Monitoring interactions between G-protein-coupled receptors and β-arrestins

β-Arrestins 1 and 2 are ubiquitously expressed intracellular adaptor and scaffolding proteins that play important roles in GPCR (G-protein-coupled receptor) desensitization, internalization, intracellular trafficking and G-protein-independent signalling. Recent developments in BRET (bioluminescence resonance energy transfer) technology enable novel insights to be gained from real-time monitoring of GPCR–β-arrestin complexes in live cells for prolonged periods. In concert with confocal microscopy, assays for studying internalization and recycling kinetics such as ELISAs, and techniques for measuring downstream signalling pathways such as those involving MAPKs (mitogen-activated protein kinases), investigators can now use a range of experimental tools to elucidate the ever-expanding roles of β-arrestins in mediating GPCR function. Introduction GPCRs (G-protein-coupled receptors) interact with a range of proteins, either directly or indirectly, in order to function as agonist-modulated signalling complexes. This mini-review will focus on the interaction between GPCRs and theβ-arrestin intracellular adaptor and scaffolding proteins, why this interaction is of interest and why it is desirable to carry out prolonged real-time monitoring of this interaction in live cells using BRET (bioluminescence resonance energy transfer) in addition to utilizing more established techniques such as confocal microscopy and ELISAs. GPCR desensitization Agonist binding leads to stabilization of an active receptor conformation that couples to a G-protein(s), thereby initiating downstream signalling cascades (G-protein-dependent signalling). One active receptor can activate multiple individual G-protein heterotrimers and so the regulation of this process is critically important. One of the major mechanisms of ‘desensitizing’ GPCRs to further G-protein coupling involves a two-step process of agonist-dependent receptor serine/threonine residue phosphorylation by GRKs (GPCR kinases), followed by binding of β-arrestin to the active and phosphorylated receptor [1]. β-Arrestin sterically hinders further coupling to G-protein, thereby resulting in a substantial reduction in G-protein-dependent signalling.