Differential Effect of Membrane Cholesterol Removal on μ- and δ-Opioid Receptors A PARALLEL COMPARISON OF ACUTE AND CHRONIC SIGNALING TO ADENYLYL CYCLASE
Differential Effect of Membrane Cholesterol Removal on μ- and δ-Opioid Receptors A PARALLEL COMPARISON OF ACUTE AND CHRONIC SIGNALING TO ADENYLYL CYCLASE
复制标题
DOI：
10.1074/jbc.m109.030411
复制
发表时间：
2009-08-14
影响因子：
4.8
通讯作者：
Traynor, John R.
中科院分区：
文献类型：
--
作者：
Levitt, Erica S.;Clark, Mary J.;Traynor, John R.
根据脂筏理论，质膜含有富含胆固醇和鞘脂的小结构域，这些结构域可以作为组织膜蛋白的平台。利用甲基-β-环糊精(M-β-CD)来清除膜胆固醇，许多G蛋白偶联受体被证明依赖于假定的脂筏来适当地传递信号。在这里，我们检验了一种假设，即用MβCD处理稳定表达标志标记的u阿片受体(HEK FLAG-MU)或增量阿片受体(HEK FLAG-DELTA)的HEK293细胞将减少阿片受体对腺苷酸环化酶的信号。Mu-阿片激动剂[D-Ala(2)，N-Me-Phe(4)，Gly(5)-ol]脑啡肽对腺酰环化酶有明显的抑制作用，或在慢性治疗后引起腺酰环化酶敏化。这些效应是由于胆固醇的去除，因为胆固醇的补充使[D-Ala(2)，N-Me-Phe(4)，Gly(5)-ol]脑啡肽反应恢复到对照组，并在内源性表达Mu-阿片受体的SH-SY5Y细胞中得到证实。MβCD的作用可能是由于Mu受体与G蛋白的解偶联，而不是由于受体数量的减少，也不是通过细胞骨架的破坏来模拟的。与HEK FLAG-MU细胞的实验结果相反，MβCD处理HEK FLAG-Delta细胞不影响Delta阿片激动剂对腺苷环化酶的急性抑制或增敏作用。Mu-阿片激动剂和Delta-阿片激动剂对胆固醇耗竭的不同反应表明，Mu-阿片受体比Delta受体更依赖于胆固醇来实现有效的信号传递，部分原因是Mu-阿片受体而不是Delta-阿片受体在胆固醇和小窝蛋白富集膜结构域中的定位。
According to the lipid raft theory, the plasma membrane contains small domains enriched in cholesterol and sphingolipid, which may serve as platforms to organize membrane proteins. Using methyl-beta-cyclodextrin (M beta CD) to deplete membrane cholesterol, many G protein-coupled receptors have been shown to depend on putative lipid rafts for proper signaling. Here we examine the hypothesis that treatment of HEK293 cells stably expressing FLAG-tagged mu-opioid receptors (HEK FLAG-mu) or delta-opioid receptors (HEK FLAG-delta) with M beta CD will reduce opioid receptor signaling to adenylyl cyclase. The ability of the mu-opioid agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol] enkephalin to acutely inhibit adenylyl cyclase or to cause sensitization of adenylyl cyclase following chronic treatment was attenuated with M beta CD. These effects were due to removal of cholesterol, because replenishment of cholesterol restored [D-Ala(2),N-Me-Phe(4),Gly(5)-ol] enkephalin responses back to control values, and were confirmed in SH-SY5Y cells endogenously expressing mu-opioid receptors. The effects of M beta CD may be due to uncoupling of the mu receptor from G proteins but were not because of decreases in receptor number and were not mimicked by cytoskeleton disruption. In contrast to the results in HEK FLAG-mu cells, M beta CD treatment of HEK FLAG-delta cells had no effect on acute inhibition or sensitization of adenylyl cyclase by delta-opioid agonists. The differential responses of mu- and delta-opioid agonists to cholesterol depletion suggest that mu-opioid receptors are more dependent on cholesterol for efficient signaling than delta receptors and can be partly explained by localization of mu- but not delta-opioid receptors in cholesterol- and caveolin-enriched membrane domains.