Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a primary determinant of β-arrestin (βarr) recruitment and trafficking. For several GPCRs such as the vasopressin receptor subtype 2 (V₂R), agonist-stimulation first drives the translocation of βarrs to the plasma membrane, followed by endosomal trafficking, which is generally considered to be orchestrated by multiple phosphorylation sites. We have previously shown that mutation of a single phosphorylation site in the V₂R (i.e., V₂R^T360A) results in near-complete loss of βarr translocation to endosomes despite robust recruitment to the plasma membrane, and compromised ERK1/2 activation. Here, we discover that a synthetic intrabody (Ib30), which selectively recognizes activated βarr1, efficiently rescues the endosomal trafficking of βarr1 and ERK1/2 activation for V₂R^T360A. Molecular dynamics simulations reveal that Ib30 enriches active-like βarr1 conformation with respect to the inter-domain rotation, and cellular assays demonstrate that it also enhances βarr1-β₂-adaptin interaction. Our data provide an experimental framework to positively modulate the receptor-transducer-effector axis for GPCRs using intrabodies, which can be potentially integrated in the paradigm of GPCR-targeted drug discovery.

激动剂诱导的 G 蛋白偶联受体 (GPCRs) 磷酸化是 β-arrestin (βarr) 募集和运输的主要决定因素。对于一些 GPCR，例如加压素受体亚型 2 (V ₂ R)，激动剂刺激首先驱动 βarr 易位至质膜，然后是内体运输，这通常被认为是由多个磷酸化位点协调的。_{我们之前已经表明 V 2} R中单个磷酸化位点的突变（即 V ₂ R ^T360A) 导致几乎完全丧失 βarr 易位到内体，尽管对质膜进行了强劲的募集，并损害了 ERK1/2 的激活。在这里，我们发现选择性识别激活的 βarr1 的合成内体 (Ib30) 有效地挽救了 βarr1 的内体运输和 V ₂ R ^T360A的 ERK1/2 激活。分子动力学模拟表明，Ib30 在域间旋转方面富集了活性样 βarr1 构象，细胞分析表明它也增强了 βarr1- _β2-适应素相互作用。我们的数据提供了一个实验框架，可以使用内部抗体正向调节 GPCR 的受体 - 转导 - 效应轴，这可以潜在地整合到 GPCR 靶向药物发现的范例中。