Abstract
G protein-coupled receptors (GPCRs) represent ∼30% of current drug targets. Ligand binding to these receptors activates G proteins and arrestins, which function in different signaling pathways. Given that functionally selective or biased ligands preferentially activate one of these two groups of pathways, they may be superior medications for certain disease states. The identification of such ligands requires robust drug screening assays for both G protein and arrestin activity. This unit describes protocols for assays that monitor reversible arrestin recruitment to GPCRs in living cells using either bioluminescence resonance energy transfer (BRET) or nanoluciferase complementation (NanoLuc). Two types of assays can be used: one configuration directly measures arrestin recruitment to a GPCR fused to a protein tag at its intracellular C-terminus, whereas the other configuration detects arrestin translocation to the plasma membrane in response to activation of an unmodified GPCR. Together, these assays are powerful tools for studying dynamic interactions between GPCRs and arrestins. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Receptor-arrestin BRET assay to measure ligand-induced recruitment of arrestin to receptors Basic Protocol 2: Receptor-arrestin NANOBIT assay to measure ligand-induced recruitment of arrestin to receptors Alternative Protocol 1: BRET assay to measure ligand-induced recruitment of arrestin to the plasma membrane Alternative Protocol 2: NANOBIT assay to measure ligand-induced recruitment of arrestin to the plasma membrane Support Protocol 1: Optimization of polyethylenimine (PEI) concentration for transfection.