Predescu Sanda A, Predescu Dan N, Timblin Barbara K, Stan Radu V, Malik Asrar B
Department of Pharmacology, University of Illinois College of Medicine, Chicago, Illinois 60612, USA.
Mol Biol Cell. 2003 Dec;14(12):4997-5010. doi: 10.1091/mbc.e03-01-0041. Epub 2003 Sep 5.
Intersectin, a multiple Eps15 homology and Src homology 3 (SH3) domain-containing protein, is a component of the endocytic machinery in neurons and nonneuronal cells. However, its role in endocytosis via caveolae in endothelial cells (ECs) is unclear. We demonstrate herein by coimmunoprecipitation, velocity sedimentation on glycerol gradients, and cross-linking that intersectin is present in ECs in a membrane-associated protein complex containing dynamin and SNAP-23. Electron microscopy (EM) immunogold labeling studies indicated that intersectin associated preferentially with the caveolar necks, and it remained associated with caveolae after their fission from the plasmalemma. A cell-free system depleted of intersectin failed to support caveolae fission from the plasma membrane. A biotin assay used to quantify caveolae internalization and extensive EM morphological analysis of ECs overexpressing wt-intersectin indicated a wide range of morphological changes (i.e., large caveolae clusters marginated at cell periphery and pleiomorphic caveolar necks) as well as impaired caveolae internalization. Biochemical evaluation of caveolae-mediated uptake by ELISA showed a 68.4% inhibition by reference to control. We also showed that intersectin interaction with dynamin was important in regulating the fission and internalization of caveolae. Taken together, the results indicate the crucial role of intersectin in the mechanism of caveolae fission in endothelial cells.
相交蛋白（Intersectin）是一种含有多个Eps15同源结构域和Src同源结构域3（SH3）的蛋白质，是神经元和非神经元细胞内吞机制的一个组成部分。然而，其在内皮细胞（ECs）中通过小窝介导的内吞作用中的作用尚不清楚。我们在此通过免疫共沉淀、甘油梯度速度沉降和交联实验证明，相交蛋白存在于内皮细胞中，且与一种包含发动蛋白和SNAP-23的膜相关蛋白复合物相结合。电子显微镜（EM）免疫金标记研究表明，相交蛋白优先与小窝颈部相关联，并且在小窝从质膜分裂后仍与小窝保持关联。一个耗尽相交蛋白的无细胞系统无法支持小窝从质膜的分裂。一种用于量化小窝内化的生物素测定法以及对过表达野生型相交蛋白的内皮细胞进行的广泛EM形态学分析表明，存在广泛的形态学变化（即大的小窝簇在细胞周边边缘化以及多形性的小窝颈部）以及小窝内化受损。通过ELISA对小窝介导的摄取进行的生化评估显示，与对照相比抑制率为68.4%。我们还表明，相交蛋白与发动蛋白的相互作用在调节小窝的分裂和内化中很重要。综上所述，结果表明相交蛋白在内皮细胞小窝分裂机制中起着关键作用。
