Abstract

We demonstrated recently that opioid-induced activation of phospholipase D2 (PLD2) enhances μ- (MOPr) and δ-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.