Signaling By Nuclear G-Protein Coupled Receptors

描述(申请人提供)：核钙离子在许多细胞功能中起着关键作用，尽管其调节方式(S)尚不清楚。最近，我们发现代谢性谷氨酸受体，mGlu5，不依赖于细胞内钙离子的调节来动员细胞核内的钙离子。免疫细胞化学、超微结构和亚细胞分离技术表明，代谢性谷氨酸受体mGlu5定位于异种细胞以及中脑和皮质神经元的核膜上。异种细胞中表达的mGlu5受体可以结合激动剂，分离的细胞核对激动剂的反应是快速、振荡的[Ca~(2+)]升高，但被拮抗剂阻断。由于这些结果挑战了关于细胞内受体作用的现有范式，并在细胞内信号转导中具有重要的分支，因此它们适用于R21机制的目的。目前应用的目标是建立一个本地的生理系统，用来进一步测试这些新概念。由于初步结果表明mGluR5受体在纹状体培养的核膜和质膜上都有表达，这些准备工作将被用来检验核mGlu5受体可以调节神经元核钙离子的假设。具体地说，培养物中将装载钙指示剂，其时空分布将在共聚焦显微镜中进行分析。在用特定的激动剂和拮抗剂处理后，培养物将被固定、染色和现场重新定位，以确定核振荡反应是否与核mGlu5受体相关。原位分离的纹状体核将以类似的方式进行分析。随后的实验将解决什么配体激活核受体以及它是如何做到这一点的。鉴于mGlu5受体在突触可塑性、神经元发育和突触传递的调节中起着关键作用，它们的作用机制至关重要。直接核内钙调节代表了一种新的信号策略，通过这种策略，细胞内受体如mGlu5可能在产生和塑造细胞内钙信号方面发挥关键作用

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