Quantitative analysis of metabotropic glutamate receptor activation and modulation

项目摘要 G蛋白偶联受体是人类最大的膜受体家族，具有 成为最大的药物靶标家族，超过35%的药物在市场上发挥作用 通过GPCRs。尽管它们是可取的，但到目前为止，许多gpcr家庭仍然没有被下药，部分原因是缺乏 关于它们的激活和调节的机械性知识。代谢性谷氨酸受体(MGluRs) 是C类GPCRs的成员，是谷氨酸信号转导的关键调节器。由于它们的广泛存在 在组织中的表达及其中心作用它们是最有希望的神经学药物靶点之一 如脆性X综合征、癫痫、焦虑、精神分裂症以及一些癌症。最新进展 在过去的二十年里，蛋白质工程和功能、结构和计算方法 提供了对mGluRs的体系结构、信号和表达模式的见解。然而，一位将军 配体如何改变mGluR的形状以及这种构象变化如何在 膜和超过12 nm激活特定的信号通路是鲜为人知的。在这项研究中，我们将 开发一种新技术，使我们能够观察单个mGluR蛋白在体内发挥作用 生理状况，实时显示。这将使我们能够量化不同领域的运动 参与信号传递的受体。接下来，我们将使用这种方法来研究如何 合成的 调制器 的 MGluR 发信号 影响 蛋白 构象 和 动力学 至 胆固醇和 情感受体 发信号。这是一项多学科的建议，其中最先进的体外单分子FRET(SmFRET) 光谱学与活细胞成像、蛋白质工程、生物物理和生化相互补充。 方法：研究方法。一旦完成，拟议的研究将为合理设计提供关键的一步 有效的药物，不良反应较少。此外，这些研究将提供一个大致的路线图 用于哺乳动物膜蛋白的定量高分辨率结构-功能研究。

Project Summary G protein-coupled receptors (GPCRs) are the largest family of membrane receptors in human and have emerged as the largest family of drug targets with more than 35% of all drugs on the market functioning through GPCRs. Despite their desirability, to date, many GPCR families remain undrugged, partly due to lack of mechanistic knowledge about their activation and modulation. Metabotropic glutamate receptors (mGluRs) are members of class C GPCRs and are critical modulators of glutamate signaling. Due to their widespread expression in tissue and their central role they are among the most promising drug targets for the neurological disorders such as fragile X syndrome, epilepsy, anxiety, schizophrenia as well as some cancers. Advances in protein engineering and functional, structural and computational methods in the past twenty years have provided insights into the architecture, signaling and expression patterns of mGluRs. However, a general model of how ligands change the shape of mGluRs and how this conformational change is relayed across the membrane and over 12 nm to activate specific signaling pathways is poorly understood. In this research we will develop a novel technology that will allow us to watch a single mGluR protein while functioning in physiological conditions, in real time. This will allow us to quantify the motions of different domains of the receptor that are involved in signaling. Next, we will employ this approach to study how synthetic modulators of mGluR signaling affect protein conformation and dynamics to cholesterol and affect receptor signaling. This is a multi-disciplinary proposal where state-of-the-art in vitro single-molecule FRET (smFRET) spectroscopy is complemented by live-cell imaging, protein engineering and biophysical and biochemical methods. Once accomplished, the proposed research could provide a critical step towards rational design of efficient drugs with fewer undesirable side effects. Furthermore, these studies will provide a general roadmap for quantitative high-resolution structure-function studies of mammalian membrane proteins.