CYTOSKELETAL TARGETS OF NEURONAL SIGNALING

这项建议涉及神经元细胞骨架的分子控制。 通过细胞外信号传递蛋白质。了解神经元是如何 细胞骨架的调控很重要，因为有几个疾病过程， 包括阿尔茨海默氏症和精神分裂症在内的疾病 细胞骨架，从而改变神经元功能。在成长过程中， 发育和突触发生，神经元的形态受到控制 受生长因子和神经递质等局部环境因素的影响。 成体神经元的微结构也被认为在 对突触或神经体液活动的反应。我们建议研究 神经元形态可塑性的细胞基础 微管的磷酸化状态和功能的调节- 相关蛋白MAP2。MAP2被认为在 ITS对树枝状形态的发育和维持 与微管和其他细胞骨架元素的相互作用。多重 已知蛋白激酶和蛋白磷酸酶可修饰MAP2 磷酸化和MAP2在体外发挥作用，但对MAP2的作用知之甚少 MAP2在体内的磷酸化特性。我们之前的研究 揭示了一个改变完整的MAP2磷酸化的信号 神经元是兴奋性氨基酸受体的激活。Map2成为 作为N-甲基-D-脱氧核糖核酸的结果快速和选择性地脱磷 天冬氨酸受体激活。这种效果似乎与一部小说有关 NMDA受体的信号转导途径。在拟议的研究中， 我们将定义信号转导通路来调节 海马片和培养脑片中MAP2的磷酸化状态 海马神经元。~(32)P-代谢性标记海马神经元 正磷酸盐将被用来确定机制，受体 兴奋性氨基酸诱导的特异性和时间特性 MAP2的去磷酸化。对MAP2磷酸化的促进作用 神经递质和生长因子依赖的蛋白激酶活性将 也要接受检查。我们将研究两种形式的MAP2：常规MAP2 在成年神经元中发现，以及一种独特的选择性剪接变异体 未成熟的神经元被称为MAP2c。这种变种缺少一个大的中间部分 ，因此包含相对较少数量的 磷酸化位点。MAP2的不成熟形式将帮助我们定义 哪些磷酸化位点受NMDA受体控制。 一旦我们确定了MAP2和MAP2c的激酶和磷酸酶途径 调节，我们将确定磷酸化的准确位置 位于较小的蛋白MAP2c上的位点。这些信息将是重要的 最终确定MAP2和MAP2c的磷酸化作用 在调节神经元形态方面。这些结果将有助于 细胞外信号调控MAP2功能的研究进展 活着。更广泛地说，这些实验将允许一种 神经元跨膜调节的分子模型 细胞骨架。

This proposal addresses the molecular control of neuronal cytoskeletal proteins by extracellular signals. Understanding how the neuronal cytoskeleton is regulated is important because several disease processes, including Alzheimer's disease and schizophrenia are thought to affect the cytoskeleton and thereby alter neuronal function. During outgrowth, development, and synaptogenesis, the morphology of neurons is controlled by local environmental cues like growth factors and neurotransmitters. The microstructure of adult neurons is also thought to be modified in response to synaptic or neurohumoral activity. We propose to examine the cellular basis for morphological plasticity in neurons by focusing on the regulation of the phosphorylation state and function of the microtubule- associated protein MAP2. MAP2 is thought to play an important role in the development and maintenance of dendritic morphology via its interaction with microtubules and other cytoskeletal elements. Multiple protein kinases and protein phosphatases are known to modify MAP2 phosphorylation and MAP2 function in vitro, but little is known about the properties of MAP2 phosphorylation in vivo. Our previous studies revealed that one signal which alters MAP2 phosphorylation in intact neurons is activation of excitatory amino acid receptors. MAP2 becomes rapidly and selectively dephosphorylated as a result of N-methyl-D- aspartate receptor activation. This effect appears to involve a novel signal transduction pathway for NMDA receptors. In the proposed studies, we will define the signal transduction pathways which modulate the phosphorylation state of MAP2 in hippocampal slices and cultured hippocampal neurons. Hippocampal cells metabolically labeled with 32P- orthophosphate will be used to determine the mechanism, receptor specificity and temporal properties of excitatory amino acid-induced dephosphorylation of MAP2. The stimulation of MAP2 phosphorylation by neurotransmitter and growth factor-dependent protein kinase activity will also be examined. Two forms of MAP2 will be studied: conventional MAP2 found in adult neurons, and an alternatively spliced variant unique to immature neurons called MAP2c. This variant lacks a large middle portion of adult MAP2, and thus contains a relatively small number of phosphorylation sites. The immature form of MAP2 will help us to define which phosphorylation sites are under the control of NMDA receptors. Once we identify the kinase and phosphatase pathways for MAP2 and MAP2c regulation, we will determine the precise location of phosphorylation sites on the smaller protein MAP2c. Such information will be important in eventually determining the role of phosphorylation of MAP2 and MAP2c in regulating neuronal morphology. These results will contribute to an understanding of how extracellular signals regulate MAP2 function in vivo. More generally, these experiments will allow formulation of a molecular model for transmembrane regulation of the neuronal cytoskeleton.