Single-molecule analysis of receptor-β-arrestin interactions in living cells

β-arrestin plays a key role in G protein-coupled receptor (GPCR) signaling and desensitization. Despite recent structural advances, the mechanisms that govern receptor–β-arrestin interactions at the plasma membrane of living cells remain elusive. Here, we combine single-molecule microscopy with molecular dynamics simulations to dissect the complex sequence of events involved in β-arrestin interactions with both receptors and the lipid bilayer. In contrast to the currently widely accepted model, we show that β-arrestin spontaneously inserts into the lipid bilayer and transiently interacts with receptors via lateral diffusion on the plasma membrane. Moreover, we show that following receptor interaction, the plasma membrane stabilizes β-arrestin in a membrane-bound, active-like conformation, allowing it to diffuse to clathrin coated pits separately from the activating receptor. These results challenge our current understanding of β-arrestin function at the plasma membrane, revealing a new critical role for β-arrestin pre-association with the lipid bilayer in facilitating its interactions with receptors and subsequent activation.