内啡肽同工型在与 N 型钙通道和动力蛋白 I 的相互作用中具有不同的特征。
Endophilin isoforms have distinct characteristics in interactions with N-type Ca2+ channels and dynamin I.
机构信息
Center for Neurobiology, Zhongshan School of Medicine, Guangzhou, 510080, China.
出版信息
Neurosci Bull. 2012 Oct;28(5):483-92. doi: 10.1007/s12264-012-1257-z. Epub 2012 Jul 13.
OBJECTIVE
Formation of the endophilin II-Ca(2+) channel complex is Ca(2+)-dependent in clathrin-mediated endocytosis. However, little is known about whether the other two endophilin isoforms have the same features. The present study aimed to investigate the characteristics of the interactions of all three isoforms with Ca(2+) channels and dynamin I.
METHODS
N-type Ca(2+) channel C-terminal fragments (NCFs) synthesized with a (3)H-leucine-labeled kit, were incubated with endophilin-GST fusion proteins, followed by pull-down assay. Results were counted on a scintillation counter. In addition, the different endophilin isoforms were each co-transfected with dynamin I into 293T cells, followed by flow cytometry and co-immunoprecipitation assay. Immunostaining was performed and an image analysis program was used to evaluate the overlap coefficient of cells expressing endophilin and dynamin I.
RESULTS
All three isoforms interacted with NCF. Endophilins I and II demonstrated clear Ca(2+)-dependent interactions with NCF, whereas endophilin III did not. Co-immunoprecipitation showed that, compared to endophilin I/II, the interaction between endophilin III and dynamin I was significantly increased. Similar results were obtained from flow cytometry. Furthermore, endophilin III had a higher overlap coefficient with dynamin I in co-transfected 293T cells.
CONCLUSION
Endophilin isoforms have distinct characteristics in interactions with NCF and dynamin I. Endophilin III binding to NCF is Ca(2+)-independent, implying that it plays a different role in clathrin-mediated endocytosis.
目的
网格蛋白介导的胞吞作用中，内收蛋白 II-Ca(2+)通道复合物的形成依赖于 Ca(2+)。然而，对于其他两种内收蛋白同种型是否具有相同的特征，人们知之甚少。本研究旨在探讨这三种同种型与 Ca(2+)通道和动力蛋白 I 相互作用的特点。
方法
用(3)H-亮氨酸标记试剂盒合成 N 型 Ca(2+)通道 C 末端片段(NCFs)，与内收蛋白-GST 融合蛋白孵育，然后进行下拉测定。在闪烁计数器上计数结果。此外，将不同的内收蛋白同种型分别与动力蛋白 I 共转染 293T 细胞，然后进行流式细胞术和共免疫沉淀测定。进行免疫染色，并使用图像分析程序评估表达内收蛋白和动力蛋白 I 的细胞的重叠系数。
结果
所有三种同种型都与 NCF 相互作用。内收蛋白 I 和 II 与 NCF 表现出明显的 Ca(2+)-依赖性相互作用，而内收蛋白 III 则没有。免疫共沉淀显示，与内收蛋白 I/II 相比，内收蛋白 III 与动力蛋白 I 的相互作用显著增加。流式细胞术也得到了类似的结果。此外，在共转染的 293T 细胞中，内收蛋白 III 与动力蛋白 I 的重叠系数更高。
结论
内收蛋白同种型在与 NCF 和动力蛋白 I 的相互作用方面具有不同的特征。内收蛋白 III 与 NCF 的结合不依赖于 Ca(2+)，这表明它在网格蛋白介导的胞吞作用中发挥着不同的作用。
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