Abstract
Desensitization and alternative signaling pathways of G protein-coupled receptors (GPCRs) are largely mediated by β-arrestins (arr). While most GPCRs recruit arrestin through phosphorylated C-terminal tails, many lack a canonical tail and instead rely on phosphorylation sites within long third intracellular loops (ICL3). Structural information on such complexes is largely missing, and the molecular details of arrestin engagement remain unknown. Here, we dissect the interaction between the muscarinic acetylcholine receptor M2 (M (2) R) and β-arrestins directly in living cells using genetically encoded crosslinkers and derive crosslinking-guided atomistic models of the M (2) R-arrestin complex, supported by unbiased molecular dynamics simulations. Our results show that the M (2) R ICL3 wraps around a positively charged belt on the arrestin N-domain, whereas the receptor core engages the arrestin central crest through a highly dynamic interface. This core contact is essential for efficient arrestin binding and is tuned by the topology of phosphorylation sites, with markedly stronger engagement when the sites reside in a C-terminal tail rather than in ICL3. Our work provides previously inaccessible structural insight into GPCR-arrestin complexes that rely on ICL3-mediated recruitment and suggests that the topology of the interaction, rather than sequence motifs alone, governs the functional dynamics of arrestin coupling.