Endophilin isoforms have distinct characteristics in interactions with N-type Ca2+ channels and dynamin I
Endophilin isoforms have distinct characteristics in interactions with N-type Ca2+ channels and dynamin I
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内亲素亚型在与 N 型 Ca2 通道和动力 I 的相互作用中具有独特的特征
DOI：
10.1007/s12264-012-1257-z
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发表时间：
2012-07
影响因子：
5.6
通讯作者：
Yuan Chen
中科院分区：
文献类型：
--
作者：
Qi Tian;Jifeng Zhang;Jinjin Fan;Zhihong Song;Yuan Chen
目的：在网格蛋白介导的内吞作用中，内亲素Ⅱ-Ca（2+）通道复合物的形成依赖于Ca（2+）。然而，很少有人知道其他两个内啡肽亚型是否具有相同的功能。方法：用3 H-leucine标记试剂盒合成N型Ca（2+）通道C端片段（NCF），与endophilin-GST融合蛋白共同孵育，采用pull-down法检测NCF与Ca 2+通道和发动蛋白I的相互作用。在闪烁计数器上计数结果。此外，将不同的内啡肽同种型各自与发动蛋白I共转染到293 T细胞中，随后进行流式细胞术和免疫共沉淀测定。免疫组化染色和图像分析程序被用来评估重叠系数的细胞表达的内啡肽和发动蛋白I。结果：所有三个异构体与NCF相互作用。Endophilin I和II与NCF表现出明显的Ca（2+）依赖性相互作用，而Endophilin III则没有。免疫共沉淀结果表明，与endophilin I/II相比，endophilin III和dynamin I之间的相互作用显著增加。从流式细胞术获得类似的结果。结论：Endophilin Ⅲ与NCF和dynamin Ⅰ的相互作用具有明显的特点。内皮素III与NCF的结合是非Ca（2+）依赖性的，这意味着它在网格蛋白介导的内吞作用中发挥着不同的作用。
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.
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影响因子：
7.8
作者：
Hill, E;van Der Kaay, J;Downes, C P;Smythe, E
通讯作者：
Smythe, E
影响因子：
16.2
作者：
Chowdhury, Shoaib;Shepherd, Jason D.;Worley, Paul F.
通讯作者：
Worley, Paul F.
影响因子：
4.8
作者：
Micheva, KD;Kay, BK;McPherson, PS
通讯作者：
McPherson, PS
影响因子：
4.4
作者：
Giachino, C;Lantelme, E;Migone, N
通讯作者：
Migone, N
影响因子：
21.3
作者：
Simpson, F;Hussain, NK;Schmid, SL
通讯作者：
Schmid, SL