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Role for C-tail residues in delta opioid receptor downregulation
N Trapaidze1, S Cvejic, R N Nivarthi
1Department of Pharmacology, New York University School of Medicine, New York 10016, USA.
Abstract:
The delta opioid receptor, a member of the G-protein-coupled receptor superfamily, was used as a model system to characterize opioid receptor downregulation. Metabolic labeling followed by immunoprecipitation resulted in the isolation of the epitope-tagged mouse delta opioid receptor as a approximately 60-kDa protein. Prolonged agonist treatment with 100 nM d-Ala2, d-Leu5-enkephalin (DADLE) caused significant (approximately 60%) reduction in the level of receptor. The delta opioid receptor contains a number of phosphorylatable residues in the C tail. Point mutations of the majority of Ser/Thr sequences did not affect the level of downregulation, whereas mutation of Thr353 to Ala did. In order to test if phosphorylation at this site is involved in receptor downregulation, we generated a Thr353Glu mutant that would mimic the phosphorylated Thr at this site. This mutant exhibited a significantly higher extent of downregulation than the Thr353Ala mutant. In order to critically evaluate the requirement of Thr353 in receptor downregulation, we examined the downregulation of wildtype rat delta receptor (which does not contain Ala353) and an Ala353Thr point-mutant rat delta receptor. The wild-type receptor exhibited poor agonist-mediated downregulation, whereas Ala353Thr mutant exhibited increased downregulation. These results and results from additional studies with rat/mouse chimeric receptors support a role for phosphorylation of sites within the C tail in efficient downregulation of delta opioid receptors.
Insights
Opioid receptor downregulation was studied using the delta opioid receptor. Phosphorylation at a specific C-tail site (Thr353) is crucial for efficient agonist-induced receptor reduction.
Area of Science:
- Pharmacology
- Molecular Biology
- Neuroscience
Background:
- The delta opioid receptor is a G-protein-coupled receptor involved in pain modulation.
- Understanding opioid receptor regulation, like downregulation, is key to developing targeted therapeutics.
- Agonist-induced downregulation affects receptor sensitivity and signaling.
Purpose of the Study:
- To investigate the mechanisms underlying delta opioid receptor downregulation.
- To identify specific amino acid residues involved in the downregulation process.
- To elucidate the role of C-terminal phosphorylation in receptor regulation.
Main Methods:
- Metabolic labeling and immunoprecipitation to isolate and quantify the delta opioid receptor.
- Site-directed mutagenesis to alter specific phosphorylatable residues in the receptor's C-tail.
- Agonist treatment (DADLE) to induce receptor downregulation.
- Comparison of downregulation levels between wild-type and mutant receptors.
Main Results:
- Prolonged agonist treatment significantly reduced delta opioid receptor levels by approximately 60%.
- Mutation of Thr353 to Alanine (Thr353Ala) impaired downregulation, while mimicking phosphorylation (Thr353Glu) enhanced it.
- Wild-type rat delta opioid receptors showed poor downregulation, whereas an Ala353Thr mutant exhibited increased downregulation, supporting Thr353's role.
Conclusions:
- Phosphorylation of threonine at position 353 (Thr353) in the C-tail is critical for efficient agonist-mediated downregulation of delta opioid receptors.
- These findings highlight the importance of C-terminal phosphorylation in G-protein-coupled receptor regulation.
- The study provides insights into the molecular basis of opioid receptor desensitization and tolerance.