Agonist - G protein-coupled receptor interactions, recycling and cell-surface targeting in neuroendocrine systems

激动剂 - 神经内分泌系统中 G 蛋白偶联受体相互作用、回收和细胞表面靶向

• 批准号: RGPIN-2016-06096
• 负责人: Stroh, Thomas
• 金额: $ 2.04万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=616097
• 关键词: Agonist; protein; coupled; receptor; interactions

My research program is geared towards the elucidation of the mechanisms regulating cell-surface targeting and sub-cellular localization of G protein-coupled receptors (GPCRs) in neuroendocrine cells and neurons. Both these processes are key to the normal functioning of cells, in particular of nerve cells which due to their complexity face particular challenges in maintaining correct intracellular targeting and positioning of their key component proteins. To this end we use classical and cutting-edge cell biological techniques and collaborate with laboratories to create genetically modified variants of cells and laboratory mice by the new technique of genome editing using CRISPR/Cas9 technology. Moreover, the recent advent of new microscopic techniques surmounting the resolution limit of light microscopy based on the clever exploitation of the photo-chemical properties of many fluorophores, referred to as super-resolution microscopy (SRM), promises the direct visualization of protein interactions regulating these processes. Much like transmission electron microscopy, the new SRM techniques have specific requirements for sample preparation and data analysis requiring substantial research to establish suitable experimental protocols and intense training of HQP to perform the procedures. Thanks to the access to a SRM microscope that is based on the point-localization, bi-plane principle of SRM provided by the MNI Microscopy Core Facility, we recently visualized for instance the sub-cellular localization of sst2A receptors at clusters of gephyrin, a hallmark structural component of the post-synaptic density at inhibitory synapses in the central nervous system at 50 nm resolution. This is at least 5 times better than the best standard light microscope can do and approaches the size of individual protein molecules. Therefore, the present application builds on my expertise in super-resolution microscopy and the cell biology of GPCRs to further my research and training program by a) integrating the new SRM techniques with established protocols in cell biology, confocal and electron microscopy and b) using all of these approaches to elucidate the open questions around the regulation of intracellular trafficking and targeting of GPCRs in neuroendocrine and neuronal cells.

我的研究计划是面向调节神经内分泌细胞和神经元中G蛋白偶联受体（GPCRs）的细胞表面靶向和亚细胞定位的机制的阐明。这两个过程都是细胞，特别是神经细胞正常功能的关键，由于其复杂性，神经细胞在维持其关键组分蛋白质的正确细胞内靶向和定位方面面临特别的挑战。为此，我们使用经典和尖端的细胞生物学技术，并与实验室合作，通过使用CRISPR/Cas9技术的基因组编辑新技术来创建细胞和实验室小鼠的遗传修饰变体。此外，最近出现的新的显微技术，超越了光学显微镜的分辨率限制的基础上巧妙利用的光化学性质的许多荧光团，被称为超分辨率显微镜（SRM），承诺直接可视化的蛋白质相互作用调节这些过程。很像透射电子显微镜，新的SRM技术对样品制备和数据分析有特定的要求，需要大量的研究来建立合适的实验方案，并对HQP进行严格的培训来执行这些程序。由于访问SRM显微镜，这是基于点定位，双平面原则的SRM提供的MNI显微镜核心设施，我们最近可视化，例如，sst 2A受体的亚细胞定位在集群的gephyrin，一个标志性的结构组成部分的突触后密度在抑制性突触在中枢神经系统在50 nm的分辨率。这比最好的标准光学显微镜至少好5倍，接近单个蛋白质分子的大小。因此，本申请建立在我在超分辨率显微术和GPCR的细胞生物学方面的专业知识的基础上，以通过a）将新的SRM技术与细胞生物学、共聚焦和电子显微术中已建立的方案相结合，以及B）使用所有这些方法来阐明围绕神经内分泌和神经元细胞中GPCR的细胞内运输和靶向的调节的开放性问题，来进一步推进我的研究和培训计划。