NEUROTROPHIN RECEPTOR INTERACTIONS

75 kD（p75）和 将测试酪氨酸激酶（TRK）神经营养因子受体。模型 提出p75的功能形式是二聚体， 受磷酸化调节，神经营养因子作用于p75和trk 受体独立，神经营养因子激活p75， 一致构象转变的Monod模型， p75的活性构象优先结合trk二聚体， TRK受体二聚化和活化。此外，p75 通过调节逆行轴突信号传导促进逆行轴突信号传导 将检测活性神经营养素/TrK复合物的转运。结合 p75受体表达的研究和生物化学研究， 将进行非神经元和神经元细胞系以检查 受体二聚化、磷酸化和阳性表达之间的关系 协同神经营养因子结合，以及这些特性之间的关系 p75增强TRK受体活化的能力将是 考察p75的体外诱变，随后在非- 神经元和神经元细胞系，将用于定义重要的 参与p75功能的结构元件-特别关注 胞质结构域中推定的调节磷酸化位点，和 跨膜结构域内的半胱氨酰残基，其可能是必需的 用于产生功能性受体二聚体。p75的作用 携带神经营养素的内吞囊泡的细胞内运输， 将在非神经元细胞和神经元轴突中检测trk受体。 培养的神经元。免疫组化技术在电子 将进行显微镜水平表征 p75和trk受体。共焦 将使用pH敏感染料进行荧光显微镜检查， 确定鸡轴突内核内体酸化的时间 培养的胚胎交感神经和感觉轴突。这些信息， 再加上研究神经营养素的pH依赖性的结果， 与受体结合，将揭示是否具有功能活性 神经营养因子/受体复合物可以从神经完整地运输 末端的神经元细胞体，以传递逆行营养 信号.这些研究将描述基本的过程， 神经营养因子塑造发育中的 大脑和周围神经系统，并通过其再生的 神经系统受到刺激。

A specific model for the functional interaction of 75 kD (p75) and tyrosine kinase (trk) neurotrophin receptors will be tested. The model proposes that the functional form of p75 is dimeric, that dimerization is regulated by phosphorylation, that neurotrophins act upon p75 and trk receptors independently, that activation of p75 by neurotrophins follows the Monod model for concerted conformational transitions, and that the active conformation of p75 preferentially binds trk dimers, facilitating trk receptor dimerization and activation. Also, the possibility that p75 facilitates retrograde axonal signaling by regulating retrograde axonal transport of active neurotrophin/trk complexes will be examined. Binding studies and biochemical studies of p75 receptors expressed in various non-neuronal and neuronal cell lines will be performed to examine the relationship of receptor dimerization, phosphorylation, and positively cooperative neurotrophin binding, and relationship of these properties to the ability of p75 to enhance trk receptor activation will be examined. In vitro mutagenesis of p75, followed by expression in non- neuronal and neuronal cell lines, will be employed to define important structural elements involved in p75 function - particularly focusing on putative regulatory phosphorylation sites in the cytoplasmic domain, and a cysteinyl residue within the transmembrane domain which may be required for generation of functional receptor dimers. The effects of p75 on intracellular trafficking of endocytic vesicles bearing neurotrophins and trk receptors will be examined, in non-neuronal cells, and in axons of neurons in culture. Immunohistochemical techniques at the electron microscopic level will be performed to characterize the distribution of p75 and trk receptors among intracellular membrane pools. Confocal fluorescence microscopy, with pH-sensitive dyes, will be performed to determine the timing of acidification of endosomes within axons of chick embryo sympathetic and sensory axons in culture. This information, coupled with the results of examining the pH-dependence of neurotrophin binding to receptors, will reveal whether functionally active neurotrophin/receptor complexes may be transported intact from the nerve terminus to the neuronal cell body in order to convey retrograde trophic signals. These studies will characterize processes which are fundamental to the mechanisms by which neurotrophic factors shape the developing brain and peripheral nervous system, and by which regeneration of the injured nervous system is encouraged.