Role of Phospholipase D2/Phosphatidic Acid Signal Transduction in μ- and δ-Opioid Receptor Endocytosis

Liquan Yang,Thomas Koch,Anja Seifert,Helmut Schröder,Volker Höllt,Lars O Brandenburg,Daifei Wu,Xiaoqian Wang,Vladan Rankovic

doi:10.1124/mol.109.063107

Abstract

We demonstrated recently that opioid-induced activation of phospholipase D2 (PLD2) enhances mu- (MOPr) and delta-opioid receptor endocytosis/recycling and thus reduces the development of opioid receptor desensitization and tolerance. However, the mechanistic basis for the PLD2-mediated induction of opioid receptor endocytosis is currently unknown. Here we show that PLD2-generated phosphatidic acid (PA) might play a key role in facilitating the endocytosis of opioid receptors. However, PLD2-derived PA is known to be further converted to diacylglycerol (DAG) by PA phosphohydrolase (PPAP2). In fact, blocking of PA phosphohydrolase activity by propranolol or PPAP2-short interfering RNA (siRNA) transfection significantly attenuated agonist-induced opioid receptor endocytosis. The primary importance of PA-derived DAG in the induction of opioid receptor endocytosis was further supported by the finding that increasing the DAG level by inhibiting the reconversion of DAG into PA with the DAG kinase inhibitor 3-[2-(4-[bis-(4-fluorophenyl)methylene]-1-piperidinyl)ethyl]-2,3-dihydro-2-thioxo-4(1H)quinazolinone (R59949) or the addition of the synthetic cell-permeable DAG analog 1,2-dioctanoyl-sn-glycerol (DOG), further increased the agonist-induced opioid receptor endocytosis. Moreover, the addition of DOG bypasses the PLD2-siRNA- or PPAP2-siRNA-mediated impairment of DAG synthesis and resulted in a restoration of agonist-induced opioid receptor internalization. Further studies established a functional link between PA-derived DAG and the activation of p38 mitogen-activated protein kinase (MAPK) and the subsequent phosphorylation of the Rab5 effector early endosome antigen 1, which has been demonstrated recently to be required for the induction of MOPr endocytosis. Taken together, our results revealed that the regulation of opioid receptor endocytosis by PLD2 involves the conversion of its product PA to DAG resulting in an activation of the p38 MAPK pathway.

Highlights

Our previous study found that the pigment epithelium-derived factor (PEDF) gene can be expressed in aqueous humor and lens epithelial cells, and that the expression level decreased significantly with increasing age
Co-expression of green fluorescent protein (GFP) with the small hairpin RNA (shRNA) constructs allowed for the evaluation of the efficiency of gene transfer to primary lens epithelium in culture
Vimentin and αB-crystallin expression are modulated by small hairpin RNA directed against pigment epithelium-derived factor (PEDF): Given the known changes in PEDF expression in catarctous lenses, we looked at the changes in key lens structural proteins implicated in the formation of cataract following the downregulation of PEDF expression by shRNA

Summary

Introduction

Our previous study found that the PEDF gene can be expressed in aqueous humor and lens epithelial cells, and that the expression level decreased significantly with increasing age. Those results suggested PEDF may regulate or protect. One study of the molecular characteristics of lens epithelial cells from patients with senile cataract by cDNA microarray technique found that PEDF was strongly downregulated (by 5.9-fold) in senile cataract [9]. One study suggested that aB-crystallin may be an important component of the cellular machinery involved in maintaining genomic stability [23]. The reduced thermal stability and the dominant negative effects of the mutant αB-crystallin may be the direct cause of cataract because αB-crystallin null mice have clear lenses [24]

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