Single-molecule and ensemble imaging of GPCR-G protein complexes in live cells

描述(由申请人提供)：G蛋白偶联受体(GPCRs)是最大的细胞表面受体家族，是所有处方和滥用药物的相当大一部分的靶标。GPCRs主要通过激活细胞内异源三聚体GTP结合蛋白(G蛋白)来改变细胞生理。G蛋白激活的步骤包括配体结合、受体激活，最终在配体、活性受体和非活性G蛋白(LR*GGDP)之间形成“偶联”的复合体，直接导致G蛋白激活。长期以来，人们一直假设，在“预偶联”或“预组装”的RGGDP复合体中，非活性的GPCRs和G蛋白在受体激活之前相互联系。然而，这些复合体在细胞中的存在和相关性一直很难证明，这主要是因为还没有研究膜蛋白之间瞬时相互作用的方法。最近，我们成功地在活细胞中检测到GPCRs和GQ异源三聚体之间的预组装复合体，并证明了它们的生理意义。然而，这些研究留下了一些重要的问题没有得到回答，部分原因是它们完全依赖于整体测量。一个关键的问题是非活性状态预组装受体-G蛋白(RGGDP)复合体的寿命。系综实验表明，预组装的RGGDP络合物的寿命比活性状态的耦合络合物长，但这两种寿命都不能用现有的方法确定。长寿命的预先组装的RGGDP复合体将允许受体“自支架”G蛋白，即保持高局部浓度的异源三聚体准备激活。在这里，我们建议使用活细胞中的定量系综和单分子成像来研究RGGDP复合体。整体成像将使我们能够确定预组装是否作为几种不同GPCR和G蛋白异三聚体的自我支架机制。单分子成像将使我们能够直接观察非活性状态预组装的RGGDP络合物的形成和解离。因此，我们将能够定量评估对G蛋白信号重要的大分子复合体的寿命。这些实验对直接领域的影响将是确定预组装是G蛋白激活途径上的重要一步，还是罕见的副反应。关于一般的大分子相互作用，该项目的影响将包括改进系综和单分子成像技术，以检测活细胞中的膜蛋白相互作用，包括表达系统、染料、标记、固定化、成像和分析策略。

DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors, and are the targets of a substantial fraction of all prescribed ad abused drugs. GPCRs change cellular physiology primarily by activating intracellular heterotrimeric GTP-binding proteins (G proteins). The steps involved in G protein activation include ligand binding, receptor activation, and ultimately the formation of a "coupled" complex between a ligand, an active receptor and an inactive G protein (LR*GGDP), leading directly to G protein activation. It has long been hypothesized that inactive GPCRs and G proteins associate with each other prior to receptor activation in "precoupled" or "preassembled" RGGDP complexes. However, the existence and relevance of these complexes in cells have been difficult to document, largely because methods to study transient interactions between membrane proteins have not been available. We recently succeeded in detecting preassembled complexes between GPCRs and Gq heterotrimers in living cells, and in demonstrating their physiological significance. However, these studies left a number of important questions unanswered, in part because they relied exclusively on ensemble measurements. One critical question is the lifetime of inactive-state preassembled receptor-G protein (RGGDP) complexes. Ensemble experiments suggest that the lifetime of a preassembled RGGDP complex is long compared to the active-state, coupled complex, but neither of these lifetimes can be determined using existing methods. Long-lived preassembled RGGDP complexes would allow receptors to "self-scaffold" G proteins, i.e. maintain a high local concentration of heterotrimers ready for activation. Here we propose to study RGGDP complexes using quantitative ensemble and single-molecule imaging in living cells. Ensemble imaging will allow us to determine if preassembly serves as a self-scaffolding mechanism for several different GPCRs and G protein heterotrimers. Single-molecule imaging will allow us to directly observe the formation and dissociation of inactive-state preassembled RGGDP complexes. We will thus be able to quantitatively assess the lifetimes of the macromolecular complexes important for G protein signaling. The impact of these experiments on the immediate field will be to determine if preassembly is a significant step along the pathway to G protein activation, or alternatively if itis a rare side-reaction. With respect to macromolecular interactions in general, the impact of this project will include refinement of ensemble and single-molecule imaging technology to detect membrane protein interactions in living cells, including expression systems, dyes, labeling, immobilization, imaging and analysis strategies.