Signaling By Nuclear G-Protein Coupled Receptors

描述（由申请人提供）：核Ca2+在许多细胞功能中起关键作用，尽管其调节模式尚不清楚。最近，我们已经表明，代谢性谷氨酸受体mGlu5，动员核Ca2+独立于细胞质Ca2+调节。免疫细胞化学、超微结构和亚细胞分离技术显示，代谢性谷氨酸受体mGlu5定位于异源细胞、中脑和皮质神经元的核膜上。在异源细胞中表达的核mGlu5受体可以结合激动剂，并且分离的细胞核对激动剂有快速的，振荡的[Ca2+]升高反应，被拮抗剂阻断。由于这些结果挑战了关于细胞内受体作用的现有范式，并且对细胞内信号传导具有重要影响，因此它们适用于R21机制的目的。当前应用程序的目标是建立一个原生的生理系统，以进一步测试这些新概念。由于初步结果表明，在纹状体培养中，mGluR5受体在核膜和质膜上都有表达，这些制备将用于检验核mGluR5受体可以调节神经元核Ca2+的假设。具体来说，培养将装载钙指示剂，其时空分布将在共聚焦显微镜下分析。在使用特异性激动剂和拮抗剂治疗后，培养物将被固定、染色并重新定位，以确定核振荡反应（如果有的话）是否与核mGlu5受体相关。纹状体核，就地分离，将以类似的方式进行分析。后续的实验将探讨是什么配体激活了核受体，以及它是如何激活的。鉴于mGlu5受体在突触可塑性、神经元发育和突触传递调节中起着关键作用，它们的作用机制至关重要。直接核钙调控是一种新的信号策略，通过这种策略，细胞内受体如mGlu5可能在细胞内Ca2+信号的产生和形成中起关键作用

DESCRIPTION (provided by applicant): Nuclear Ca2+ plays a critical role in many cellular functions although its mode (s) of regulation is unclear. Recently, we have shown that the metabotropic glutamate receptor, mGlu5, mobilizes nuclear Ca2+ independent of cytosolic Ca2+ regulation. Immunocytochemical, ultrastructural, and subcellular fractionation techniques revealed that the metabotropic glutamate receptor, mGlu5, was localized to nuclear membranes in heterologous cells as well as midbrain and cortical neurons. Nuclear mGlu5 receptors expressed in heterologous cell types could bind agonist and isolated nuclei responded to agonist with rapid, oscillatory [Ca2+] elevations that were blocked by antagonist. Because these results challenge existing paradigms as to the role of intracellular receptors and have important ramifications in intracellular signaling, they are applicable to the purpose of the R21 mechanism. The goal of the current application is to establish a native, physiological system with which to test these novel concepts further. Because preliminary results indicate that mGluR5 receptors are expressed on both nuclear and plasma membranes in striatal cultures, these preparations will be used to examine the hypothesis that nuclear mGlu5 receptors can regulate nuclear Ca2+ in neurons. Specifically, cultures will be loaded with a calcium indicator whose spatio-temporal distribution will be analyzed in the confocal microscope. Following treatment with specific agonists and antagonists, cultures will be fixed, stained, and field re-located to determine whether nuclear oscillatory responses, if any, are associated with nuclear mGlu5 receptors. Striatal nuclei, isolated in situ, will be analyzed in a similar fashion. Subsequent experiments will address what ligand is activating nuclear receptors and how it is doing so. Given that mGlu5 receptors play pivotal roles in synaptic plasticity, neuronal development and modulation of synaptic transmission, the mechanisms by which they do so are of critical importance. Direct nuclear calcium regulation represents a novel signaling strategy by which intracellular receptors such as mGlu5 may play a pivotal role in generating and shaping intracellular Ca2+ signals