Matthew L. Andria

Tyrosine phosphorylation of the δ-opioid receptor: Evidence for its role in mitogen-activated protein kinase activation and receptor internalization

The internalization of G-protein-coupled receptors (GPCRs), including the delta opioid receptor (delta-OR), has been shown to involve the phosphorylation of serine and threonine residues. However, recent studies suggest that these residues may not be the only ones phosphorylated in response to prolonged opioid exposure. Tyrosines also appear important for delta-OR signalling, but it remains unclear whether they undergo phosphorylation. We examined whether the delta-OR, stably expressed in Chinese hamster ovary (CHO-K1) cells, was tyrosine-phosphorylated during prolonged agonist treatment. The epitope-tagged delta-OR was purified by immunoprecipitation, and the presence of phosphorylated tyrosines was detected using anti-phosphotyrosine antibodies. Tyrosine residues in the delta-OR were phosphorylated after exposure to the high-affinity agonist [d-Thr(2)]-Leu-enkephalin-Thr (DTLET) in a time- and concentration-dependent manner. Tyrosine phosphorylation of the delta-OR appeared to require the actions of a Src-like protein tyrosine kinase, since the Src inhibitor 4-amino-5-(4-methylphenyl)-7-(t-butyl)-pyrazolo-[3,4-d]-pyrimidine (PP1) attenuated this response. PP1 also attenuated the DTLET-mediated activation of mitogen-activated protein kinase, as well as rapid delta-OR internalization, but not receptor down-regulation. Finally, only opioid agonists that induce receptor internalization via the clathrin-dependent endosomal pathway stimulated significant tyrosine phosphorylation of the delta-OR protein. Evidence is presented that the delta-OR is tyrosine-phosphorylated, and we suggest how this may have an active role in opioid receptor signalling and regulation.

Delta Opioid activation of the Mitogen-activated protein kinase cascade does not require transphosphorylation of Receptor Tyrosine Kinases

BackgroundIn this study, we investigated the mechanism(s) by which delta opioids induce their potent activation of extracellular signal-regulated protein kinases (ERKs) in different cell lines expressing the cloned δ-opioid receptor (δ-OR). While it has been known for some time that OR stimulation leads to the phosphorylation of both ERK isoforms, the exact progression of events has remained elusive.ResultsOur results indicate that the transphosphorylation of an endogenous epidermal growth factor receptor (EGFR) in the human embryonic kidney (HEK-293) cell line does not occur when co-expressed δ-ORs are stimulated by the δ-opioid agonist, D-Ser-Leu-enkephalin-Thr (DSLET). Moreover, neither pre-incubation of cultures with the selective EGFR antagonist, AG1478, nor down-regulation of the EGFR to a point where EGF could no longer activate ERKs had an inhibitory effect on ERK activation by DSLET. These results appear to rule out any structural or catalytic role for the EGFR in the δ-opioid-mediated MAPK cascade. To confirm these results, we used C6 glioma cells, a cell line devoid of the EGFR. In δ-OR-expressing C6 glioma cells, opioids produce a robust phosphorylation of ERK 1 and 2, whereas EGF has no stimulatory effect. Furthermore, antagonists to the RTKs that are endogenously expressed in C6 glioma cells (insulin receptor (IR) and platelet-derived growth factor receptor (PDGFR)) were unable to reduce opioid-mediated ERK activation.ConclusionTaken together, these data suggest that the transactivation of resident RTKs does not appear to be required for OR-mediated ERK phosphorylation and that the tyrosine-phosphorylated δ-OR, itself, is likely to act as its own signalling scaffold.

Mutation of tyrosine 318 (Y318F) in the delta-opioid receptor attenuates tyrosine phosphorylation, agonist-dependent receptor internalization, and mitogen-activated protein kinase activation

Opioid receptors are known for their ability to activate diverse second messenger systems. Previously, we showed that selective delta-opioid agonists were able to induce the rapid tyrosine phosphorylation of delta-opioid receptors (delta-ORs) through Src. Src-dependent tyrosine phosphorylation of delta-ORs appears to be important for activation of the mitogen-activated protein kinase cascade and for receptor sequestration into clathrin-coated endosomes, as the Src antagonist, PP1, inhibited both. In an attempt to clarify the role of tyrosine phosphorylation in delta-OR signalling and regulation, we constructed a mutant receptor in which the tyrosine located in the conserved NPXXY motif of the C-terminus was replaced by a phenylalanine (Y318F-delta-OR). Mutation of Y318 resulted in a receptor that was comparable to the wild type in its expression level in HEK-293 cells and in its affinity for opioid ligands. Both receptors showed effective coupling to G proteins and were capable of inhibiting forskolin-stimulated cAMP accumulation with similar potencies. However, the mutant receptor was able to stimulate (35)S-GTPgammaS binding with a lower EC(50) than the wild type receptor. The stimulation of tyrosine phosphorylation in delta-ORs by [D-Thr(2)]-Leu-enkephalin-Thr (DTLET) was significantly less in cells expressing the Y318F-delta-OR than in cells expressing the wild type. In addition, both rapid receptor internalization and down-regulation were markedly attenuated in the mutant. Finally, the mutant receptor was unable to induce a robust activation of the MAPK pathway, suggesting that tyrosine phosphorylation of the delta-OR protein is important for this signalling pathway. These findings implicate tyrosine phosphorylation of Y318 in receptor signalling and agonist-mediated regulation.

Identification of a neurorestrictive suppressor element (NRSE) in the human μ-opioid receptor gene

Analysis of the DNA sequence of the human mu-opioid receptor gene (MOR) revealed that a region overlapping the start codon was substantially homologous to a DNA element named the neurorestrictive suppressor element (NRSE) or restrictive element 1 (RE-1). Transient transfection experiments in the L929 and HEK non-neural cell lines showed that expression of a MOR promoter/reporter gene construct was suppressed in non-neural cell lines by inclusion of this MOR NRSE. Expression from a thymidine kinase promoter was also suppressed when the MOR NRSE was inserted upstream or downstream of the reporter gene. The MOR NRSE did not suppress expression of the reporter gene in neural derived cell lines, IMR-32 and Neuro 2a. The transcription factor REST which binds NRSE thereby enacting the suppression of transcription, was encoded in a plasmid and co-transfected into the IMR-32 cells. The REST co-transfected neuronal derived (IMR-32) cells became sensitive to the MOR NRSE mediated suppression of reporter gene expression. Electrophoretic mobility shift experiments revealed that oligonucleotides containing the MOR NRSE were bound by a factor from nuclear extracts of non-neural cell lines, HeLa and Jurkat. This binding was specifically competed by oligonucleotides containing NRSE sequences previously shown to suppress transcription through REST. Thus an NRSE element overlapping the human MOR start codon suppresses gene expression in non-neural cell lines and may help direct neural tissue specific expression of MOR.

A member of the heat shock protein 40 family, hlj1, binds to the carboxyl tail of the human mu opioid receptor.

A yeast two-hybrid screen, using the carboxyl tail of the human mu opioid receptor as bait and a human brain cDNA library as target, indicated that the carboxyl terminal portion of hlj1, a member of the human heat shock protein 40 family, interacts with the carboxyl tail of the human mu opioid receptor. To determine if direct in vitro binding occurs between these two proteins, we performed overlay experiments. Results from the overlay experiments showed that binding occurs between the His fusion protein of hlj1 and the GST fusion protein of the carboxyl tail of the human mu opioid receptor. In contrast, no binding with the His fusion protein of hlj1 occurred with GST alone or the GST fusion protein of the third cytoplasmic loop of the human mu opioid receptor. Results from co-immunoprecipitation studies, carried out in whole HEK cell lysates, confirmed in vivo binding between these two proteins. Immunofluorescent studies, using laser scanning confocal microscopy, showed significant co-localization between hlj1 and the human mu opioid receptor in the cell membrane. The function of this protein-protein interaction and its physiological relevance in animal and human brain is yet to be determined.