Molecular insights into atypical modes of β-arrestin interaction with seven transmembrane receptors

β-arrestins are multifunctional proteins that are critically involved in regulating spatio-temporal aspects of GPCR signaling. The interaction of β-arrestins with GPCRs is typically conceptualized in terms of receptor activation and phosphorylation primarily in the carboxyl-terminus. Interestingly however, there are several GPCRs that harbor majority of phosphorylation sites in their 3rd intracellular loop (ICL3) instead of carboxyl-terminus but still robustly engage β-arrestins. Moreover, there are several 7TMRs that are now characterized as intrinsically-biased, β-arrestin-coupled receptors (ACRs) due to lack of functional G-protein-coupling but robust β-arrestin binding leading to functional outcomes. The molecular basis of β-arrestin interaction and activation upon binding to these types of 7TMRs is currently elusive, and it represents a major knowledge gap in our current understanding of this signaling system. Here, we present seven cryo-EM structures of β-arrestins in basal state, activated by the muscarinic M2 receptor (M2R) through its ICL3, and a β-arrestin-coupled receptor known as decoy D6 receptor (D6R). These structural snapshots combined with biochemical, cellular, and biophysical experiments including HDX-MS and MD simulation provide novel insights into the ability of β-arrestins to preferentially select specific phosphorylation patterns in the receptors, and also illuminate the structural diversity in 7TMR-β-arrestin interaction. Surprisingly, we also observe that the carboxyl-terminus of β-arrestin2 but not β-arrestin1 undergoes structural transition from a β-strand to α-helix upon activation by D6R, which may preclude the core-interaction with the activated receptor. Taken together, our study elucidates previously unappreciated aspects of 7TMR-β-arrestin interaction, and provides important mechanistic clues about how the two isoforms of β-arrestins can recognize and regulate a large repertoire of GPCRs. ### Competing Interest Statement The authors have declared no competing interest.