NanoLuc-Based Methods to Measure β-Arrestin2 Recruitment to G Protein-Coupled Receptors

Abstract

Cytosolic β-arrestins are key regulators of G protein-coupled receptors (GPCRs) by sterically uncoupling G protein activation, facilitating receptor internalization, and/or acting as G protein-independent signaling scaffolds. The current awareness that GPCR ligands may display bias toward G protein signaling or β-arrestin recruitment makes β-arrestin recruitment assays important additions to the drug discovery toolbox. This chapter describes two NanoLuc-based methods to monitor β-arrestin2 recruitment to the human histamine H₁ receptor by measuring bioluminescence resonance energy transfer and enzyme-fragment complementation in real-time on living cells with reasonable high throughput. In addition to the detection of agonism, both assay formats can be used to qualitatively evaluate the binding kinetics of antihistamines on the human histamine H₁ receptor.

Keywords: Bioluminescence resonance energy transfer (BRET); Enzyme-fragment complementation (EFC); GPCR; Luciferase; Protein-protein interaction (PPI); β-Arrestin.