Differentiation of Opioid Drug Effects by Hierarchical Multi-Site Phosphorylation

TLDR

Opioid drugs differ in their ability to promote μ‑opioid receptor endocytosis, a factor linked to tolerance and dependence. The study aims to show that a conserved 10‑residue sequence in the receptor’s C‑terminal tail determines drug‑specific internalization. Opioids phosphorylate S375 first, then sequentially add phosphates to T370, T376, and T379, and this multi‑phosphorylation is required for endocytosis. The study found that GRK2/3‑dependent phosphorylation of T370, T376, and T379 drives receptor internalization in neurons, revealing a biochemical mechanism that differentiates opioid drugs’ endocytic activity.

Abstract

Differences in the ability of opioid drugs to promote regulated endocytosis of <i>μ</i>-opioid receptors are related to their tendency to produce drug tolerance and dependence. Here we show that drug-specific differences in receptor internalization are determined by a conserved, 10-residue sequence in the receptor's carboxyl-terminal cytoplasmic tail. Diverse opioids induce receptor phosphorylation at serine (S)375, present in the middle of this sequence, but opioids differ markedly in their ability to drive higher-order phosphorylation on flanking residues [threonine (T)370, T376, and T379]. Multi-phosphorylation is required for the endocytosis-promoting activity of this sequence and occurs both sequentially and hierarchically, with S375 representing the initiating site. Higher-order phosphorylation involving T370, T376, and T379 specifically requires GRK2/3 isoforms, and the same sequence controls opioid receptor internalization in neurons. These results reveal a biochemical mechanism differentiating the endocytic activity of opioid drugs.