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，代谢型谷氨酸受体，mGlu5，调动细胞核Ca 2+独立的胞质Ca 2+调节。免疫细胞化学，超微结构和亚细胞分级技术显示，代谢型谷氨酸受体，mGlu5，定位于核膜在异源细胞以及中脑和皮层神经元。异源细胞类型中表达的核mGlu5受体可以结合激动剂，并且分离的细胞核对激动剂的反应是快速的、振荡的[Ca 2 +]升高，其被拮抗剂阻断。由于这些结果挑战了现有的范例，细胞内受体的作用，并在细胞内信号传导的重要分支，它们适用于R21机制的目的。本申请的目标是建立一个天然的生理系统，用它来进一步测试这些新概念。由于初步结果表明，mGluR5受体表达的核和质膜在纹状体文化，这些制剂将被用来检查的假设，核mGlu5受体可以调节核Ca 2+在神经元。具体而言，培养物将加载钙指示剂，其时空分布将在共聚焦显微镜中分析。在用特异性激动剂和拮抗剂处理后，将培养物固定、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。将以类似的方式分析原位分离的纹状体核。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关键作用，它们这样做的机制至关重要。直接核钙调节是一种新的信号转导策略，通过这种策略，细胞内受体如mGlu5可能在产生和塑造细胞内Ca 2+信号中发挥关键作用

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