Structural snapshots uncover a lock-and-key type conserved activation mechanism of β-arrestins by GPCRs

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a key determinant for the binding and activation of multifunctional regulatory proteins known as β-arrestins (βarrs). Although the primary sequence and phosphorylation pattern of GPCRs are poorly conserved, the downstream functional responses mediated by βarrs such as receptor desensitization, endocytosis and signaling are broadly applicable across GPCRs. A conserved principle of βarr activation, if any, upon their interaction with different GPCRs harboring divergent phosphorylation patterns remains to be visualized, and it represents a major knowledge gap in our current understanding of GPCR signaling and regulatory paradigms. Here, we present four structural snapshots of activated βarrs, in complex with distinct phosphorylation patterns derived from the carboxyl-terminus of three different GPCRs, determined using cryogenic-electron microscopy (cryo-EM). These structures of activated βarrs elucidate a “lock-and-key” type conserved mechanism of βarr activation wherein a P-X-P-P phosphorylation pattern in GPCRs interacts with a spatially organized K-K-R-R-K-K sequence in the N-domain of βarrs. Interestingly, the P-X-P-P pattern simultaneously engages multiple structural elements in βarrs responsible for maintaining the basal conformation, and thereby, leads to efficient βarr activation. The conserved nature of this lock-and-key mechanism is further illustrated by a comprehensive sequence analysis of the human GPCRome, and demonstrated in cellular context with targeted mutagenesis including “loss-of-function” and “gain-of-function” experiments with respect to βarr activation measured by an intrabody-based conformational sensor. Taken together, our findings uncover previously lacking structural insights, which explain the ability of distinct GPCRs to activate βarrs through a common mechanism, and a key missing link in the conceptual framework of GPCR-βarr interaction and resulting functional outcomes. ### Competing Interest Statement The authors have declared no competing interest.