Agonist-induced, G Protein-dependent and -independent Down-regulation of the μ Opioid Receptor

The μ opioid receptor (MOR) has been shown to desensitize after 1 h of exposure to the opioid peptide, [ d -Ala 2, N-MePhe 4, Gly-ol 5 ]enkephalin (DAMGO), largely by the loss of receptors from the cell surface and receptor down-regulation. We have previously shown that the Thr 394 in the carboxyl tail is essential for agonist-induced early desensitization, presumably by serving as a primary phosphorylation site for G protein-coupled receptor kinase. Using a T394A mutant receptor, we determined that Thr 394 was also responsible for μ opioid receptor down-regulation. The T394A mutant receptor displayed 50% reduction of receptor down-regulation (14.8%) compared with wild type receptor (34%) upon 1 h of exposure to DAMGO. Agonist-induced T394A receptor down-regulation was unaffected by pertussis toxin treatment, indicating involvement of a mechanism independent of G protein function. Interestingly, pertussis toxin-insensitive T394A receptor down-regulation was completely inhibited by a tyrosine kinase inhibitor, genistein. Tyrosine kinase inhibition blocked wild type MOR down-regulation by 50%, and the genistein-resistant wild type MOR down-regulation was completely pertussis toxin-sensitive. Following DAMGO stimulation, MOR was shown to be phosphorylated at tyrosine residue(s), indicating that the receptor was a direct substrate for tyrosine kinase action. Mutagenesis of the four intracellular tyrosine residues resulted in complete inhibition of the G protein-insensitive MOR internalization. Therefore, agonist-induced MOR down-regulation appears to be mediated by two distinct cellular signal transduction pathways. One is G protein-dependent and GRK-dependent, which can be abolished by pertussis toxin treatment of wild type MOR or by mutagenesis of Thr 394. The other novel pathway is G protein-independent but tyrosine kinase-dependent, blocked by genistein treatment, and one in which Thr 394 has no regulatory role but phosphorylation of tyrosine residues appears essential.