STRUCTURE AND FUNCTION OF NERVE GROWTH FACTOR

神经生长因子（NGF）是参与神经生长的重要蛋白质。 感觉和交感神经元的发育和维持。 虽然 关于活性β亚基的蛋白质结构已知很多， 蛋白质的7S寡聚体形式， 发育，以及其刺激分化的细胞生物学， 反应性细胞，对结构-功能关系知之甚少 β-神经生长因子本身 该项目旨在确定一些 13，000 Da多肽链的特征， 与细胞受体的相互作用以及对细胞的影响 形态学 其具体目标是：（1）利用特定场地 诱变以获得β-NGF变体;（2）将这些变化关联起来 具有生物学和蛋白质性质;（3）获得激动剂和部分激动剂， β-NGF激动剂;（4）产生针对 针对合成寡肽;以及（5）将这些寡肽的作用与 抗体对β-NGF生物学特性的影响。 将采用两种方法来实现这些目标。 (a)免疫化学。 对应于β-NGF中特定序列的寡肽 分子将被合成，并且针对它们的单特异性抗体将 制作。 现有单克隆抗体识别的决定簇 将被确定。 这些抗体将用于研究功能性 β-NGF分子的不同部分的作用。 (b)分子 工程. 我们已经将β-NGF的cDNA插入E.杆菌 表达系统 关键氨基酸的位点特异性置换 将产生残留物，以确定这些残留物在 生物功能。 抗体和改变的β-NGF的作用将在 嗜铬细胞瘤（PC 12）细胞和感觉神经元的神经突生长， 结合PC 12细胞的细胞受体，在早期细胞反应中， 在β亚基与其他两个亚基的相互作用中， 7S寡聚体和β-NGF的二聚化。 效应对 重组β-NGF的构象将通过 光谱技术 这些实验的结果应该提供 用于发育研究的β-NGF的有用的新激动剂和拮抗剂， 以及允许在分子水平上的批判性理解 神经生长因子与神经细胞相互作用和刺激的必要特征。

Nerve Growth Factor (NGF) is an important protein involved in the development and maintenance of sensory and sympathetic neurons. Although much is known about the protein structure of the active beta-subunit and the 7S oligomeric form of the protein, the neurobiologic role during development, and the cell biology of its stimulus for differentiation of responsive cells, less is known about the structure-function relationships in the beta-NGF itself. This project is directed toward determining some of the features of the 13,000 Da polypeptide chain that are responsible for interaction with the cellular receptor and resultant effects on cell morphology. The specific aims are: (1) to utilize site-specific mutagenesis to obtain beta-NGF variants; (2) to correlate these changes with biological and protein properties; (3) to obtain agonists and partial agonists of beta-NGF; (4) to produce site-specific antibodies directed against synthetic oligopeptides; and (5) to correlate the effects of these antibodies on biological properties of beta-NGF. Two approaches will be utilized to achieve these aims. (a) Immunochemical. Oligopeptides corresponding to particular sequences in the beta-NGF molecule will be synthesized and monospecific antibodies toward them will be produced. The determinants recognized by existing monoclonal antibodies will be determined. These antibodies will be used to study the functional role of various portions of the beta-NGF molecule. (b) Molecular engineering. We have inserted the cDNA for beta-NGF into an E. coli expression system. Site specific replacements in critical amino acid residues will be produced to determine the role of these residues in biological function. The effects of the antibodies and altered beta-NGF will be assessed in neurite outgrowth in Pheochromocytoma (PC12) cells and sensory neurons, in binding to cellular receptor of PC12 cells, in early cellular responses to NGF, in the interaction of the beta subunit with the other two subunits of the 7S oligomer, and on the dimerization of beta-NGF. Effects on the conformation of the recombinant beta-NGF will be determined by spectroscopic techniques. The results of these experiments should provide useful new agonists and antagonists of beta-NGF for developmental studies, as well as allow a critical understanding at the molecular level of the requisite features for interaction and stimulation of neuronal cells by NGF.