A phosphorylation-regulated brake mechanism controls the initial endocytosis of opioid receptors but is not required for post-endocytic sorting to lysosomes.

The delta -opioid receptor (DOR) can undergo proteolytic down-regulation by endocytosis of receptors followed by sorting of internalized receptors to lysosomes. Although phosphorylation of the receptor is thought to play an i mportant role in controlling receptor downregulation, previous studies disa gree on whether phosphorylation is actually required for the agonist-induce d endocytosis of opioid receptors. Furthermore, no previous studies have de termined whether phosphorylation is required for subsequent sorting of inte rnalized receptors to lysosomes. We have addressed these questions by exami ning the endocytic trafficking of a series of mutant versions of DOR expres sed in stably transfected HEK 293 cells. Our results confirm that phosphory lation is not required for agonist-induced endocytosis of truncated mutant receptors that lack the distal carboxyl-terminal cytoplasmic domain contain ing sites of regulatory phosphorylation. However, phosphorylation is requir ed for endocytosis of full-length receptors. Mutation of all serine/threoni ne residues located in the distal carboxyl-terminal tail domain of the full -length receptor to alanine creates functional mutant receptors that exhibi t no detectable agonist-induced endocytosis. Substitution of these residues with aspartate restores the ability of mutant receptors to undergo agonist -induced endocytosis. Studies using green fluorescent protein-tagged versio ns of arrestin-3 suggest that the distal tail domain, when not phosphorylat ed, inhibits receptor-mediated recruitment of beta -arrestins to the plasma membrane. Biochemical and radioligand binding studies indicate that, after endocytosis occurs, phosphorylation-defective mutant receptors traffic to lysosomes with similar kinetics as wild type receptors. We conclude that ph osphorylation controls endocytic trafficking of opioid receptors primarily by regulating a "brake" mechanism that prevents endocytosis of full-length receptors in the absence of phosphorylation. After endocytosis occurs, subs equent steps of membrane trafficking mediating sorting and transport to lys osomes do not require receptor phosphorylation.