Regulation of synaptic actin reorganization by signal transmission with drebrin family

Drebrins是一种肌动蛋白结合蛋白，其表达与脊柱形态密切相关。因此，我们假设Drebrin家族蛋白在膜受体到肌动蛋白细胞骨架的突触后信号传递中起适配蛋白的作用。在先前的研究中，我们建立了皮质神经元的原代培养，在这种培养中，神经元可以发育，树突棘似乎完全成熟。利用这种培养体系，我们用反义寡核苷酸抑制了Drebrin的表达，并证明了突触功能蛋白的活性依赖积累发生了改变。其次，我们假设除了肌动蛋白之外，其他Drebrin结合蛋白在神经元的突触后信号传递中发挥着重要作用。我们使用酵母双杂交系统克隆了Drebrin家族蛋白(Drebrin E、Drebrin A和SH3P7)的结合蛋白，其中一个克隆命名为DRAP1，编码一个新的Drebrin结合蛋白的全长尾巴。然后，我们进行了它的生化鉴定，发现它的N-末端半部分实际上与DREBRIN分子的N-末端区域结合。然后，利用基因工程技术表达的DRAP1多肽和DRAP1片段，制备抗DRAP1的多克隆抗血清，构建编码GFP-DRAP1融合蛋白的表达载体。当我们用表达载体转化成纤维细胞时，我们观察到它们的细胞核中有GFP荧光。

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.