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 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 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 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.
Whistler
J L
JL
Department of Neurology, Ernest Gallo Clinic and Research Center, University of California, San Francisco, Emeryville, California 94608, USA. shooz2@itsa.ucsf.edu
Tsao
P
P
von Zastrow
M
M
D000262:Adenylyl Cyclases / Q000378:metabolism; D000701:Analgesics, Opioid / Q000494:pharmacology; D019390:Arrestins / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004705:Endocytosis; D005048:Etorphine / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011957:Receptors, Opioid / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D000071557:beta-Arrestins
D000701:Analgesics, Opioid; D019390:Arrestins; D018076:DNA, Complementary; D008164:Luminescent Proteins; D011957:Receptors, Opioid; D011993:Recombinant Fusion Proteins; C082333:arrestin3; D000071557:beta-Arrestins; D049452:Green Fluorescent Proteins; D013912:Threonine; D005048:Etorphine; D012694:Serine; D001710:Biotin; D000262:Adenylyl Cyclases