Src-dependent phosphorylation of 2-adaptin dissociates the -arrestin-AP-2 complex

-arrestins are known to act as endocytic adaptors by recruiting the clathrin adaptor protein 2 (AP-2) complex to G-protein-coupled receptors (GPCRs), linking them to clathrin-coated pits (CCPs) for internalization. They also act as signaling molecules connecting GPCRs to different downstream effectors. We have previously shown that stimulation of the angiotensin II (Ang II) type 1 receptor (AGTR1, hereafter referred to as AT1R), a member of the GPCR family, promotes the formation of a complex between -arrestin, the kinase Src and AP-2. Here, we report that formation of such a complex is involved in the AT1R-mediated tyrosine phosphorylation of 2-adaptin, the subunit of AP-2 involved in binding -arrestin. We identify a crucial tyrosine residue in the ear domain of 2-adaptin and show in vitro that the phosphorylation of this site regulates the interaction between -arrestin and 2-adaptin. Using fluorescently tagged proteins combined with resonance energy transfer and image cross-correlation spectroscopy approaches, we show in live cells that 2-adaptin phosphorylation is an important regulatory process for the dissociation of -arrestin-AP-2 complexes in CCPs. Finally, we show that 2-adaptin phosphorylation is involved in the early steps of receptor internalization. Our findings not only unveil 2-adaptin as a new Src target during AT1R internalization, but also support the role of receptor-mediated signaling in the control of clathrin-dependent endocytosis of receptors.