The conserved isoleucine-valine-phenylalanine motif couples activation state and endocytic functions of β-arrestins

Beta-arrestins (βarrs) play a central role in the regulation of G-protein-coupled receptors (GPCRs). Their binding to phosphorylated activated GPCRs induces a conformational transition to an active state resulting in the release of their flexible C-terminal tail. Binding sites for clathrin and the adaptor protein (AP)-2 clathrin adaptor complex are then unmasked, which drive the recruitment of βarrs-GPCR complexes into clathrin-coated pits (CCPs). A conserved isoleucine-valine-phenylalanine (IVF) motif of the C-terminal tail controls βarr activation through intramolecular interactions. Here, we provide structural, biochemical and functional evidence in living cells that the IVF motif also controls binding to AP-2. While the F residue is directly involved in AP-2 binding, substitutions of I and V residues, markedly enhanced affinity for AP-2 resulting in active βarr mutants, which are constitutively targeted to CCPs in the absence of any GPCR activation. Conformational change and endocytic functions of βarrs thus appear to be coordinated via the complex molecular interactions established by the IVF motif.