Elsevier

European Journal of Pharmacology

Volume 519, Issue 3, 20 September 2005, Pages 212-214
European Journal of Pharmacology

Short communication
Morphine promotes phosphorylation of the human δ-opioid receptor at serine 363

https://doi.org/10.1016/j.ejphar.2005.07.024Get rights and content

Abstract

After prolonged stimulation, the δ-opioid receptor becomes desensitized by regulatory mechanisms such as receptor phosphorylation, internalization and down-regulation. In this study, we demonstrate that morphine treatment causes phosphorylation of S363 in the C-terminus of the human δ-opioid receptor. Morphine-mediated phosphorylation reached 53 ± 8% of maximum deltorphin II-mediated phosphorylation. Phosphorylation of S363 may contribute to δ-opioid receptor desensitization by morphine.

Introduction

Morphine is a commonly used pharmaceutical agent that produces its effects by activating three types of opioid receptors. After repeated or prolonged stimulation, the opioid receptors become less responsive (desensitized)— an adaptation which is thought to contribute to opioid tolerance. Desensitization of most G protein-coupled receptors involves a cascade of regulatory mechanisms, such as receptor phosphorylation, internalization and down-regulation. Phosphorylation of the δ-opioid receptor has been implicated in rapid desensitization (Pei et al., 1995) as well as in β-arrestin- and clathrin-mediated internalization (Law et al., 2000). In contrast to other opioid agonists, morphine elicits minimal δ-opioid receptor phosphorylation and β-arrestin translocation (Zhang et al., 1999), and does not stimulate rapid δ-opioid receptor internalization (Keith et al., 1996). Overexpression of G protein-coupled receptor kinase 2 facilitated morphine-mediated δ-opioid receptor phosphorylation (Zhang et al., 1999), suggesting that the morphine-activated δ-opioid receptor is a sub-optimal substrate for G protein-coupled receptor kinases (Von Zastrow et al., 2003).

The peptide agonist, DADLE ([d-Ala2, d-Leu5]enkephalin) was shown to phosphorylate several residues in the C-terminus of the rodent δ-opioid receptor, with S363 as the primary phosphorylation site (Kouhen et al., 2000). It is presently not known whether morphine is able to phosphorylate the same residues as peptide agonists. Therefore, in this study, we investigated phosphorylation of S363 in the human δ-opioid receptor upon morphine or deltorphin II treatment. Using an antibody specific for S363-phosphorylated δ-opioid receptor, we demonstrate that in Chinese hamster ovary cells (CHO) expressing the human δ-opioid receptor (Malatynska et al., 1996) morphine promotes phosphorylation of S363.

Section snippets

Materials and methods

To induce receptor phosphorylation, CHO cells expressing the human δ-opioid receptor were treated with morphine (30 μM) (Sigma) or deltorphin II (100 nM) (Tocris) for 30 min at 37 °C in a serum-free medium. Optimal agonist concentrations and treatment times to achieve maximal receptor phosphorylation were determined in preliminary experiments. After agonist treatment, the cells were rinsed and harvested in homogenization buffer (20 mM Tris, 4 mM EGTA, 2 mM EDTA, pH 7.4) containing 1% protease

Results

In samples from agonist-treated CHO cells expressing the human δ-opioid receptor the anti-phospho-S363 antibody recognized a diffuse band in the molecular weight range of 50–70 kDa, presumably corresponding to differently glycosylated forms of the S363-phosphorylated human δ-opioid receptor (Fig. 1, lanes 2, 3). Quantitative analyses of the 50–70 kDa bands show that morphine (30 μM)-mediated S363 phosphorylation was 53 ± 8% of maximum phosphorylation mediated by deltorphin II (100 nM). Both

Discussion

Our findings indicate that, like a full δ-selective opioid agonist deltorphin II, morphine is also able to promote phosphorylation of S363 in the C-terminus of the human δ-opioid receptor. Phosphorylation of S363 is the initial step in δ-opioid receptor regulation and is a prerequisite for phosphorylation of other residues in the C-terminus including T358, T361 (Kouhen et al., 2000). Additional sites may also be phosphorylated in other intracellular domains. Since total morphine-mediated

Acknowledgements

The authors thank Michelle Thatcher and Carol Haussler for maintaining the cell cultures. This work was supported by grants from the National Institutes of Health.

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