A high-resolution analysis of arrestin2 interactions responsible for CCR5 endocytosis

Clathrin-mediated endocytosis (CME) is crucial for regulating G protein-coupled receptors (GPCRs) via phosphorylation-dependent arrestin interactions. The interplay between receptor phosphorylation and arrestin coupling to the CME machinery has only been explored for a few GPCRs. Here we have investigated the CME of the chemokine receptor CCR5 induced by native and engineered CCL5 ligands. Upon CCL5 agonist stimulation, arrestin2 translocates to CCR5 at the plasma membrane forming a long-lived CCR5-arrestin2 complex that is internalized but does not reach the lysosomes. Solution NMR revealed that arrestin2 interacts weakly with clathrin through a single binding site, independent of arrestin2 activation by phosphorylated CCR5 C-terminal tail peptides. In contrast, the arrestin2-AP2 interaction is stronger and requires arrestin2 activation depending quantitatively on the CCR5 C-terminal tail phosphorylation. The in vitro results are corroborated by cellular assays, which establish a crucial role of the arrestin2 interaction with AP2, but not with clathrin for CCR5 internalization. These findings give insights how receptor phosphorylation regulates arrestin-mediated GPCR internalization.