Received for publication August 24, 2006.
Revised February 9, 2007.
Accepted for publication February 21, 2007.

Quantitative evaluation of human delta opioid receptor desensitization using the Operational Model of Drug Action

Abstract

Agonist-mediated desensitization of the opioid receptors is thought to function as a protective mechanism against sustained opioid signaling and therefore may prevent the development of opioid tolerance. However, the exact molecular mechanism of opioid receptor desensitization remains unresolved, due to difficulties in measuring and interpreting receptor desensitization. In the present study, we investigated deltorphin II-mediated rapid desensitization of the human delta opioid receptors (hDOR) by measuring GTP[³⁵S] binding and inhibition of cAMP accumulation. We developed a mathematical analysis based on the Operational Model of Agonist Action (Black et al., 1985) to calculate the proportion of desensitized receptors. This approach permits a correct analysis of the complex process of functional desensitization by taking into account receptor-effector coupling and the time dependence of agonist pre-treatment. Finally, we compared hDOR desensitization with receptor phosphorylation at S363, the translocation of -arrestin2, and hDOR internalization. We found that in Chinese hamster ovary (CHO) cells expressing the hDOR, deltorphin II treatment leads to phosphorylation of S363, translocation of & [beta]-arrestin2 to the plasma membrane, receptor internalization and uncoupling from G proteins. Interestingly, mutation of the primary phosphorylation site S363 to alanine had virtually no effect on agonist- induced -arrestin2 translocation and receptor internalization, yet significantly attenuated receptor desensitization. These results strongly indicate that phosphorylation of S363 is the primary mechanism of hDOR desensitization.

Key words: Opioid, Desensitization/uncoupling, Sequestration/Internalization, GRKs, barrestins, Phosphorylation/Dephosphorylation