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Agonist-induced phosphorylation of G protein–coupled receptors (GPCRs) is a key determinant for their interaction with β-arrestins (βarrs) and subsequent functional responses. Therefore, it is important to decipher the contribution and interplay of different receptor phosphorylation sites in governing βarr interaction and functional outcomes. Here, we find that several phosphorylation sites in the human vasopressin receptor (V2R), positioned either individually or in clusters, differentially contribute to βarr recruitment, trafficking, and ERK1/2 activation. Even a single phosphorylation site in V2R, suitably positioned to cross-talk with a key residue in βarrs, has a decisive contribution in βarr recruitment, and its mutation results in strong G-protein bias. Molecular dynamics simulation provides mechanistic insights into the pivotal role of this key phosphorylation site in governing the stability of βarr interaction and regulating the interdomain rotation in βarrs. Our findings uncover important structural aspects to better understand the framework of GPCR-βarr interaction and biased signaling.

Highlights d The structure of b-arrestin 2 bound to CXCR7 phosphopeptide (C7pp) was solved d The C7pp-bound b-arrestin 2 shows small inter-domain rotation d The three C7pp phosphates bind with the positively charged residues on b-arrestin 2 d The phosphate-binding pocket around Arg148 recognizes the first phosphate of C7pp

SummaryDesensitization, signaling and trafficking of G protein-coupled receptors (GPCRs) are critically regulated by multifunctional adaptor proteins, β-arrestins (βarrs). The two isoforms of βarrs (βarr1 and 2) share a high degree of sequence and structural similarity, still however, they often mediate distinct functional outcomes in the context of GPCR signaling and regulation. A mechanistic basis for such a functional divergence of βarr isoforms is still lacking. Using a set of complementary approaches including antibody fragment based conformational sensors, we discover structural differences between βarr1 and 2 upon their interaction with activated and phosphorylated receptors. Interestingly, domain swapped chimeras of βarrs display robust complementation in functional assays thereby, linking the structural differences between the receptor-bound βarr1 and 2 with their divergent functional outcomes. Our findings reveal important insights into the ability of βarr isoforms to drive distinct functional outcomes, and underscore the importance of integrating this aspect in the current framework of biased agonism.