Role for C-Tail Residues in Delta Opioid Receptor Downregulation

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 60-kDa protein. Prolonged agonist treatment with 100 nM d-Ala2, d-Leu5-enkephalin (DADLE) caused significant (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 wildtype 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.