Multisite phosphorylation is required for sustained interaction with GRKs and arrestins during rapid μ-opioid receptor desensitization

G protein receptor kinases (GRKs) and beta-arrestins are key regulators of mu-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 Ser(375) and is not sufficient to drive substantial receptor internalization. We report how specific multisite phosphorylation controlled the dynamics of GRK and beta-arrestin interactions with MOR and show how such phosphorylation mediated receptor desensitization. We showed that GRK2/3 was recruited more quickly than was beta-arrestin to a DAMGO-activated MOR. beta-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 (370)TREHPSTANT(379) motif. Our results also suggested that other residues outside this motif participated in the initial and transient recruitment of GRK and beta-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.