Characterization of two distinct modes of endophilin in clathrin-mediated endocytosis.
Characterization of two distinct modes of endophilin in clathrin-mediated endocytosis.
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DOI：
10.1016/j.cellsig.2012.06.006
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发表时间：
2012-11
影响因子：
4.8
通讯作者：
Jifeng Zhang;Jin-jin Fan;Qi Tian;Zhihong Song;Ji-fang Zhang;Yuan Chen
中科院分区：
文献类型：
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作者：
Jifeng Zhang;Jin-jin Fan;Qi Tian;Zhihong Song;Ji-fang Zhang;Yuan Chen
内啡肽是网格蛋白介导的内吞作用的主要辅助蛋白之一，通过其SH 3结构域与其他内吞蛋白如发动蛋白相互作用。我们之前报道过电压门控Ca 2+通道通过与内啡肽的相互作用成为突触囊泡（SV）内吞机制的组成部分。内亲素通道复合物的形成是Ca 2+依赖性的。内亲素中的谷氨酸残基E264是通道-内亲素复合物的Ca 2+依赖性形成的主要Ca 2+传感器的一部分。我们提出，endophilin存在于两个不同的模式（构象），开放模式的情况下的Ca 2+，和封闭模式的存在下的Ca 2+。Ca 2+的结合将内亲和素从其开放模式切换到闭合模式，导致内亲和素与其他蛋白质的解离。本研究旨在通过构建两个内啡肽突变体E264 A和E264 R，分别模拟内啡肽的永久开放模式和永久封闭模式，以了解内啡肽在两种不同模式下的功能作用。在这里，我们表明，这两种模式的内啡肽有不同的影响如何与其他蛋白质，如发动蛋白或β1-肾上腺素能受体的相互作用。在活细胞中，处于永久封闭模式的内亲素对激动剂诱导的β1-肾上腺素能受体内化没有明显影响。Endophilin在其永久开放模式下，增强了培养的海马神经元的短时程突触抑制，部分原因是其在Ca 2+存在下不能与Ca 2+通道解离。我们的研究结果表明，模式切换由Ca 2+允许endophilin调节，更有效地，网格蛋白介导的SV在神经末梢的内吞作用。
Endophilin, one of the main accessory proteins involved in clathrin-mediated endocytosis, interacts with other endocytic proteins, such as dynamin, by its SH3 domain. We previously reported that voltage-gated Ca2+channels are an integral part of the synaptic vesicle (SV) endocytosis machinery through their interaction with endophilin. Formation of the endophilin–channel complex is Ca2+dependent. A glutamate residue, E264, in endophilin is part of the primary Ca2+sensor for Ca2+-dependent formation of the channel–endophilin complex. We proposed that endophilin exists in two distinct modes (conformations), an open mode in the absence of Ca2+, and a closed mode in the presence of Ca2+. Binding of Ca2+switches endophilin from its open mode to the closed mode, resulting in dissociation of endophilin from other proteins. The present study is aimed at understanding the functional roles of endophilin in its two different modes, by creating two endophilin mutants, E264A and E264R, to mimic endophilin in its permanent open mode and permanent closed mode respectively. Here, we show that these two modes of endophilin have different effects on how endophilin interacts with other proteins, such as dynamin or β1-adrenergic receptors. In living cells, endophilin in its permanent closed mode does not show obvious effects on agonist-induced internalization of β1-adrenergic receptors. Endophilin, when in its permanent open mode, enhances the short-term synaptic depression in cultured hippocampal neurons, due partly to its failure to dissociate from Ca2+channels in the presence of Ca2+. Our results show that modal switching by Ca2+allows endophilin to regulate, more effectively, the clathrin-mediated endocytosis of SV at the nerve terminal.