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Agonist-specific down-regulation of the human delta-opioid receptor
Takashi Okura1, Eva V Varga, Yoshiaki Hosohata
1Department of Pharmacology, The University of Arizona Health Sciences Center, Tucson, AZ 85724, USA.
Abstract:
Down-regulation of the delta-opioid receptor contributes to the development of tolerance to delta-opioid receptor agonists. The involvement of the carboxy terminus of the mouse delta-opioid receptor in peptide agonist-mediated down-regulation has been established. In the present study, we examined the down-regulation of the truncated human delta-opioid receptor by structurally distinct delta-opioid receptor agonists. Chinese hamster ovary (CHO) cells, expressing the full-length or truncated epitope-tagged human delta-opioid receptors were incubated with various delta-opioid receptor agonists (100 nM, 24 h), and membrane receptor levels were determined by [(3)H]naltrindole saturation binding. Each delta-opioid receptor agonist tested down-regulated the full-length receptor. Truncation of the carboxy terminus abolished down-regulation by all delta-opioid receptor agonists, except SNC80 ((+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]N,N-diethylbenzamide). In addition, truncation of the C-terminus completely attenuated [D-Pen(2)-D-Pen(5)]enkephalin (DPDPE), but not SNC80-mediated [32P] incorporation into the protein immunoreactive with an anti-epitope-tagged antibody. These findings suggest that SNC80-mediated phosphorylation and down-regulation of the human delta-opioid receptor involves other receptor domains in addition to the carboxy terminus. Pertussis toxin treatment did not block SNC80-mediated down-regulation of the truncated Et-hDOR, indicating that the down-regulation is independent of G(i/o) protein activation and subsequent downstream signaling.
Insights
Down-regulation of the delta-opioid receptor is key to tolerance. The receptor's C-terminus is crucial, but SNC80 acts differently, suggesting other domains are involved in its effects.
Area of Science:
- Pharmacology
- Molecular Biology
- Neuroscience
Background:
- Delta-opioid receptor (DOR) down-regulation contributes to tolerance against DOR agonists.
- The mouse DOR carboxy terminus is implicated in peptide agonist-induced down-regulation.
Purpose of the Study:
- To investigate the down-regulation of the truncated human DOR by distinct DOR agonists.
- To elucidate the role of the C-terminus in human DOR down-regulation and signaling.
Main Methods:
- Chinese hamster ovary (CHO) cells expressing full-length or truncated epitope-tagged human DOR were used.
- Receptor levels were quantified using [(3)H]naltrindole saturation binding after agonist incubation.
- Agonist-mediated [(32)P] incorporation into the receptor was assessed.
Main Results:
- All tested DOR agonists down-regulated the full-length human DOR.
- Truncation of the C-terminus abolished down-regulation by most agonists, except SNC80.
- SNC80 induced C-terminal truncation-resistant phosphorylation and down-regulation, independent of G(i/o) proteins.
Conclusions:
- SNC80-mediated human DOR down-regulation and phosphorylation involve domains beyond the C-terminus.
- The C-terminus is essential for down-regulation by certain DOR agonists but not SNC80.
- SNC80's effects on truncated DOR are independent of G(i/o) protein signaling pathways.