Elliott Michael H, Fliesler Steven J, Ghalayini Abboud J
Dean A. McGee Eye Institute, Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
Biochemistry. 2003 Jul 8;42(26):7892-903. doi: 10.1021/bi027162n.
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.
有证据表明，小窝蛋白是一种分子量为21 - 24 kDa的胆固醇结合蛋白，通常存在于特殊的抗去污剂膜微区中，起到信号支架的作用。先前已证明，从视杆细胞外段（ROS）分离出的抗去污剂膜含有光感受器G蛋白——转导素。在本报告中，我们通过亚细胞分级分离表明，小窝蛋白-1是纯化的ROS的真实组成成分。我们证明，ROS中的小窝蛋白-1几乎完全存在于低浮力密度、富含胆固醇、抗去污剂的膜中，使用甲基-β-环糊精（MCD）消耗胆固醇可破坏这些膜。还观察到胆固醇消耗会从ROS膜中提取出一部分转导素α（Tα）。用抗小窝蛋白-1进行免疫沉淀揭示了在没有Tβγ的情况下Tα的关联。用MCD处理ROS导致抗小窝蛋白-1免疫沉淀中Tα的回收率降低了2倍。当ROS在鸟苷5'-O-(3-硫代三磷酸)（GTPγS，一种不可水解的GTP类似物）存在下暴露于光时，这种相互作用也会完全被破坏。此外，基于小窝蛋白-1支架结构域的一级序列的肽会破坏免疫复合物中的小窝蛋白-1/Tα关联。最后，我们通过免疫荧光显微镜证实了小窝蛋白-1和Tα在光感受器中的共定位。这些结果强烈表明，Tα与小窝蛋白-1之间的关联发生在富含胆固醇、抗去污剂的膜中，并且可能取决于Tα的激活状态。
