Role for C-tail residues in delta opioid receptor downregulation

The delta opioid receptor, a member of the G-protein-coupled receptor super family, was used as a model system to characterize opioid receptor downregu lation, Metabolic labeling followed by immunoprecipitation resulted in the isolation of the epitope-tagged mouse delta opioid receptor as a similar to 60-kDa protein. Prolonged agonist treatment with 100 nM d-Ala(2), d-leu(5) -enkephalin (DADLE) caused significant (similar to 60%) reduction in the le vel of receptor. The delta opioid receptor contains a number of phosphoryla table residues in the C tail. Point mutations of the majority of Ser/Thr se quences did not affect the level of downregulation, whereas mutation of Thr 353 to Ala did. In order to test if phosphorylation at this site is involve d in receptor downregulation, we generated a Thr353Glu mutant that would mi mic the phosphorylated Thr at this site. This mutant exhibited a significan tly higher extent of downregulation than the Thr353Ala mutant. In order to critically evaluate the requirement of Thr353 in receptor downregulation, w e examined the downregulation of wildtype rat delta receptor (which does no t contain Ala353) and an Ala353Thr point-mutant rat delta receptor. The wil dtype receptor exhibited poor agonist-mediated downregulation, whereas Ala3 53Thr mutant exhibited increased downregulation. These results and results from additional studies with rat/mouse chimeric receptors support a role fo r phosphorylation of sites within the C tail in efficient downregulation of delta opioid receptors,.