ADP-ribosylation factor-dependent phospholipase D2 activation is required for agonist-induced μ-opioid receptor endocytosis
ADP-ribosylation factor-dependent phospholipase D2 activation is required for agonist-induced μ-opioid receptor endocytosis
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DOI：
10.1074/jbc.m206709200
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发表时间：
2003-03-14
影响因子：
4.8
通讯作者：
Höllt, V
中科院分区：
文献类型：
--
作者：
Koch, T;Brandenburg, LO;Höllt, V
激动剂暴露于许多G蛋白偶联受体后，受体迅速磷酸化，并与G蛋白解偶联。这些脱敏受体的再增敏需要内吞作用和随后的去磷酸化作用。通过酵母双杂交筛选，发现大鼠Mu-阿片受体(MOR1，又称MOP)与磷脂酶D2(PLD2)有关，磷脂酶D2是一种位于质膜上的磷脂专一性磷酸二酯酶，与内吞囊泡的形成有关。在共表达MOR1和PLD2的HEK293细胞中的免疫共沉淀实验证实了MOR1与PLD2的结构性相互作用。用Mu受体激动剂DAMGO([D-Ala 2，Me Phe(4)，Glyol(5)]Enkephalin)处理后，PLD2活性增加，而不诱导MOR1受体内化的吗啡不能诱导PLD2激活。DAMGO介导的PLD2激活可被ADP核糖化因子(ARF)的抑制剂布雷菲尔丁A抑制，但不被蛋白激酶C(PKC)抑制剂calphostin C抑制，这表明阿片受体介导的PLD2激活是ARF依赖的，而不是PKC依赖的。此外，佛波酯对PLD2的异源刺激导致在DAMGO和吗啡暴露后mU-阿片受体的内化加速。相反，正丁醇抑制PLD2介导的磷脂酸的形成或过表达PLD2的负性突变体可以阻止激动剂介导的MOR1内吞作用。综上所述，这些数据表明PLD2在激动剂诱导的阿片受体内吞作用的调节中起着关键作用。
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.