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.

激动剂诱导的 G 蛋白偶联受体 (GPCR) 磷酸化是其与 β-抑制蛋白 (βarrs) 相互作用和随后的功能反应的关键决定因素。因此，重要的是破译不同受体磷酸化位点在控制 βarr 相互作用和功能结果中的贡献和相互作用。在这里，我们发现人血管加压素受体 (V ₂ R) 中的几个磷酸化位点，单独或成簇定位，对 βarr 募集、运输和 ERK1/2 激活有不同的贡献。_{即使是 V 2}中的单个磷酸化位点R，适当地定位以与 βarr 中的关键残基串扰，在 βarr 募集中具有决定性的贡献，其突变导致强烈的 G 蛋白偏差。分子动力学模拟为这一关键磷酸化位点在控制 βarr 相互作用的稳定性和调节 βarr 中的域间旋转中的关键作用提供了机理见解。我们的研究结果揭示了重要的结构方面，以更好地理解 GPCR-βarr 相互作用和偏向信号的框架。