Dataset Information

ClickArr: a novel, high-throughput assay for evaluating β-arrestin isoform recruitment.

ABSTRACT: Background: Modern methods for quantifying signaling bias at G protein-coupled receptors (GPCRs) rely on using a single β-arrestin isoform. However, it is increasingly appreciated that the two β-arrestin isoforms have unique roles, requiring the ability to assess β-arrestin isoform preference. Thus, methods are needed to efficiently screen the recruitment of both β-arrestin isoforms as they compete for a target GPCR in cells. Methods: We used molecular cloning to develop fusion proteins of the δ-opioid receptor (δOR), β-arrestin 1, and β-arrestin 2 to fragments of click beetle green and click beetle red luciferases. In this assay architecture, recruitment of either β-arrestin 1 or 2 to the δOR generates a spectrally distinct bioluminescent signal, allowing us to co-transfect all three constructs into cells prior to agonist challenge. Results: We demonstrate that our new assay, named "ClickArr," is a live-cell assay that simultaneously reports the recruitment of both β-arrestin isoforms as they compete for interaction with the δOR. We further find that the partial δOR agonist TAN67 has a significant efficacy bias for β-arrestin 2 over β-arrestin 1 when recruitment is normalized to the reference agonist leu-enkephalin. We confirm that ClickArr reports this bias when run either as a high-throughput endpoint or high-throughput kinetic assay, and cross-validate this result using the PathHunter assay, an orthogonal commercial assay for reporting β-arrestin recruitment to the δOR. Conclusion: Our results suggest that agonist:GPCR complexes can have relative β-arrestin isoform bias, a novel signaling bias that may potentially open up a new dimension for drug development.

SUBMITTER: French AR

PROVIDER: S-EPMC10662323 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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ClickArr: a novel, high-throughput assay for evaluating β-arrestin isoform recruitment.

French Alexander R AR Meqbil Yazan J YJ van Rijn Richard M RM

Frontiers in pharmacology 20231107

<b>Background:</b> Modern methods for quantifying signaling bias at G protein-coupled receptors (GPCRs) rely on using a single β-arrestin isoform. However, it is increasingly appreciated that the two β-arrestin isoforms have unique roles, requiring the ability to assess β-arrestin isoform preference. Thus, methods are needed to efficiently screen the recruitment of both β-arrestin isoforms as they compete for a target GPCR in cells. <b>Methods:</b> We used molecular cloning to develop fusion pro ...[more]

PMID: 38027002

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Project description:Background: Adenosine A1 receptor (A1AR) plays a prominent role in neurological and cardiac diseases and inflammatory processes. Its endogenous ligand adenosine is known to be one of the key players in the sleep-wake cycle. Like other G protein-coupled receptors (GPCRs), stimulation of A1AR leads to the recruitment of arrestins in addition to the activation of G proteins. So far, little is known about the role of these proteins in signal transduction and regulation of A1AR compared to the activation of G proteins. In this work, we characterized a live cell assay for A1AR-mediated β-arrestin 2 recruitment. We have applied this assay to a set of different compounds that interact with this receptor. Methods: Based on NanoBit® technology, a protein complementation assay was developed in which the A1AR is coupled to the large part of the nanoluciferase (LgBiT), whereas its small part (SmBiT) is fused to the N-terminus of β-arrestin 2. Stimulation of A1AR results in the recruitment of β-arrestin 2 and subsequent complementation of a functional nanoluciferase. For comparison, corresponding data on the effect of receptor stimulation on intracellular cAMP levels were collected for some data sets using the GloSensor™ assay. Results: The assay gives highly reproducible results with a very good signal-to-noise ratio. Capadenoson, in contrast to adenosine, CPA, or NECA, shows only partial agonism in this assay with respect to the recruitment of β-arrestin 2, whereas it shows full agonism in the case of the inhibitory effect of A1AR on cAMP production. By using a GRK2 inhibitor, it becomes clear that the recruitment is at least partially dependent on the phosphorylation of the receptor by this kinase. Interestingly, this was also the first time that we demonstrate the A1AR-mediated recruitment of β-arrestin 2 by stimulation with a valerian extract. Conclusion: The presented assay is a useful tool for the quantitative study of A1AR-mediated β-arrestin 2 recruitment. It allows data collection for stimulatory, inhibitory, and modulatory substances and is also suitable for more complex substance mixtures such as valerian extract.

Project description:IntroductionDihydroergotamine mesylate (DHE) is an established effective acute therapy for migraine and is often characterized by its broad receptor pharmacology. Knowledge of DHE pharmacology largely comes from studies employing older methodologies.ObjectiveTo assess DHE receptor activity using high-throughput methods to screen for functional ß-arrestin activity at G protein-coupled receptors (GPCRs).MethodsFunctional receptor activities of DHE and sumatriptan succinate (both 10 μM) were screened against 168 GPCRs using the gpcrMAX assay. Agonist and antagonist effects were considered significant if receptor activity was >30% or inhibited by >50%, respectively. Radiolabeled ligand binding assays were performed for DHE (0.01-300 nM for 5-HT3 and 4E; 0.3-10,000 nM for 5-HT1B, α-adrenergic2B [i.e., α2B-adrenoceptor], D2, and D5) to assess specific binding to select receptors.ResultsDHE (10 μM) exhibited agonist activity at α-adrenergic2B, CXC chemokine receptor 7 (CXCR7), dopamine (D)2/5, and 5-hydroxytryptamine (5-HT)1A/1B/2A/2C/5A receptors and antagonist activity at α-adrenergic1B/2A/2C (i.e., α1B/2A/2C-adrenoceptors), calcitonin receptor-receptor activity modifying protein 2 (CTR-RAMP2) or amylin 2 (AMY2), D1/3/4/5, and 5-HT1F receptors. Sumatriptan succinate (10 μM) exhibited agonist activity at the 5-HT1B/1E/1F/5A receptors. DHE demonstrated a half-maximal inhibitory concentration (IC50) of 149 nM at the 5-HT1F receptor and a half-maximal effective concentration (EC50) of 6 μM at the CXCR7 receptor. DHE did not bind to the 5-HT3 receptor at concentrations up to 300 nM and bound poorly to 5-HT4E and D5 receptors (IC50 of 230 and 370 nM, respectively). DHE bound strongly to the D2, 5-HT1B, and α-adrenergic2B receptors (IC50 of 0.47, 0.58, and 2.8 nM, respectively).ConclusionBy using a high-throughput β-arrestin recruitment assay, this study confirmed the broad receptor profile of DHE and provided an update on DHE receptor pharmacology as it relates to migraine.

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Dataset Information

ClickArr: a novel, high-throughput assay for evaluating β-arrestin isoform recruitment.

Publications

ClickArr: a novel, high-throughput assay for evaluating β-arrestin isoform recruitment.

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