Abstract
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.
Highlights
G protein-coupled receptors (GPCRs)1 are regulated by multiple mechanisms
Many GPCRs undergo a process of rapid desensitization within seconds to minutes after ligand-induced activation, mediated by ligand-dependent phosphorylation of receptors followed by the association of phosphorylated receptors with arrestins
To examine whether phosphorylation was required for endocytosis of full-length receptors, we created a full-length mutant version of DOR in which all serine and threonine residues located in the carboxyl-terminal cytoplasmic tail were mutated to alanine (DOR5A mutant receptor)
Summary
Human embryonic kidney cells (HEK293) cells were maintained and passaged in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (University of California, San Francisco Cell Culture Facility). A stable cell line expressing FLAG-tagged DOR (DOR5) was generated as described previously [7]. The previously described truncated mutant DOR (DOR344T) cell line was constructed by engineering a stop codon following residue 344 in the coding sequence of the FLAG-tagged murine DOR and introducing the cDNA into HEK293 cells via calcium phosphate precipitation and G418 selection [21]. Stable cell lines of these constructs were generated by introducing them into HEK293 cells via calcium phosphate precipitation and Zeocin selection. Double stable cell lines expressing GFP-arrestin and receptor were generated by cotransfection with both constructs using calcium phosphate precipitation followed by selection with both G418 and Zeocin. Relative receptor expression levels were quantified using immunofluorescence microscopy and/or radioligand binding using [3H]diprenorphine (see below for methods)
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