay platforms mandatory for other applications. Stable, robust, cell-free signaling assemblies comprising receptor and appropriate molecular switching components will form the basis of future GPCR assay platforms adaptable for such applications as microarrays. The authors review current cell-free GPCR assay technologies and molecular biological approaches for construction of novel, functional GPCR assays.     

G protein-coupled receptor-mediated ERK1/2 phosphorylation: towards a generic sensor of GPCR activation

The development of new analytical methods, aimed at profiling G protein-coupled receptor (GPCR) ligands, regardless of the G protein-coupling pattern of their respective receptor, remains a key goal in drug discovery. Considerable evidence has recently revived the central role that could be played by extracellular-signal-regulated kinase (ERK), the cornerstone protein kinase of the first tyrosine kinase receptor-mediated pathway identified, in response to the activation of various types of GPCRs. Here we reveal a conceptual study in which the potential of ERK phosphorylation is evaluated as a generic readout in response to three different receptors activating three main classes of G proteins: Galphas, Galphai and Galphaq. GPCR-mediated ERK phosphorylation was compared with different readouts such as GTPgammaS, CAMP, or Ca2 +. We propose the measurement of GPCR-activated ERK phosphorylation as an alternative assay to better understand the molecular pharmacology of ligands of promiscuous GPCRs.     

A novel method for analyzing [Ca2+] flux kinetics in high-throughput screening

Driven by multiparameter fluorescence readouts and the analysis of kinetic responses from biological assay systems, the amount and complexity of high-throughput screening data are constantly increasing. As a consequence, the reduction of data to a simple number, reflecting a percentage activity/inhibition, is no longer an adequate approach because valuable additional information, for example, about compound-or process-induced artifacts, is lost. Time series data such as the transient calcium flux observed after activation of Gq-coupled G protein-coupled receptors (GPCRs), are especially challenging with respect to quantity of data; typically, responses are followed for several minutes. Based on measurements taken on the fluorometric imaging plate reader, the authors have introduced a mathematical model to describe the time traces of cellular calcium fluxes mediated by the activation of GPCRs. The model describes the time series using 13 parameters, reducing the amount of data by 90% while guiding the detection of compound-induced artifacts as well as the selection of compounds for further characterization.     

A Fluorescent Reporter Assay for the Detection of Ligands Acting Through GI Protein-Coupled Receptors

Abstract

Accompanying the advances in basic biology of G protein-coupled receptors (GPCRs) is the practical need among biopharmaceutical companies for sensitive assays to assess GPCR function, particularly formats that are compatible with high-throughput drug screening. Here we describe a novel cell-based assay format for the high-throughput detection of ligands for G, protein-coupled receptors. Two G_i-GPCRs, μ-opioid receptor (μ-OPR) and 5-hydroxytryptamine receptor la (5HTlaR) are employed as model receptor targets. The key feature of this assay system is the isolation of stable, clonal Chinese hamster ovary (CHO) cell lines that carry three separate expression plasmids: (1) a chimeric G_q/i5 protein (which re-directs a negative G_i-type signal to a positive Gq-type response), (2) a given G_i-GPCR, and (3) a β-lactamase (βla) reporter gene responsive to G_i-GPCR signaling. Cell-based assays built using this format show appropriate rank order of potency among a reference set of receptor agonist and antagonist compounds. Such assays are also robust, reliable, and can be used for industrial-scale applications such as high-throughput screening for drug leads.