Role of cGKII in AMPA Receptor Transport

描述(申请人提供)：AMPA受体产生触发动作电位形成的主要去极化电流。突触上受体的插入和移除速度的改变可以改变受体突触的丰度和控制突触的强度。AMPA受体C末端结构域的磷酸化被认为是受体与控制转运的蛋白质相互作用的主要调节因素。突触将与突触活动相关的离子流转化为控制激酶和CTD磷酸化的生化信号。位于GluR1CTD中间的丝氨酸845上的GluR1亚单位被PKA磷酸化，与质膜突触外部位的GluR1水平相关。我们将使用分子和电生理学的方法来研究环状GMP调节的蛋白激酶cGKII对丝氨酸845磷酸化的新机制。CGKII受NMDA受体控制，通过NMDAR激活nNOS，产生一氧化氮，激活可溶性鸟苷环化酶，产生cGMP，从而诱导cGKII的发生。本课题的目的是：1.分析cGKII肉豆蔻酰化、二聚化和激活在GluR1转运和生理过程中的作用。我们将分析cGKII的结构如何参与GluR1的调节。目的2.探讨S845磷酸化增加细胞质膜GluR1水平的机制。我们将确定细胞内cGKII磷酸化GluR1的位置，并对GluR1的运输产生影响。我们将尝试区分cGKII是否调节GluR1的胞吐作用，或者GluR1是否通过S845磷酸化稳定在质膜上。我们将分离对S845磷酸化有反应的因子，并控制GluR1表面水平。目的：研究cGKII对GluR4和GluR2L转运的调控作用。GluR4和GluR2L是AMPAR亚基，它们在结构上与GluR1相关，这表明它们也可能受cGKII控制。我们将分析cGKII在这些亚基运输中的作用，以确定是否更普遍地使用NMDAR-nNOS-cGKII途径。该项目将提供有关突触功能活动依赖控制机制的新信息，并可能有助于确定一氧化氮在这些机制中的作用。与公共健康相关：学习和记忆取决于大脑中神经元之间连接强度的变化。这个项目将研究一种新的通路，它可以让神经元对自己的活动做出反应，并改变它们之间的联系强度。这项工作与记忆形成机制和学习障碍的起源有关。它可以为设计帮助保持记忆或减轻学习障碍的新药提供基础。

DESCRIPTION (provided by applicant): AMPA receptors generate the major depolarizing currents that trigger the formation of action potentials. Alterations of the rates of insertion and removal of receptors at synapses can modify receptor synaptic abundance and control synaptic strength. AMPA receptor C-terminal domain phosphorylation is thought to be a major regulator of receptor interaction with proteins that control trafficking. Synapses convert ionic fluxes associated with synapse activity into biochemical signals that control kinases and CTD phosphorylation. Phosphorylation of the GluR1 subunit on serine 845, which lies near the middle of the GluR1 CTD, by PKA has been correlated with the level of GluR1 at extrasynaptic sites in the plasma membrane. We will use molecular and electrophysiological approaches to investigate a new mechanism of serine 845 phosphorylation by the cyclic GMP regulated kinase cGKII. cGKII is under the control of the NMDA receptor through NMDAR activation of nNOS, leading to the production of nitric oxide, activation of soluble guanylate cyclase and production of cGMP, which induces cGKII. The Aims of this project are: Aim 1. To analyze the roles of cGKII myristoylation, dimerization and activation in GluR1 trafficking and physiology. We will analyze how the structure of cGKII contributes to GluR1 regulation. Aim 2. To distinguish the mechanism of S845 phosphorylation in increasing GluR1 levels on the plasma membrane. We will determine the place within the cell that cGKII phosphorylates GluR1 and exerts its effect on GuR1 trafficking. We will attempt to distinguish whether cGKII regulates GluR1 exocytosis or if GluR1 delivered constitutively is stabilized on the plasma membrane by S845 phosphorylation. We will isolate factors that respond to S845 phosphorylation and control GluR1 surface levels. Aim 3. To study the cGKII regulated trafficking of GluR4 and GluR2L. GluR4 and GluR2L are AMPAR subunits that are structurally related to GluR1 in ways that suggest that they may also be controlled by cGKII. We will analyze the role of cGKII in the trafficking of these subunits to determine if the NMDAR-nNOS-cGKII pathway is more generally employed. This project will provide new information about mechanisms of activity dependent control of synapse function and may help to define the role of nitric oxide in these mechanisms. PUBLIC HEALTH RELEVANCE: Learning and memory depend upon changes in the strength of connections between neurons in the brain. This project will study a new pathway that can allow neurons to respond to their own activity and change the strength of their connections. This work is relevant to mechanisms of memory formation and to the origin of learning disabilities. It can provide the basis for devising new drugs to help maintain memory or lessen learning disabilities.