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

Abstract 

Objective Formation of the endophilin II-Ca2+ channel complex is Ca2+-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 Ca2+ channels and dynamin I. Methods N-type Ca2+ channel C-terminal fragments (NCFs) synthesized with a 3H-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 Ca2+-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 Ca2+-independent, implying that it plays a different role in clathrin-mediated endocytosis.

Keywords

endophilin; Ca2+; dynamin I; endocytosis; proline-rich domain; SH3 domain

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