Elucidating transsynaptic regulation of metabotropic glutamate receptors

阐明代谢型谷氨酸受体的跨突触调节

基本信息

批准号：
10508656
负责人：
William Grant LUDLAM
金额：
$ 6.68万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-01-01 至 2025-12-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10508656
关键词：
Autoreceptors Binding Biological Assay Brain Cell Adhesion Molecules Cells Cognitive Complex Coupled Cryoelectron Microscopy Deuterium Dimerization Disease Environment Epilepsy Epitopes Exposure to Family Feedback Fibronectins Foundations G-Protein-Coupled Receptors Glutamates Homeostasis Homo Hormones Hydrogen Hyperactivity In Vitro Individual Knock-out Leucine-Rich Repeat Mass Spectrum Analysis Measures Membrane Metabotropic Glutamate Receptors Modeling Molecular Motor Movement Disorders Mus Mutation Neurons Neurotransmitters Parkinson Disease Pathology Physiological Physiology Positioning Attribute Post-Traumatic Stress Disorders Proteins Receptor Signaling Regulation Resolution Role Seizures Signal Transduction Stimulus Structural Models Structure Synapses Testing base crosslink dimer experimental study extracellular grasp insight postsynaptic presynaptic receptor receptor binding reconstitution therapy design

项目摘要

Project Summary / Abstract It has recently been discovered that G-protein coupled receptors (GPCR) can be regulated by extracellular interactions. The mechanisms behind this phenomenon remain poorly understood. One such interaction is the trans-synaptic connection between presynaptic group III metabotropic glutamate receptors (mGluR) and postsynaptic cell-adhesion molecules of the extracellular leucine-rich repeat and fibronectin type III domain-containing (ELFN) family. Group III mGluRs sense glutamate release and inhibit further release in a negative feedback mechanism. A great body of evidence points to the essential role of synaptic glutamate homeostasis in a range of cognitive and motor functions with dysregulation leading to movement disorders and epilepsy. Binding and stabilization of mGluRs by trans-synaptic interactions with ELFN proteins is critical for regulating glutamate homeostasis. Loss of ELFN proteins in mice tremendously augments glutamate release and results in seizures and hyperactivity. Understanding how these proteins interact in the synaptic environment will provide valuable insight into how neurons maintain control over glutamate levels in the synapse and has implications for physiology and disease. Importantly, both mGluRs and ELFN proteins have been shown to form homo- and hetero-dimers. This proposal aims to test the hypothesis that apart from positioning and stabilizing group III mGluRs at the synapses, ELFN proteins act to allosterically modulate mGluR activity in part by influencing the dimerization dynamics of mGluR subunits. To test this hypothesis, I will build a structural model of the ELFN-mGluR interaction using crosslinking coupled with mass spectrometry, hydrogen/deuterium exchange, and cryo-electron microscopy to understand what binding determinants govern the ELFN-mGluR binding interaction. Additionally, I will investigate the mechanisms of mGluR allosteric modulation by ELFN proteins using a variety of cell-based signaling assays. Together, these experiments will provide a clear foundation for understanding how the brain maintains glutamate homeostasis inside the synapse.

项目概要/摘要最近发现G蛋白偶联受体(GPCR)可以通过以下途径调节：细胞外相互作用。这种现象背后的机制仍然知之甚少。这样的一位相互作用是突触前 III 族代谢型谷氨酸受体之间的跨突触连接 (mGluR) 和细胞外富含亮氨酸重复序列和纤连蛋白类型的突触后细胞粘附分子包含 III 结构域 (ELFN) 的家族。 III 组 mGluR 感知谷氨酸释放并抑制进一步释放负反馈机制。大量证据表明突触谷氨酸的重要作用一系列认知和运动功能的稳态，失调导致运动障碍和癫痫。 mGluRs 通过与 ELFN 蛋白的跨突触相互作用的结合和稳定对于调节谷氨酸稳态。小鼠 ELFN 蛋白的缺失极大地增加了谷氨酸的释放并导致癫痫发作和多动症。了解这些蛋白质如何在突触环境中相互作用将为神经元如何维持对突触中谷氨酸水平的控制提供有价值的见解，并具有对生理学和疾病的影响。重要的是，mGluR 和 ELFN 蛋白都已被证明可以形成同源二聚体和异源二聚体。该提案旨在检验以下假设：除了定位和稳定之外突触处的 III 组 mGluR，ELFN 蛋白部分通过变构调节 mGluR 活性影响 mGluR 亚基的二聚化动力学。为了检验这个假设，我将建立一个结构模型使用交联结合质谱、氢/氘分析 ELFN-mGluR 相互作用交换和冷冻电子显微镜，以了解哪些结合决定因素控制 ELFN-mGluR 结合相互作用。此外，我将研究 ELFN 的 mGluR 变构调节机制使用各种基于细胞的信号转导测定来检测蛋白质。总之，这些实验将提供一个清晰的为理解大脑如何维持突触内谷氨酸稳态奠定了基础。