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J. Biol. Chem., Vol. 276, Issue 36, 34331-34338, September 7, 2001
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From the The
A Phosphorylation-regulated Brake Mechanism Controls the
Initial Endocytosis of Opioid Receptors but Is Not Required for
Post-endocytic Sorting to Lysosomes*
§,
**, and§§
Department of Neurology, Ernest Gallo
Clinic and Research Center, University of California, San Francisco,
Emeryville, California 94608, the ¶ Program in Cell Biology,
Departments of Biochemistry and Biophysics,
Psychiatry, and
§§ Cellular and Molecular Pharmacology,
University of California, San Francisco, California 94143-0984
-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 down-regulation, 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 
-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.
*
This work was supported in part by Research Grant DA12864
from the National Institutes of Health.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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