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首页> 外文期刊>Biochemistry >Cholesterol-dependent association of caveolin-1 with the transducin alpha subunit in bovine photoreceptor rod outer segments: Disruption by cyclodextrin and guanosine 5 '-O-(3-thiotriphosphate)
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Cholesterol-dependent association of caveolin-1 with the transducin alpha subunit in bovine photoreceptor rod outer segments: Disruption by cyclodextrin and guanosine 5 '-O-(3-thiotriphosphate)

机译：胆甾醇-1与牛感光受体外部片段中的转导蛋白α亚基的胆固醇依赖性缔合：环糊精和鸟苷5'-O-（3-硫代三磷酸）的破坏

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Evidence suggests that caveolins, 21-24 kDa cholesterol-binding proteins that generally reside in specialized detergent-resistant membrane microdomains, act as signaling scaffolds. Detergent-resistant membranes isolated from rod outer segments (ROS) have been previously shown to contain the photoreceptor G-protein, transducin. In this report we show, by subcellular fractionation, that caveolin-1 is an authentic component of purified ROS. We demonstrate that caveolin-1 in ROS almost exclusively resides in low-buoyant-density, cholesterol-rich, detergent-resistant membranes that can be disrupted by cholesterol depletion using methyl-beta-cyclodextrin (MCD). Cholesterol depletion was also observed to extract a pool of transducin alpha (Talpha) from ROS membranes. Immunoprecipitation with anti-caveolin-1 revealed the association of Talpha in the absence of Tbetagamma. Treatment of ROS with MCD resulted in a 2-fold decrease in recovery of Talpha in anti-caveolin-1 immunoprecipitates. This interaction was also completely disrupted when ROS were exposed to light in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), a nonhydrolyzable GTP analogue. In addition, caveolin-1/Talpha association in the immune complex was disrupted by a peptide based on the primary sequence of the caveolin-1 scaffolding domain. Finally, we confirm the colocalization of caveolin-1 and Talpha in photoreceptors by immunofluorescence microscopy. These results strongly suggest that the association between Talpha and caveolin-1 occurs in cholesterol-rich, detergent-resistant membranes and is likely to be dependent upon the activation state of Talpha. [References: 64]
机译：有证据表明，通常位于特定的耐去污剂的膜微区中的21-24 kDa胆固醇结合蛋白小窝蛋白起信号传递支架的作用。从杆外段（ROS）分离的耐洗涤剂膜先前已显示含有感光G蛋白，转导蛋白。在本报告中，我们通过亚细胞分级显示，caveolin-1是纯化的ROS的可靠成分。我们证明了ROS中的caveolin-1几乎只存在于低浮力密度，富含胆固醇，耐洗涤剂的膜中，该膜可被使用甲基-β-环糊精（MCD）的胆固醇消耗所破坏。还观察到胆固醇消耗从ROS膜上提取了一组转导蛋白α（Talpha）。用抗caveolin-1进行的免疫沉淀揭示了在缺乏Tbetagamma的情况下Talpha的相关性。用MCD处理ROS可使抗caveolin-1免疫沉淀物中Talpha的回收率降低2倍。当在不可水解的GTP类似物鸟苷5'-O-（3-硫代三磷酸）（GTPgammaS）存在下将ROS暴露在光下时，这种相互作用也被完全破坏。另外，基于caveolin-1支架结构域的一级序列的肽破坏了免疫复合物中的caveolin-1 / Talpha缔合。最后，我们通过免疫荧光显微镜证实小窝蛋白1和Talpha在感光器中的共定位。这些结果强烈表明，Talpha和Caveolin-1之间的缔合发生在富含胆固醇，耐去污剂的膜中，并且可能取决于Talpha的活化状态。 [参考：64]

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