MECHANISM(S) OF ACTION OF NERVE GROWTH FACTOR

神经生长因子（NGF）在神经系统发育和 神经系统的维持是有据可查的。 虽然我们知道很多 关于它的化学和结构， 但在理解神经生长因子参与的 NGF的作用机制。 本提案的总体目标是 是研究受体生物化学中的两个主要问题， 关于NGF：（I）是一种神经生长因子的内化和随后的加工， 神经生长因子-受体复合物的必要性，为引发的生理 对这种激素的反应（即，NGF）？以及（II）在这种情况下， 如果将其引入靶细胞， 通过一种绕过其天然受体（NGF）的机制， 受体）？ 实验方法将利用“嵌合” 毒素技术”，这是一种新的强大的方法，将用于 该项目用于选择具有改变的NGF受体的细胞变体， 功能协调发展的 大鼠嗜铬细胞瘤来源的克隆细胞系PC-12， 通过广泛的神经突生长和获得 神经元样分化特性将用作组织培养物 这些研究的模型系统。 要回答第一个问题，一个“嵌合体” 或由NGF和纯化的高毒性蓖麻毒素A组成的杂合缀合物 将建立链。 这种受体特异性缀合物（NGF-A）将 可用于选择对以下抗性的变体PC-12系（克隆）： 被毒素杀死 两种变异表型，NGF受体阴性 (R-)和NGF受体阳性-内化阴性（R+I-），将是 通过标准受体结合测定区分。 然后将检测内化阴性变体（R+I-），以确定 如果单独的NGF结合足以引起对NGF的反应。 回答 第二个问题是，由NGF和 纯化的蓖麻毒素B链，无毒的半乳糖结合亚基， 构建，并用于通过蓖麻毒素将NGF导入（R-）细胞 受体。 然后，将对NGF-B缀合物进行测试。 受体阴性变体（R-）。 这些克隆体将被直接用于 测试神经生长因子受体的重要性，在细胞的反应， 通过测定神经生长因子依赖的现象，如神经突生长和 已知的多型反应。

The importance of nerve growth factor (NGF) in the development and maintenance of the nervous system is well documented. While much is known about its chemistry and structure and about the biological processes in which NGF participates, little progress has been made toward understanding the mechanism(s) by which NGF acts. The overall objective of this proposal is to investigate two major questions in hormone-receptor biochemistry with respect to NGF: (I) is the internalization and subsequent processing of an NGF-receptor complex necessary for elicitation of the physiological responses to this hormone (i.e., NGF)? and (II) can a hormone, in this case NGF, exert its physiological responses if it is introduced into a target cell by a mechanism which by-passes its native receptor (the NGF receptor)? The experimental approach will take advantage of "chimeric toxin technology", a new and powerful methodology, which will be used in this project for the selection of cell variants with altered NGF-receptor functions. A rat pheochromocytoma-derived clonal cell line, PC-12, which responds to NGF by extensive neurite outgrowth and acquisition of neuron-like differentiated properties will be used as a tissue culture model system for these studies. To answer the first question, a "chimeric" or hybrid conjugate consisting of NGF and purified, highly-toxic ricin A chain will be constructed. This receptor-specific conjugate (NGF-A) will be used to select variant PC-12 lines (clones) which are resistant to killing by toxin. Two resulting variant phenotypes, NGF-receptor negative (R-) and NGF-receptor positive-internalization negative (R+I-), will be distinguished by standard receptor-binding assays. Internalization-negative variants (R+I-) will then be tested to determine if NGF binding alone is sufficient to elicit responses to NGF. To answer the second question, a "chimeric" conjugate (NGF-B) consisting of NGF and the purified B chain of ricin, the non-toxic galactose-binding subunit will be constructed, and used to introduce NGF into (R-) cells via ricin receptors. The NGF-B conjugate will then be tested on the receptor-negative variants (R-). These clones will be used to directly test for the importance of the NGF-receptor in cellular responsiveness to NGF, by assaying for NGF-dependent phenomena such as neurite outgrowth and known pleiotypic responses.