The two non-visual arrestin isoforms, arrestin2 and arrestin3 recognize and bind hundreds of G protein-coupled receptors (GPCRs) with different phosphorylation patterns leading to distinct functional outcomes. The impact of phosphorylation on arrestin interactions has been well studied only for very few GPCRs. Here we have characterized the interactions between the phosphorylated CC chemokine receptor 5 (CCR5) and arrestin2. We detected several new CCR5 phosphorylation sites, which are necessary for stable complex formation with arrestin2. Crystal structures of arrestin2 in apo form and in complexes with CCR5 C-terminal phosphopeptides together with NMR spectroscopy, biochemical and functional assays revealed three phosphoresidues in a pXpp motif that are essential for the arrestin2 interactions and activation. The same phosphoresidue cluster is present in other receptors, which form stable complexes with arrestin2. We propose that the identified pXpp motif is responsible for robust arrestin2 recruitment in many GPCRs. An analysis of available sequences, structural and functional information on other GPCR•arrestin interactions suggests that a particular arrangement of phosphoresidues within the GPCR intracellular loop 3 and C-terminal tail determines arrestin2 and 3 isoform specificity. Taken together, our findings demonstrate how multi-site phosphorylation controls GPCR•arrestin interactions and provide a framework to probe the intricate details of arrestin activation and signaling.
A structural and functional analysis of arrestin2 in apo form and complexes with several CCR5 phosphopeptides reveals key phosphorylation sites responsible for stable GPCR•arrestin interactions and their contributions to CCR5-arrestin2 function.