Abstract
The mu 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 mu 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.
Highlights
The opioid receptor (MOR) has been shown to desensitize after 1 h of exposure to the opioid peptide, [D-Ala2, N-MePhe4, Gly-ol5]enkephalin (DAMGO), largely by the loss of receptors from the cell surface and receptor down-regulation
It is tempting to speculate that MOR down-regulation, which is partially blocked by the T394A mutation, may be dependent on G protein activation and GRKdependent phosphorylation, whereas the residual down-regulation seen in the T394A mutant or after pertussis toxin treatment may be independent from G protein activation and, further, independent from G protein-coupled receptor kinase (GRK)-dependent phosphorylation
The recognition sites for the G protein-coupled receptor kinases have been identified as repeated serines and threonines mostly in the carboxyl tail, by phosphopeptide sequencing of high pressure liquid chromatography purified peptides derived from proteolysis of phosphorylated receptors [21, 22]
Summary
Generation of Mutants and Stable Cell Lines Expressing Wild Type or Mutant Receptors—The full-length cDNA for the rat MOR was cloned into the mammalian expression vector pRC/CMV (InVitrogen, San Diego, CA). Desensitization Conditions and Adenylyl Cyclase Assay—CHO cells expressing wild type or mutant MORs at confluent monolayer were treated with either medium alone or 1 M DAMGO (final concentration) and incubated for 1 h at 37 °C in 5% CO2. Membrane Preparation and Radioligand Binding Assays—Cells were grown until apparent confluence and washed twice with 12 ml of ice-cold PBS, harvested, and centrifuged at 100 ϫ g for 10 min. Pretreatment Conditions of Stably Transfected Cells with Various Drugs—To cause the down-regulation of receptors, CHO cells expressing wild type or mutant MOR at confluent monolayer were treated with either medium alone or 1 M DAMGO (final concentration) and incubated for 1 h at 37 °C in 5% CO2. The results were considered significantly different when the probability of randomly obtaining a mean difference was Ͻ0.05 using the paired Student’s t test
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