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

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 -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. ]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 -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 -opioid receptor. Phospholipase D (PLD)1 is a widely distributed phospholipid-specific diesterase that hydrolyzes phosphatidylcholine (PC) to phosphatidic acid (PA) and choline and is assumed to play an important function in cell regulation (1, 2). 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). Pathways leading to PLD activation include protein serine/threonine kinases, e.g. protein kinase C, small GTPases, e.g. ADP-ribosylation factor (ARF), RhoA and Ral, phosphatidylinositol 4,5-bisphosphate (PIP2), and tyrosine kinases (4 -6). Recently two mammalian PLDs (PLD1 and PLD2) have been identified (7-10). Subcellular fractionation studies have demonstrated the presence of PLD1 in intracellular membranes, e.g. ER, Golgi, and vesicular compartment (9, 11), whereas PLD2 was largely associated with the plasma membrane (9). After stimulation with serum, redistribution of PLD2 from the plasma membrane into submembraneous endocytotic vesicles (early endosomes) was observed (9). Another study revealed that PLD2 is associated with the EGF receptor (12). Interestingly, 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 NH 2 terminus of PLD2. We therefore investig...

Ral and Phospholipase D2-Dependent Pathway for Constitutive Metabotropic Glutamate Receptor Endocytosis

“…Ral may also regulate EGF receptor endocytosis through its interaction with phospholipase D1 (PLD1) and PLD2 (Shen et al, 2001). However, a role for Ral in GPCR endocytosis has yet to be described, and it is not clear what functional role PLD2 plays in regulating clathrin-coated vesicle-mediated endocytosis (Shen et al, 2001;Koch et al, 2003Koch et al, , 2004Du et al, 2004). Here, we show that both Ral and PLD2 bind to class 1 mGluRs and act as proximal adaptor proteins, the activity of which is specifically required to promote constitutive mGluR endocytosis.…”

Lysophosphatidylcholine suppresses apoptosis and induces neurite outgrowth in PC12 cells through activation of phospholipase D2

“…LPC has been shown to play a crucial role in the Ca 2+-dependent activation of PLD2 in L1210 and COS-7 cells (Kim et al, 1999b). PLD2 activity has been reported to be stimulated by PKC-dependent phosphorylation, interaction with ARF, and several unsaturated fatty acids (Lopez et al, 1998;Kim et al, 1999a;Sung et al, 1999;Han et al, 2002;Kim et al, 2003b;Koch et al, 2003;Chen and Exton, 2004). However, LPC could not activate directly the enzymatic activity of PLD2 in vitro (Kim et al, 1999b).…”

Chronic morphine treatment up-regulates mu opioid receptor binding in cells lacking filamin A

“…There are many examples of association of the GPCRs, including MOP with other proteins, such as beta-arrestins, (Lefkowitz, 1998), GRKs (Pitcher et al, 1998) and small G proteins (Mitchell et al, 1998). Different laboratories, including ours, have reported interaction of MOP C-tail with different proteins, such as periplakin (Feng et al, 2003), Phospholipase D2 (Koch et al, 2003), mPKCI (Guang et al, 2004), HSP40 {Ancevska-Taneva, 2006 #5292)and Filamin A (Onoprishvili et al, 2003).…”