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Divers pathways mediate delta-opioid receptor down regulation within the same cell
1The Mauerberger Chair in Neuropharmacology, Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, 69978, Tel-Aviv, Israel.
Abstract:
Various mechanisms have been proposed for opioid receptor down regulation in different experimental preparations. The present study was aimed to test whether distinct mechanisms can mediate opioid receptor down regulation within the same cell. For this purpose we transfected HEK-293 cells with rat delta-opioid receptor (DOR). We exposed the cells to the opioid agonist etorphine in the absence or presence of various pharmacological agents and measured the binding of the opioid ligand [(3)H]diprenorphine to either isolated cell membranes or whole cells. We found that internalization of the receptors into the cell was mediated by clathrin coated pits and that the internalized receptors were degraded either in lysosomes or by proteosomes. Down regulation involved phosphorylation and at least two different kinases, a tyrosine kinase (TK) and MAPK kinase (MEK), mediated DOR down regulation in parallel routes. G-protein-coupled receptor kinase (GRK) was found to have only a minor role in DOR down regulation in HEK-293 cells. On the other hand, in N18TG2 cells that endogenously express delta-opioid receptors, GRK was the predominant kinase mediating DOR down regulation, with only a minor role for TK and MEK. We conclude that down regulation can take place via divers pathways within the same cell, and that in different cells down regulation is mediated by different mechanisms, depending on the kinase profile of the cells and the compartmentalization of the receptors within the cells.
Insights
Opioid receptor down regulation involves distinct mechanisms within the same cell, utilizing pathways like clathrin-coated pits and various kinases. Different cell types employ different kinase profiles for delta-opioid receptor regulation.
Area of Science:
- Pharmacology
- Cell Biology
- Neuroscience
Background:
- Opioid receptor down regulation is crucial for understanding opioid tolerance and dependence.
- Previous studies proposed various mechanisms for opioid receptor down regulation in different experimental systems.
Purpose of the Study:
- To investigate if distinct mechanisms mediate opioid receptor down regulation within the same cell.
- To elucidate the specific roles of different kinases and cellular pathways in delta-opioid receptor (DOR) down regulation.
Main Methods:
- HEK-293 cells transfected with rat DOR were treated with etorphine and pharmacological agents.
- Opioid receptor binding was measured using [(3)H]diprenorphine in isolated membranes and whole cells.
- Internalization, degradation, and phosphorylation pathways were analyzed, including the roles of clathrin-coated pits, lysosomes, proteasomes, tyrosine kinase (TK), MAPK kinase (MEK), and G-protein-coupled receptor kinase (GRK).
Main Results:
- Receptor internalization occurred via clathrin-coated pits, followed by degradation in lysosomes or proteasomes.
- DOR down regulation in HEK-293 cells involved phosphorylation mediated by TK and MEK in parallel pathways, with a minor role for GRK.
- In N18TG2 cells, GRK was the primary kinase for DOR down regulation, with minor roles for TK and MEK.
Conclusions:
- Opioid receptor down regulation can occur through diverse pathways within a single cell.
- Different cell types utilize distinct kinase profiles and receptor compartmentalization for mediating opioid receptor down regulation.