Methods to Monitor the Trafficking of β-Arrestin/G Protein-Coupled Receptor Complexes Using Enhanced Bystander BRET.

Abstract

β-Arrestins are adaptors that regulate the signaling and trafficking of G protein-coupled receptors (GPCRs). Bioluminescence resonance energy transfer (BRET) is a sensitive and versatile method for real-time monitoring of protein-protein interactions and protein kinesis within live cells, such as the recruitment of β-arrestins to activated receptors at the plasma membrane (PM) and the trafficking of GPCR/β-arrestin complexes to endosomes. Trafficking of receptor/β-arrestin complexes can be assessed by BRET through tagging β-arrestins with the donor luciferase from Renilla reniformis (Rluc) and anchoring the acceptor green fluorescent protein from the same species (rGFP) in distinct cell compartments (e.g., PM or endosomes) to generate highly efficient bystander BRET (referred to as enhanced bystander BRET (EbBRET)) upon re-localization of β-arrestins to these compartments following receptor activation. Here, we outline the protocol for quantitatively monitoring β-arrestin recruitment to agonist-activated Angiotensin II type 1 receptor (AT1R) and β2-adrenergic receptor (β2AR) at the PM and the trafficking of receptor/β-arrestin complexes into endosomes using EbBRET-based biosensors.