STRUCTURE-FUNCTION DYNAMICS OF NGF RECEPTORS

一种蛋白质之间存在基本的三方关系 结构、其功能以及相对于 它必须与之相互作用的分子。这项建议集中在 这种关系对于神经生长因子受体(NGFR)所起的作用 在神经发育和修复中起着重要作用。NGFR在这两种情况下都存在 高亲和力和低亲和力的形式。低拷贝数高亲和力形式是 功能所必需的。最近，第二个组件gp140trk已经 识别，这是高亲和力结合所必需的。 我们已经证明gp75是均匀分布和自由扩散的。 在无反应细胞上，但在反应细胞上聚集和固定 细胞。我们的假设是gp140trk与gp75形成络合物，形成 高亲和力NGFR，负责固定和聚集 反应细胞上的gp75。为了检验这一假设，我们将问： 1.gp140trk的扩散和分布特性是什么？一个。 Gp140trk的分布和扩散性与 响应性？B.添加NGF是否改变了分布或 Gp140trk？C.gp75与哪些结构有关 和/或反应细胞中的gp140trk复合体？D.是扩散吗 高亲和力和低亲和力NGFR gp75和gp140trk的性质不同。 将通过以下方式研究gp140trk的成分分布 荧光显微镜。扩散测量将使用 光漂白后荧光恢复(FRAP)。 2.高亲和力的NGFR是gp75和gp140trk的复合体吗？(a.)会吗？ 这个复合体是预先存在的，还是响应NGF而形成的？(B)在那里吗 这个建筑群中有不止一个gp75或gp140trk吗？ NGFR及其成分gp75和gp140trk的相对量将 在有反应和无反应的细胞上用放射性测定 约束性分析。然后将使用三种方法来解决 聚合问题：(1.)Frap；(2.)受体增溶作用；以及 (3)荧光共振能量转移(FRET)。 3.gp75-gp140trk络合是造成大规模的原因吗？ 在反应细胞上观察到gp75的聚集？我们将进一步 开发一种使用视频成像FRET显微镜来确定 Gp75-gp140trk复合体在细胞表面的空间分布。 4.gp75和gp140trk的哪些结构域控制功能和物理 NGFR的特性？使用NGFR突变体，我们会问，a.是 Gp75的胞质结构域是形成复合体所必需的，gp75- Gp140trk固定化？B.是gp140trk的胞内结构域 Gp75-gp140trk复合体的形成和固定所需的？C.IS 需要一个功能激活域吗？D.是以下项目的共识序列 是否需要与涂层坑相关联？

There is a fundamental tripartite relationship between a proteins's structure, its function, and its ability to diffuse relative to the molecules with which it must interact. This proposal concentrates on this relationship for the nerve growth factor receptor (NGFR) which plays an important role in neural development and repair. NGFR exists in both high- and low-affinity forms. The low copy number high-affinity form is required for function. Recently, a second component, gp140trk, has been identified, which is essential for high-affinity binding. We have shown that gp75 is homogeneously distributed and diffuses freely on nonresponsive cells, but is aggregated and immobilized on responsive cells. Our hypothesis is that gp140trk complexes with gp75 to form the high affinity NGFR and is responsible for immobilizing and aggregating gp75 on responsive cells. To test this hypothesis we will ask: 1. What are the diffusion and distribution properties of gp140trk? a. How does gp140trk distribution and diffusibility relate to responsiveness? b. Does addition of NGF alter the distribution or diffusion of gp140trk? c. What structures are associated with gp75 and/or gp140trk complexes in responsive cells? d. Are the diffusion properties of high- and low-affinity NGFR gp75 and gp140trk different. Distribution of the components of gp140trk will be studied by fluorescence microscopy. Diffusion measurements will be made using fluorescence recovery after photobleaching (FRAP). 2. Is the high-affinity NGFR a complex of gp75 and gp140trk? (a.) Does the complex pre-exist or does it form in response to NGF? (b.) Is there more than one gp75 or gp140trk in this complex? The relative amounts of NGFR and its components gp75 and gp140trk will be determined on responsive and nonresponsive cells by radioactive binding analysis. Three methods will then be used to address the question of aggregation: (1.) FRAP; (2.) receptor solubilization; and (3.) fluorescence resonance energy transfer (FRET). 3. Is gp75-gp140trk complexing responsible for the large scale aggregation of gp75 observed on responsive cells? We will further develop a new method using video imaging FRET microscopy to determine the spatial distribution of gp75-gp140trk complexes on the cell surface. 4. Which domains of gp75 and gp140trk control the function and physical properties of NGFR? Using NGFR mutants we will ask, a. Is the cytoplasmic domain of gp75 required for complex formation and gp75- gp140trk immobilization? b. Is the intracellular domain of gp140trk required for gp75-gp140trk complex formation and immobilization? c. Is a functional kinase domain required? d. Is the consensus sequence for association with coated pits required?