An arginine switch drives the stepwise activation of β-arrestin

β-arrestins (βarrs) play a crucial role in regulating G protein-coupled receptor (GPCR) signaling and trafficking. Canonically, interactions of βarr with the phosphorylated intracellular GPCR-tail induce a multi-step conformational transition that results in the activation of βarr. Depending on the specific interaction pattern with the receptor, βarrs adopt multiple conformational states, each tightly linked to a specific functional outcome of βarr recruitment. Despite its physiological relevance, the structural determinants of βarr activation remain poorly understood. Using a combination of molecular dynamics simulations, biochemical and cell-based experiments, we reveal how specific interactions with a prototypical GPCR promote the unbinding of the βarr2 C-tail—a crucial step in arrestin activation. Importantly, we observe that the expulsion of the C-tail is promoted by the displacement of a conserved arginine residue (Arg394) within the βarr polar core, which we dub “the arginine switch.” Our study uncovers a previously unknown molecular switch that, upon engagement, destabilizes the polar core as a crucial step in the GPCR-induced βarr activation.