STRUCTURE & FUNCTION OF NERVE GROWTH FACTOR

神经生长因子（NGF）是一种关键的分化和维持 感觉、交感和中枢神经元的蛋白质。 信息 关于活性β亚基的蛋白质结构 和蛋白质的7S寡聚体形式， 神经系统中的NGF，其质膜的结构 受体、受体的细胞动力学和运动 在绑定之后，以及所需信令的某些方面 用于应答细胞的分化。 信息 β-NGF分子的结构-功能关系是 对于理解这种神经营养蛋白至关重要。 该项目的总体目标是确定结构 活性β-NGF亚基的特征， 与细胞受体相互作用， 信号，使用重组DNA和免疫化学方法。 分子工程。 一个目的是获得β-NGF变体， 在分子的四个选定区域中的氨基酸替换 并将这些结构变化与生物学特性联系起来。 在前一个赠款期间，生物活性NGF已被 在E.大肠杆菌系统利用克隆的 小鼠cDNA。 关键氨基酸的定点突变 现在将允许确定单个残基的作用， 生物功能。 β-NGF的拮抗剂和部分激动剂 也可用于生物学研究。 免疫化学。 另一个目的是确定NGF分子的表面表位 通过产生位点特异性的 针对合成的NGF寡肽的抗体 蛋白 对应于特定序列的寡肽， β-神经生长因子分子正在合成，位点特异性 它们的抗体就会产生， 用于确定表面部分的功能作用， β-NGF分子。 这些突变的神经生长因子蛋白和位点特异性抗体将被 通过大鼠嗜铬细胞瘤（PC 12）细胞中的轴突生长研究 和鸡背根感觉神经元，在受体结合， PC 12细胞中的细胞运输，细胞信号传导和基因 调节，在β-NGF的自我二聚化，在形成 7S寡聚体的构象分析， 重组蛋白 这些研究的结果将进一步促进我们的 基本结构要求的知识， 神经生长因子与神经元靶点相互作用及其分子机制 以及神经元分化和存活的细胞方面。 新的类似物的神经生长因子将产生，应该是有用的， 发展研究 这些信息最终会导致 更好地了解神经发育问题和相关的 阿尔茨海默氏症和亨廷顿氏症等疾病。

Nerve Growth Factor (NGF) is a key differentiation and maintenance protein for sensory, sympathetic, and central neurons. Information is available about the protein structure of the active beta-subunit and the 7S oligomeric form of the protein, the developmental role of NGF in the nervous system, the structure of its plasma membrane receptor, the cellular dynamics and movement of the receptor subsequent to binding, and some aspects of the signalling required for differentiation of responsive cells. Information about the structure-function relationships of the beta-NGF molecule is essential for understanding such neuronotrophic proteins. The overall goal of this project is to determine structural features of the active beta-NGF subunit that are responsible for interaction with the cellular receptor and resultant intracellular signalling, using recombinant DNA and immunochemical approaches. Molecular engineering. One aim is to obtain beta-NGF variants with amino acid replacements in four selected regions of the molecule and correlate these structural changes with biological properties. In the previous grant period biologically active NGF has been successfully expressed in an E. coli system utilizing the cloned moused cDNA. Site specific mutagenesis of critical amino acids will now allow determination of the role of individual residues in biological function. Antagonists and partial agonists of beta-NGF may also be obtained for biological studies. Immunochemistry. Another aim is to determine surface epitopes of the NGF molecule involved in receptor recognition by producing site-specific antibodies directed against synthetic oligopeptides of the NGF protein. Oligopeptides corresponding to particular sequences in the beta-NGF molecule are being synthesized, site specific antibodies toward them will be produced, and the antibodies will be used to ascertain the functional role of surface portions of the beta-NGF molecule. These mutant NGF proteins and site specific antibodies will be studied by neurite outgrowth in rat Pheochromocytoma (PC12) cells and chick dorsal root sensory neurons, in receptor binding and cellular trafficking in PC12 cells, in cellular signalling and gene regulation, in the self-dimerization of beta-NGF, in the formation of the 7S oligomer, and analysis of the conformation of the recombinant protein. The results of these studies will further our knowledge of the fundamental structural requirements for the interaction of NGF with neuronal targets and elucidate molecular and cellular aspects of neuronal differentiation and survival. Novel analogues of NGF will be produced that should be useful in developmental studies. this information will eventually lead to better understanding of neurodevelopmental problems and related diseased such as Alzheimer's and Huntington's diseases.