ADP-ribosylation Factor-dependent Phospholipase D2 Activation Is Required for Agonist-induced μ-Opioid Receptor Endocytosis

Thomas Koch,Lars-Ove Brandenburg,Stefan Schulz,Yingjian Liang,Jochen Klein,Volker Höllt

doi:10.1074/jbc.m206709200

Abstract

Agonist exposure of many G protein-coupled receptors induces a rapid receptor phosphorylation and uncoupling from G proteins. Resensitization of these desensitized receptors requires endocytosis and subsequent dephosphorylation. Using a yeast two-hybrid screen, the rat mu-opioid receptor (MOR1, also termed MOP) was found to be associated with phospholipase D2 (PLD2), a phospholipid-specific phosphodiesterase located in the plasma membrane, which has been implicated in the formation of endocytotic vesicles. Coimmunoprecipitation experiments in HEK293 cells coexpressing MOR1 and PLD2 confirmed that MOR1 constitutively interacts with PLD2. Treatment with the mu receptor agonist DAMGO ([d-Ala(2), Me Phe(4), Glyol(5)]enkephalin) led to an increase in PLD2 activity, whereas morphine, which does not induce MOR1 receptor internalization, failed to induce PLD2 activation. The DAMGO-mediated PLD2 activation was inhibited by brefeldin A, an inhibitor of ADP-ribosylation factor (ARF) but not by the protein kinase C (PKC) inhibitor calphostin C indicating that opioid receptor-mediated activation of PLD2 is ARF- but not PKC-dependent. Furthermore, heterologous stimulation of PLD2 by phorbol ester led to an accelerated internalization of the mu-opioid receptor after both DAMGO and morphine exposure. Conversely the inhibition of PLD2-mediated phosphatidic acid formation by 1-butanol or overexpression of a negative mutant of PLD2 prevented agonist-mediated endocytosis of MOR1. Together, these data suggest that PLD2 play a key role in the regulation of agonist-induced endocytosis of the mu-opioid receptor.

Highlights

IntroductionSignal-dependent activation of Phospholipase D (PLD) was demonstrated in numerous cell types stimulated by various hormones, growth factors, cytokines, neurotransmitters, adhesion molecules, drugs, and physical stimuli (reviewed in Ref. 3)
Play an important function in cell regulation [1, 2]
MOR1 Stimulates phospholipase D2 (PLD2) Activity in HEK293 Cells—Since PLD2 activation has been previously described for various G protein-coupled receptors, the association of MOR1 and PLD2 indicated that agonist stimulation of the MOR1 might activate the PLD2

Summary

Introduction

Signal-dependent activation of PLD was demonstrated in numerous cell types stimulated by various hormones, growth factors, cytokines, neurotransmitters, adhesion molecules, drugs, and physical stimuli (reviewed in Ref. 3). Another study revealed that PLD2 is associated with the EGF receptor [12]. EGF receptor endocytosis is impaired when PLD activity is inhibited [13] suggesting a role for PLD2 in receptor trafficking. Using the yeast two-hybrid system to identify proteins that interact with the ␮-opioid receptor, we isolated a rat cDNA encoding for the NH2 terminus of PLD2. We investigated the potential role of PLD2 in the process of agonistmediated endocytosis of the ␮-opioid receptor. We show that PLD2 is constitutively associated with the ␮-opioid receptor. We provide evidence that the opioid receptormediated activation is ARF-dependent and essential for receptor endocytosis. Phospholipase D (PLD) is a widely distributed phospholipid-specific diesterase that hydrolyzes phosphatidylcholine (PC) to phosphatidic acid (PA) and choline and is assumed to

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Results

Conclusion