G protein receptor kinases (GRKs) and β-arrestins are key regulators of μ-opioid receptor (MOR) signaling and trafficking. We have previously shown that high-efficacy opioids such as DAMGO stimulate a GRK2/3-mediated multisite phosphorylation of conserved C-terminal tail serine and threonine residues, which facilitates internalization of the receptor. In contrast, morphine-induced phosphorylation of MOR is limited to Ser³⁷⁵ and is not sufficient to drive substantial receptor internalization. We report how specific multisite phosphorylation controlled the dynamics of GRK and β-arrestin interactions with MOR and show how such phosphorylation mediated receptor desensitization. We showed that GRK2/3 was recruited more quickly than was β-arrestin to a DAMGO-activated MOR. β-Arrestin recruitment required GRK2 activity and MOR phosphorylation, but GRK recruitment also depended on the phosphorylation sites in the C-terminal tail, specifically four serine and threonine residues within the ³⁷⁰TREHPSTANT³⁷⁹ motif. Our results also suggested that other residues outside this motif participated in the initial and transient recruitment of GRK and β-arrestins. We identified two components of high-efficacy agonist desensitization of MOR: a sustained component, which required GRK2-mediated phosphorylation and a potential soluble factor, and a rapid component, which was likely mediated by GRK2 but independent of receptor phosphorylation. Elucidating these complex receptor-effector interactions represents an important step toward a mechanistic understanding of MOR desensitization that leads to the development of tolerance and dependence.

G蛋白受体激酶（GRKs）和β-arrestin是μ阿片受体（MOR）信号传导和运输的关键调节因子。先前我们已经表明，高效率的阿片类药物（例如DAMGO）会刺激GRK2 / 3介导的保守C端尾部丝氨酸和苏氨酸残基的多位磷酸化，从而促进受体的内在化。相反，吗啡诱导的MOR磷酸化仅限于Ser ³⁷⁵并且不足以推动实质性的受体内在化。我们报告了特定的多位点磷酸化如何控制GRK和β-arrestin与MOR相互作用的动力学，并显示了这种磷酸化如何介导受体脱敏。我们表明，GRK2 / 3比β-arrestin更快地被DAMGO激活的MOR募集。β-Arrestin募集需要GRK2活性和MOR磷酸化，但是GRK募集还取决于C末端尾部的磷酸化位点，特别是³⁷⁰ TREHPSTANT ^379中的四个丝氨酸和苏氨酸残基主题。我们的结果还表明，该基序以外的其他残基参与了GRK和β-arrestin的初始和短暂募集。我们确定了MOR的高效激动剂脱敏的两个成分：一个持续成分，它需要GRK2介导的磷酸化和一个潜在的可溶性因子；一个快速成分，它可能由GRK2介导，但独立于受体的磷酸化。阐明这些复杂的受体-效应子相互作用代表了对MOR脱敏的机械理解的重要一步，从而导致了耐受性和依赖性的发展。