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家族蛋白在突触后信号从膜受体传递到肌动蛋白细胞骨架的过程中发挥了衔接蛋白的作用。在之前的研究中，我们建立了皮层神经元原代培养，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了drebrin的表达，并证明突触功能蛋白的活性依赖性积累发生了变化。接下来，我们假设除肌动蛋白外，还有其他drebrin结合蛋白在神经元突触后信号传递中发挥重要作用。我们使用酵母双杂交系统克隆drebrin家族蛋白（drebrin E， drebrin A和SH3P7）的结合蛋白。其中一个克隆，被命名为DRAP1，编码一种新的drebrin结合蛋白的全长尾链。然后我们对其进行了生化表征，发现它的n端一半实际上与drebrin分子的n端区域结合。接下来，利用DRAP1的多肽和基因工程技术表达的DRAP1片段，构建DRAP1的多克隆抗血清。进一步构建了编码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，我们在其细胞核中观察到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.