Cholesterol reduction by methyl-β-cyclodextrin attenuates the delta opioid receptor-mediated signaling in neuronal cells but enhances it in non-neuronal cells
Cholesterol reduction by methyl-β-cyclodextrin attenuates the delta opioid receptor-mediated signaling in neuronal cells but enhances it in non-neuronal cells
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DOI：
10.1016/j.bcp.2006.10.032
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发表时间：
2007-02-15
影响因子：
5.8
通讯作者：
Liu-Chen, Lee-Yuan
中科院分区：
文献类型：
--
作者：
Huang, Peng;Xu, Wei;Liu-Chen, Lee-Yuan
阿片受体位于富含小泡蛋白的非神经元细胞的脂筏/小泡中，并受其调节。本研究发现，在内源性表达delta阿片受体（DOR）的NG108-15细胞中，caveolin-1在大鼠脑中的表达水平非常低，且检测不到。用FLAG-mouse-DOR （CHO- flag - mdor）稳定转染的大鼠尾状壳核（CPu）膜、NG108-15细胞和CHO细胞均质化，在无洗涤剂的0.5 M Na2CO3缓冲液中超声，通过不连续或连续的蔗糖密度梯度进行分离。两种细胞系的CPu和DOR中约70%的阿片受体存在于富含胆固醇和神经节苷M1 （GM1）的低密度（5-20%蔗糖）膜结构域，这是质膜脂筏的特征。在这两个细胞中，通过纳洛酮可逆和百日咳毒素不敏感机制，可通过渗透性或非渗透性完全激动剂，而不是部分或反向激动剂刺激30分钟，将类似25%的DORs从木排中转移出去，这种机制可能经历内化。甲基- p -环糊精（MCD）处理大大降低了胆固醇，将DOR转移到更高密度的组分，并降低了DPDPE的亲和力。MCD处理可减弱dpdpe诱导的[S-35] GTR γ S在CPu和NG108-15细胞中的结合，但可增强CHO-FLAG-mDOR细胞中的结合。在CHO-FLAG-mDOR细胞中，G（α i）与caveolin-1共免疫沉淀，这被证明可以抑制G(α i/o)， MCD治疗显著降低了这种关联，导致抑制解除。因此，尽管筏中的定位和激动剂诱导的DOR的转移不依赖于小窝蛋白-1，但脂质筏在小窝蛋白缺乏的神经元细胞中维持DOR介导的信号传导，但在小窝蛋白富集的非神经元细胞中似乎抑制了它。胆固醇依赖性的小窝蛋白-1与G蛋白的关联和由此产生的抑制可能是一个促成因素。(c) 2006爱思唯尔公司版权所有。
Opioid receptors have been shown to be located in and regulated by lipid rafts/caveolae in caveolin-rich non-neuronal cells. Here, we found that caveolin-1 level was very low in rat brain and undetectable in NG108-15 cells, which endogenously express delta opioid receptors (DOR). Rat caudate putamen (CPu) membranes, NG108-15 cells and CHO cells stably transfected with FLAG-mouse-DOR (CHO-FLAG-mDOR) were homogenized, sonicated in a detergent-free 0.5 M Na2CO3 buffer and fractionated through discontinuous or continuous sucrose density gradients. About 70% of opioid receptors in CPu and DOR in both cell lines were present in low-density (5-20% sucrose) membrane domains enriched in cholesterol and ganglioside M1 (GM1), characteristics of lipid rafts in plasma membranes. In both cells, stimulation with permeable or non-permeable full agonists, but not with partial or inverse agonists, for 30 min shifted similar to 25% of DORs out of rafts, by a naloxone-reversible and pertussis toxin-insensitive mechanism, which may undergo internalization. Methyl-P-cyclodextrin (MCD) treatment greatly reduced cholesterol and shifted DOR to higher density fractions and decreased DPDPE affinities. MCD treatment attenuated DPDPE-induced [S-35] GTR gamma S binding in CPu and NG108-15 cells, but enhanced it in CHO-FLAG-mDOR cells. In CHO-FLAG-mDOR cells, G(alpha i) co-immunoprecipitated with caveolin-1, which was shown to inhibit G(alpha i/o), and MCD treatment dramatically reduced the association leading to disinhibition. Thus, although localization in rafts and agonist-induced shift of DOR are independent of caveolin-1, lipid rafts sustain DOR-mediated signaling in caveolin-deficient neuronal cells, but appear to inhibit it in caveolin- enriched non-neuronal cells. Cholesterol-dependent association of caveolin-1 with and the resulting inhibition of G proteins may be a contributing factor. (c) 2006 Elsevier Inc. All rights reserved.