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