Disruption of Cdk5-Associated Phosphorylation of Residue Threonine-161 of the δ-Opioid Receptor: Impaired Receptor Function and Attenuated Morphine Antinociceptive Tolerance

Morphine is the most commonly used and most effective analgesic in the clinic. However, its use is limited by the tolerance. Evidence indicates that the delta-opioid receptor (DOR) is essential for morphine antinociceptive tolerance; however, their underlying mechanisms are poorly understood. Here, we show that cyclin-dependent kinase 5 (Cdk5), activated in morphine antinociceptive tolerance, directly phosphorylates DOR at Thr-161 in DRG neurons. Cdk5 was found to phosphorylate Thr-161 in the second loop of DOR, but not the corresponding residue in the mu-opioid receptor (MOR). Phosphorylation at Thr-161 is required for normal cell surface expression of DOR, and the formation of DOR-MOR heterodimers. Our studies indicated that inhibition of Cdk5 activity or overexpression of a DOR mutant lacking the Cdk5 phosphorylation site displayed relatively low cell surface expression and relatively low abilities to form heterodimers of DOR and MOR; intrathecal delivery of a construct expressing the T161A mutant of DOR attenuated morphine antinociceptive tolerance in rats, suggesting that Thr-161 phosphorylation of DOR contributed to Cdk5-mediated morphine antinociceptive tolerance. Furthermore, an engineered Tat fusion-interfering peptide corresponding to the second intracellular loop of DOR (Tat-DOR-2L), reduced the cell surface expression of DOR, disrupted the formation of DOR-MOR heterodimers, and significantly attenuated the development of morphine antinociceptive tolerance after intrathecal injection. The present study indicates that Cdk5-mediated phosphorylation of DOR at Thr-161 plays a crucial role in the development of morphine tolerance and suggests the possibility of targeting DOR phosphorylation at Thr-161 to attenuate morphine antinociceptive tolerance during pain management.