Probing Supramolecular Structure, Stoichiometry, and Trafficking in Live Cells of Oligomers of G-Protein Coupled receptors

被称为G蛋白偶联受体（GPCR）的大类膜受体蛋白的成员识别并响应各种各样的信号，这些信号可能以小配体分子（如神经递质和激素）以及物理刺激（如光和机械应力）的形式到达。GPCR在体内形成寡聚体复合物的相对较新的观察结果导致了许多与寡聚体的结构和功能作用有关的有趣问题，包括它们的大小、在细胞内的分布以及对配体结合的反应。在该项目中，主要研究者将开发基于先进激光扫描显微镜的新方法，用于在单个活细胞中测量光谱分辨的Foerster共振能量转移（FRET）能量从激发的荧光分子到附近的非激发分子的非辐射转移。因此，建立的实验程序将用于探测非侵入性的时间变化的荧光标记的GPCR的寡聚复合物的结构和细胞定位的存在和不存在的天然和人工配体。作为拟议研究的一部分开发的方法将使PI有助于理解GPCR介导的信号传导的生物学及其在疾病或其他医学疾病中的功能障碍。因此，这项研究的结果不仅有望大大推进研究，而且还将为开发更有效的药物提供基础。该研究将由威斯康星大学密尔沃基分校的物理系和生物科学系提供，它将为研究生和本科生创造一个良好的环境，通过使用技术和方法来实践跨学科的科学方法。PI实验室的一些实验装置将用于他的本科和研究生课程的演示。PI还将与高中教师合作，通过在PI实验室进行的小型研究项目以及教师带回课堂的研究项目来激发高中学生对科学的兴趣。该项目由物理系生命系统物理学项目和MCB生物分子动力学，结构和功能共同支持。

Members of the large class of membrane receptor proteins called G-protein-coupled receptors (GPCRs) recognize and respond to a wide variety of signals that may arrive in the form of small ligand molecules such as neurotransmitters and hormones, and physical stimuli such as light and mechanical stress. The relatively recent observation that GPCRs form oligomeric complexes in vivo has led to many interesting questions related to the structure and functional role of the oligomers, including their size, distribution within the cell, and response to ligand binding. In this project, the Principal Investigator will develop new methodology based on advanced laser-scanning microscopy for measurements, in single live cells, of spectrally resolved Foerster Resonance Energy Transfer (FRET) a non-radiative transfer of energy from an excited fluorescent molecule to a non-excited one that resides nearby. The experimental procedures thus established will be used for probing non-invasively the temporal changes in the structure and cellular localization of oligomeric complexes of fluorescently-tagged GPCRs in the presence and absence of natural and artificial ligands. The methods developed as part of the proposed research will allow the PI to contribute to the understanding of the biology of GPCR-mediated signaling and its dysfunction in diseases or other medical disorders. The outcomes of this research are therefore expected to not only dramatically advance research but also to provide foundations for developing more efficient pharmaceutical agents. The research will be housed by the Physics Department and the Department of Biological Sciences at the University of Wisconsin-Milwaukee, and it will create an excellent environment for graduate and undergraduate students to practice an interdisciplinary approach to science, by using techniques and methods from a variety of sub-disciplines seamlessly integrated to serve a well-defined goal. Some of the experimental set-ups in the PIs laboratory will be used for demonstrations in his undergraduate- and graduate-level classes. The PI will also collaborate with a high-school teacher to stimulate the interest in science of high school students through small research projects conducted in the PI's laboratory as well as through the research projects brought by the teacher back to her class.This project is being supported jointly by the Physics of Living Systems Program in the Physics Division and by Biomolecular Dynamics, Structure and Function in MCB.