Endophilin isoforms have distinct characteristics in interactions with N-type Ca^(2+) channels and dynamin I  

作　　者：Qi Tian Ji-Feng Zhang Jinjin Fan Zhihong Song Yuan Chen 

机构地区：[1]Center for Neurobiology, Zhongshan School of Medicine [2]Department of Nephrology, The First Affiliated Hospital [3]Department of Biochemistry, Zhongshan School of Medicine, Sun-Yat Sen University, Guangzhou 510080, China

出　　处：《Neuroscience Bulletin》2012年第5期483-492,共10页神经科学通报（英文版）

基　　金：supported by grants from the National Natural Science Foundation of China (30870785);the Natural Science Foundation of Guangdong Province, China (9351008901000003)

摘　　要：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.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.

关 键 词：ENDOPHILIN Ca2＋ dynamin I ENDOCYTOSIS proline-rich domain SH3 domain 

分 类 号：Q463[生物学—生理学] Q78