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Regulation of synaptic actin reorganization by signal transmission with drebrin family

通过与drebrin家族的信号传递调节突触肌动蛋白重组

基本信息

批准号：
12480236
负责人：
SHIRAO Tomoaki
金额：
$ 9.54万
依托单位：
Gunma University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

Drebrins are actin-binding proteins, which expression is closely related to spine morphology. Therefore we hypothesized that drebrin-family proteins play adaptor proteins of postsynaptic signal transmission from membrane receptor to actin cytoskeleton.In the previous study, we established the primary culture of cortical neurons, in which neurons can develop and the dendritic spine seemed to be fully maturated. Using this culture systems, we inhibit the drebrin expression wit antisense-oligonucleotides, and demonstrated that activity-dependent accumulation of synaptic functional proteins were changed.Next, we hypothesized that other drebrin-binding proteins than actin play an important role in postsynaptic signal transmission in the neuron. We performed the yeast two hybrid system for cloning the binding proteins to drebrin-family proteins (drebrin E, drebrin A, and SH3P7).One of the clone, designated as DRAP1, encodes full length of a coda of a novel drebrin-binding protein. Then we performed its biochemical characterization and found its N-terminal half actually binds to the N-terminal region of a drebrin molecule. Next, using polypeptide of DRAP1 and DRAP1 fragment expressed by gene engineering technique, we raise polyclonal antiserum to DRAP1.Further, we constructed expression vector which encode the GFP-DRAP1 fused protein. When we transformed fibroblasts with the expression vector, we observed GFP fluorescence in their nuclei.

Drebrins是肌动蛋白结合蛋白，其表达与棘的形态密切相关。因此，我们推测drebrin家族蛋白在突触后信号从膜受体到肌动蛋白细胞骨架的传递中起着衔接蛋白的作用。在前期的研究中，我们建立了皮层神经元的原代培养体系，在此体系中神经元可以发育，树突棘似乎已经完全成熟。利用这种培养系统，我们用反义寡核苷酸抑制drebrin的表达，证明了突触功能蛋白的活性依赖性积累被改变。接下来，我们假设除了肌动蛋白之外，其他drebrin结合蛋白在神经元突触后信号传递中起重要作用。我们利用酵母双杂交系统克隆了drebrin家族蛋白（drebrin E、drebrin A和SH 3 P7）的结合蛋白，其中一个克隆（DRAP 1）编码一个新的drebrin结合蛋白的尾段全长。然后我们对其进行了生物化学表征，发现其N端的一半实际上结合到一个dreplant分子的N端区域。然后，利用基因工程技术表达DRAP 1多肽和DRAP 1片段，制备DRAP 1多克隆抗血清，并构建GFP-DRAP 1融合蛋白表达载体。当我们用表达载体转化成纤维细胞时，我们在它们的细胞核中观察到GFP荧光。