GPCR Interactions: Focus on β‑Arrestin Recruitment

When GPCRs bind to a ligand, interactions with G-proteins, GPCR kinases and β-arrestin are critical for signal propagation and attenuation. NanoLuc® technologies sensitively measure specific GPCR interactions in live cells to assess real-time kinetics and better understand GPCR signaling, activation, trafficking and biased agonism.

Measuring β-Arrestin 2 Recruitment

G protein-coupled receptors (GPCRs) are a large family of transmembrane receptors that transmit signals from the outside to the inside of the cell through specific G-protein-dependent or -independent mechanisms. Ligand-induced β-arrestin recruitment plays a critical role in GPCR regulation through receptor desensitization and internalization and also mediates G-protein independent signal transduction. The kinetics of the GPCR/β-arrestin interaction varies for different GPCR receptors, affecting downstream signaling events.

NanoBiT® PPI technology can monitor ligand-induced β-arrestin recruitment in real time. By tagging the GPCR with one NanoBiT® subunit and β-arrestin with the other subunit, luminescence can be measured when the two proteins interact. These videos show an increase in luminescence following ligand-induced membrane recruitment of β-arrestin 2 for two different classes of GPCRs, demonstrating the difference in interaction kinetics.

β2-adrenergic receptor (β2AR)

β2AR is a class A receptor with a weak β-arrestin-2 (βarr2) interaction, that rapidly dissociates during receptor trafficking. In this video, β2ARSmBiT and βarr2-LgBiT fusion vectors are transfected 1:1 in HeLa cells. The β2AR:βarr2 interaction is induced by 30µM isoproterenol, the membrane recruitment of βarr2 and rapid dissociation is evident.

Arginine vasopressin receptor 2 (V2R)

V2R is a class B receptor with a stronger β-arrestin-2 (βarr2) interaction, causing more sustained signaling and slower recycling. In this video, V2R-SmBiT and βarr2-LgBiT fusion vectors are transfected 1:1 in HeLa cells. When the V2R:βarr2 interaction is induced by 1μM arginine vasopressin, the stable interaction is seen at the membrane.

Case Study: Detecting Untraceable Synthetic Drugs Using β-Arrestin Recruitment

Synthetic "designer" drugs make up a large segment of the illegal drug market and pose a challenge to those trying to detect their use. The formulations change constantly, bypassing drug tests specific to a particular chemical target. Two classes of potent drugs, opioids and cannabinoids, exert their activity by interacting with specific GPCRs. Cannaert et al. used NanoBiT® technology to develop β-arrestin 2 recruitment assays that could be used to detect synthetic variants of opioids and cannabinoids drugs in an untargeted approach, demonstrating sensitive, real-time detection in biological samples. By detecting β-arrestin recruitment to GPCRs, this untargeted method for identifying illegal substances presents one possibility for keeping up with the ever-changing formulations of synthetic drugs.

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NanoBiT Beta-Arrestin Assay Detects Untraceable Synthetic Drugs

How can I build a NanoBiT® β-arrestin recruitment assay? See Materials

Exploring GPCR Biology using NanoLuc® Luciferase

See peer-reviewed papers that demonstrate how NanoLuc® luciferase, NanoBiT® assays and the HiBiT peptide can be used to study GPCRs and interacting partners like β-arrestin.

Antibody Binding

Soave, M. et al. (2018) A monoclonal antibody raised against a thermo-stabilised β₁-adrenoceptor interacts with extracellular loop 2 and acts as a negative allosteric modulator of a sub-set of β₁-adrenoceptors expressed in stable cell lines. Biochem. Pharmacol. 147, 38–54

β-Arrestin Recruitment

Cannaert, A. et al. (2018) Activity-based detection of cannabinoids in serum and plasma samples. Clin. Chem. 64, 918–26.

He, S.Q. et al. (2018) Oligomerization of MrgC11 and µ-opioid receptors in sensory neurons enhances morphine analgesia. Sci. Signal. 11, eaao3134.

Littmann, T., Buschauer, A. and Bernhardt, G. (2019) Split luciferase-based assay for simultaneous analyses of the ligand concentration- and time-dependent recruitment of β-arrestin2. Anal. Biochem. 573, 8–16.

Lu, J. et al. (2017) Dopamine D2 receptor and β-arrestin 2 mediate amyloid-β elevation induced by anti-parkinson’s disease drugs, levodopa and piribedil, in neuronal cells. PLoS One 12, e0173240.

Shintani, Y. et al. (2018) β-Arrestin1 and 2 differentially regulate PACAP-induced PAC1 receptor signaling and trafficking. PLoS One 13, e0196946.

Storme, J. et al. (2018) Molecular dissection of the human A3 adenosine receptor coupling with β-arrestin2. Biochem. Pharmacol. 148, 298–307.

Szpakowska, M. et al. (2018) Mutational analysis of the extracellular disulphide bridges of the atypical chemokine receptor ACKR3/CXCR7 uncovers multiple binding and activation modes for its chemokine and endogenous non-chemokine agonists. Biochem. Pharmacol. 153, 299–309.

White, C.W. et al. (2017) Using NanoBRET and CRISPR/Cas9 to monitor proximity to a genome edited protein in real-time. Sci. Reports 7, 3187.

G Protein Interactions and Signaling

Bodle, C.R. et al. (2017) Development of a bimolecular luminescence complementation assay for RGS:G protein interaction in cells. Anal. Biochem. 522, 10–17.

Laschet, C., Dupuis, N. and Hanson, J. (2019) A dynamic and screening-compatible nanoluciferase-based complementation assay enables profiling of individual GPCR-G protein interactions. J. Biol. Chem. 294, 4079–90.

Liu, P. et al. (2019) Ligand-induced activation of ERK1/2 signaling by constitutively active Gs-coupled 5-HT receptors. Acta Pharmacol. Sin. [Epub ahead of print].

Qian, Y. et al. (2018) A bioluminescent Ca²⁺ indicator based on a topological variant of GCaMP6s. Chembiochem 20, 516–20.

Yang, J. et al. (2016) Coupling optogenetic stimulation with NanoLuc-based luminescence (BRET) Ca⁺⁺ sensing. Nat. Commun. 7, 13268.

GPCR Dimerization

Wouters, E. et al. (2019) Distinct dopamine D₂ receptor antagonists differentially impact D₂ receptor oligomerization. Int. J. Mol. Sci. 20, E1686.

GPCR Internalization

Rouault, A.A.J., Lee, A.A. and Sebag, J.A. (2017) Regions of MRAP2 required for the inhibition of orexin and prokinecticin receptor signaling. Biochim. Biophys. Acta Mol. Cell. Res. 1864, 2322–9.

Additional Resources

Detect Cannabinoid GPCR Agonists with NanoBiT

Detecting Cannabinoid Receptor Agonists

This blog post describes how a NanoBiT® assay detected natural and synthetic cannabinoids.

Monitoring GPCR Trafficking

Read how the luminescent HiBiT peptide tag enables one lab to better understand appetite triggers.

GPCR Beta-arrestin research using NanoLuc

Overcoming the Challenges of Tagging GPCRs

Learn how the HiBiT bioluminescent tag can simplify receptor internalization research in this blog post.

Exploring GPCRs: Additional Research Tools

This page highlights research tools for investigating GPCR ligand binding, cAMP assays and more.