A Phosphorylation-regulated Brake Mechanism Controls the Initial Endocytosis of Opioid Receptors but Is Not Required for Post-endocytic Sorting to Lysosomes

Jennifer Whistler, Patricia Tsao, Mark Von Zastrow

Research output: Contribution to journalArticle

51 Citations (Scopus)

Abstract

The δ-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 important role in controlling receptor downregulation, previous studies disagree on whether phosphorylation is actually required for the agonist-induced endocytosis of opioid receptors. Furthermore, no previous studies have determined whether phosphorylation is required for subsequent sorting of internalized receptors to lysosomes. We have addressed these questions by examining the endocytic trafficking of a series of mutant versions of DOR expressed in stably transfected HEK 293 cells. Our results confirm that phosphorylation is not required for agonist-induced endocytosis of truncated mutant receptors that lack the distal carboxyl-terminal cytoplasmic domain containing sites of regulatory phosphorylation. However, phosphorylation is required for endocytosis of full-length receptors. Mutation of all serine/threonine residues located in the distal carboxyl-terminal tail domain of the full-length receptor to alanine creates functional mutant receptors that exhibit 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 versions of arrestin-3 suggest that the distal tail domain, when not phosphorylated, inhibits receptor-mediated recruitment of β-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 phosphorylation 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, subsequent steps of membrane trafficking mediating sorting and transport to lysosomes do not require receptor phosphorylation.

Original languageEnglish (US)
Pages (from-to)34331-34338
Number of pages8
JournalJournal of Biological Chemistry
Volume276
Issue number36
DOIs
StatePublished - Sep 7 2001
Externally publishedYes

Fingerprint

Phosphorylation
Opioid Receptors
Endocytosis
Lysosomes
Sorting
Brakes
Down-Regulation
Arrestins
Arrestin
HEK293 Cells
Threonine
Cell membranes
Green Fluorescent Proteins
Aspartic Acid
Alanine
Serine
Substitution reactions
Cell Membrane
Membranes
Mutation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

A Phosphorylation-regulated Brake Mechanism Controls the Initial Endocytosis of Opioid Receptors but Is Not Required for Post-endocytic Sorting to Lysosomes. / Whistler, Jennifer; Tsao, Patricia; Von Zastrow, Mark.

In: Journal of Biological Chemistry, Vol. 276, No. 36, 07.09.2001, p. 34331-34338.

Research output: Contribution to journalArticle

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