Distinct phosphorylation sites in a prototypical GPCR differently orchestrate β-arrestin interaction, trafficking, and signaling

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a key determinant for their interaction with beta-arrestins (beta arrs) and subsequent functional responses. Therefore, it is important to decipher the contribution and interplay of different receptor phosphorylation sites in governing beta arr 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 beta arr recruitment, trafficking, and ERK1/2 activation. Even a single phosphorylation site in V2R, suitably positioned to cross-talk with a key residue in beta arrs, has a decisive contribution in beta arr 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 beta arr interaction and regulating the interdomain rotation in beta arrs. Our findings uncover important structural aspects to better understand the framework of GPCR-beta arr interaction and biased signaling.