a | In the classical model for seven-transmembrane receptor (7TMR) activation, signalling is mediated by G proteins and desensitization is mediated by β-arrestins. b | In the current model for 7TMR activation, binding of a ligand results in activation of signalling by G proteins and β-arrestins, as well as desensitization and internalization by β-arrestins. c | In a system with biased agonism (β-arrestin-biased in this example), signalling only proceeds through one pathway. EGFR, epidermal growth factor receptor; GRK, G protein-coupled receptor kinase; MAPK, mitogen-activated protein kinase; PI3K, phosphoinositide 3-kinase.

In balanced signalling (a; green), binding of a ligand results in activation of signalling by G proteins and β-arrestins, as well as desensitization and internalization by β-arrestin alone. Two cases of biased agonism exist. In the case of a biased ligand (b), binding of a ligand (β-arrestin-biased, purple; G protein-biased, blue) to an unbiased receptor results in a biased response. In the case of a biased receptor (c), binding of an unbiased ligand to the biased receptor (β-arrestin-biased, purple; G protein-biased, blue) also results in a biased response. GRK, G protein-coupled receptor kinase.

a | One way to compare signalling between G protein- and β-arrestin-mediated pathways is to plot β-arrestin activity on the x axis and G protein activity on the y axis. Unbiased ligands would be expected to have equal levels of efficacy for β-arrestin- and G protein-mediated pathways, as shown by the green circles and the line. For biased ligands, there would be differing levels of β-arrestin- and G protein-mediated efficacies, as illustrated for β-arrestin-biased full agonists (grey), β-arrestin-biased partial agonists (yellow), G protein-biased partial agonists (blue) and G protein- biased full agonists (purple). b | Such data can also be represented in matrix form for m ligands (upper) or in terms of a bias factor for each ligand i (bottom).

a | In redistribution assays, changes in distribution of β-arrestins (green) or receptor (not shown) can be used as a response. Before stimulation, the receptor is usually localized to the membrane, whereas β-arrestins are found largely diffusely in the cytosol. In class A receptors, β-arrestins translocate to the membrane while the receptor is internalized into rapid recycling endosomes. In class B receptors, β-arrestins are internalized with the receptors into slow recycling endosomes. b | In proximity assays, the proximity between the β-arrestin and the receptor is monitored using a number of strategies. In resonance energy transfer (RET) assays, a donor probe and an acceptor probe can be attached to the receptor and β-arrestin, respectively. Upon recruitment of the β-arrestin, emission of the donor probe, by excitation with light in fluorescence RET (FRET) or with chemiluminescent substrate in bioluminescence RET (BRET), results in energy transfer to the acceptor probe. Proximity can also be assessed using reporter-based assays, such as the Tango assay. Upon recruitment of the β-arrestin, a protease — tobacco etch virus (TEV) in this example — covalently linked to the β-arrestin cleaves a site and releases a transcription factor that was attached to a modified receptor. This transcription factor then translocates to the nucleus resulting in reporter-gene expression. In assays based on enzyme complementation, the β-arrestin and the receptor are modified with fragments of an enzyme, which, upon recruitment of the β-arrestin, results in the formation of a functional enzyme. c | In conformation assays, changes in receptor or β-arrestin conformation are used to assess ligand activity and binding. In assays of receptor conformation, site-specific probes are incorporated in areas of the receptor that are thought to undergo significant structural change after ligand binding. Monitoring of FRET between these two fluorescent probes shows distinct changes associated with binding of the ligand. Similar strategies have been used to study the conformation of β-arrestin. Here, intramolecular BRET between luciferase (Luc) and yellow fluorescent protein (YFP) attached to the amino and carboxyl termini of the β-arrestin is used to monitor changes in β-arrestin conformation.