Recycling and Resensitization of Delta Opioid Receptors

Abstract

Exposure to opioids results in the activation of opioid receptors; this is follow ed by receptor endocytosis. Previously, we showed that delta opioid receptors undergo rapid agonist-mediated internalization and that mutations in the C-tail result in a substantial loss of agonist-mediated internalization. In this study, we investigated the fate of receptors following rapid internalization. We found that the majority of the wild type receptors recycled back to the surface after acute agonist treatment. The kinetics of internalization and recycling of the receptor were virtually identical to the kinetics of internalization and recycling of the radiolabeled agonist. In contrast, the kinetics of internalization and recycling of a C-tail mutant receptor were substantially altered, suggesting an involvement of the C-tail in the recycling process. It is possible that in addition to agonist-mediated internalization, opioid receptors undergo constitutive, agonist-independent internalization. We directly examined this possibility using an antibody-prebinding assay. The wild type delta opioid receptors exhibited agonist-independent internalization via the clathrincoated pit pathway. We also examined the role of receptor internalization and recycling in the modulation of its function by quantitating the level of opioid-stimulated phosphorylation of MAP kinase (MAPK) under conditions of receptor internalization and recycling. We found that agonist treatment caused a rapid increase in the level of phosphorylated MAPK that was rapidly desensitized. The removal of the agonist, which results in receptor recycling, led to the resensitization of the receptor, as evidenced by the agonist's ability to reinduce MAPK phosphorylation. Mutant receptors that underwent rapid recycling exhibited enhanced resensitization, suggesting a role for receptor recycling in the re-sensitization process. Taken together, these results indicate that agonist-mediated internalization and recycling modulate opioid receptor function and that the receptor C-tail plays an important role in both processes.

Opioid Receptor Function Is Regulated by Post-endocytic Peptide Processing

“…Although many studies have explored the fate of the endocytosed receptor using various candidate receptors, relatively few studies have focused on the fate of the agonist co-endocytosed with the receptor. Studies using peptide agonists, at least in the case of opioid receptors, have shown that the co-endocytosed agonist is recycled back to the surface or processed in an acidic compartment, depending on the length of agonist treatment (17,53). Relatively few studies have focused on the enzymes that are responsible for post-endocytic peptide agonist degradation, although a number of peptidases have been shown to be capable of hydrolyzing neuroendocrine peptides including opioid peptides (2,45,54,55).…”

“…Receptor Recycling-Recycling experiments were carried out as described previously (17). Briefly, F6, F6-ECE2, N2A-␦OR, and N2A-␦OR-ECE2 cells or primary cortical neurons (2 ϫ 10 5 cells) were seeded into each well of a 24-well polylysine-coated plate.…”

“…Receptors present at the cell surface were determined using a 1:1000 dilution (in PBS containing 1% BSA) of anti-mouse IgG coupled to horseradish peroxidase (Vector Laboratories) followed by color development as described above for ELISA. The cells were chilled to 4°C, and the total radioactivity (representing surface plus intracellular radiolabeled ligand), radioactivity in an acid wash (representing surface radiolabeled ligand), and radioactivity remaining in the cells (representing intracellular radiolabeled ligand) were determined as described previously (17). Briefly, cells were washed three times in icecold 50 mM Tris-Cl, pH 7.5, and the total bound radioactivity was measured by scintillation counting.…”

Conformation State-sensitive Antibodies to G-protein-coupled Receptors

“…In order to quantitate the level of receptors in brain regions by ELISA, a 3-fold serial dilution of CHO cells expressing receptors by adding CHO cells not expressing the receptor was carried out (the final cell number was kept constant at 3 ϫ 10 5 cells/well). Receptor number at each dilution was quantitated by ligand binding as described (27). A parallel set of wells were subjected to ELISA using the SA25 antibody.…”

Oligomerization of opioid receptors with beta 2-adrenergic receptors: A role in trafficking and mitogen-activated protein kinase activation

“…4). The levels of cell surface receptors were examined by using anti-Flag or anti-myc antibodies by ELISA (21).…”