UvA-DARE ( Digital Academic Repository ) Real-time visualization of heterotrimeric G protein Gq activation in living cells
UvA-DARE ( Digital Academic Repository ) Real-time visualization of heterotrimeric G protein Gq activation in living cells
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发表时间：
2017
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通讯作者：
M. Adjobo-Hermans;J. Goedhart;L. V. Weeren;S. Nijmeijer;E. Manders;S. Offermanns;T. Gadella
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作者：
M. Adjobo-Hermans;J. Goedhart;L. V. Weeren;S. Nijmeijer;E. Manders;S. Offermanns;T. Gadella
背景：Gq是一种异源三聚体G蛋白，在许多生理过程中发挥重要作用。为了描述通过该蛋白质的信号传导的分子机制和动力学，其活化应该在单个活细胞中可测量。最近，荧光共振能量转移（FRET）传感器已开发用于这一目的。结果如下：在本文中，我们描述了一种改进的FRET为基础的Gq活性传感器，由黄色荧光蛋白（YFP）标记的Gg 2亚基和Gaq亚基与插入单体绿松石（mTurquoise），目前可用的最好的青色荧光蛋白变体的发展。该传感器使我们能够首次确定Gq激活的kon（2/s）。此外，我们发现，鸟嘌呤核苷酸交换因子p63 RhoGEF有一个深刻的影响的Gq蛋白的数量，成为激活后刺激内源性组胺H1受体。该传感器还用于测量组胺H1受体（H1 R）的配体非依赖性激活时，添加低渗刺激。结论：我们的观察结果表明，截断的mTurquoise作为供体和YFP标记的Gg 2作为受体的FRET为基础的Gq活动传感器的应用程序大大提高了它们的动态范围。这种优化能够实时单细胞定量Gq信号动力学，辅助蛋白的影响，并允许未来的药物筛选应用凭借其灵敏度。背景异源三聚体G蛋白由Ga亚基和Gbg二聚体组成，可被G蛋白偶联受体（GPCR）激活。在结合激动剂后，受体改变其构象，并作为鸟嘌呤核苷酸交换因子（GEF）。通过诱导Ga亚基中鸟嘌呤二磷酸（GDP）与鸟嘌呤三磷酸（GTP）的交换，G蛋白变得活跃[1]。Gbg通过两个界面与Ga相互作用：开关区域和Ga亚基的N末端形成的区域。受体激活后GTP的结合破坏了开关界面，这被认为触发了Gbg与Ga的解离[2]。Ga亚基家族由四类组成;亚基Gaq、Ga 11、Ga 14和Ga 16属于Gq类。Gq类的主要靶标是磷脂酶（PL）Cb [3]。最近，RhoGEF蛋白如白血病相关Rho-鸟嘌呤核苷酸交换因子（LARG）和p63 RhoGEF已被证明与Gaq直接相互作用并受Gaq调节，表明它们可以将Gaq偶联受体与小G蛋白RhoA的激活联系起来[4-6]。研究Gaq很重要，因为这种蛋白质与心脏机械应力后心肌肥大的发展有关[7，8]。因为这是心力衰竭的触发因素之一，心力衰竭是西方世界死亡的主要原因，抑制Gaq的药物需求量很大[9]。属于Gq类的蛋白质还参与调节突触传递[10，11]、细胞生长、血小板聚集[12]、葡萄糖分泌、肌动蛋白细胞骨架重排、造血细胞分化、白细胞活化和平滑肌收缩，强调了它们在人体生理学中的重要性[13]。* 通讯地址：Th. W. J. uva.nl Swammerdam生命科学研究所，分子细胞学部，货车列文虎克高级显微镜中心，阿姆斯特丹大学，科学园904，1098 XH，阿姆斯特丹，荷兰作者信息的完整列表可在文章Adjobo-Hermans et al. BMC Biology 2011，9：32 http://www.biomedcentral.com/1741-7007/9/32 © 2011 Adjobo-Hermans et al;这是一篇开放获取的文章，根据知识共享署名许可证（http：//creativecommons.org/licenses/by/2.0）的条款分发，该许可证允许在任何媒体上无限制地使用，分发和复制，前提是原始作品被正确引用。最近，已经开发了几种荧光共振能量转移（FRET）传感器来监测GPCR激活时活细胞中特定异源三聚体G蛋白的激活状态[14-21]。在本文中，我们报告了一个高灵敏度的传感器的基础上功能mTurquoise标记的Gaq和黄色荧光蛋白（YFP）标记的Gg 2，它允许监测的位置和G蛋白激活状态的Gq在活细胞和动力学的过程。此外，我们描述了内源性和过度表达的受体的配体依赖性和配体独立性刺激的影响，同时监测效应器对传感器的行为的影响。我们选择了双发射率FRET测量补充FRET荧光寿命成像显微镜（FLIM）测量，分别监测Gq激活和FRET效率的动力学。
Background: Gq is a heterotrimeric G protein that plays an important role in numerous physiological processes. To delineate the molecular mechanisms and kinetics of signalling through this protein, its activation should be measurable in single living cells. Recently, fluorescence resonance energy transfer (FRET) sensors have been developed for this purpose. Results: In this paper, we describe the development of an improved FRET-based Gq activity sensor that consists of a yellow fluorescent protein (YFP)-tagged Gg2 subunit and a Gaq subunit with an inserted monomeric Turquoise (mTurquoise), the best cyan fluorescent protein variant currently available. This sensor enabled us to determine, for the first time, the kon (2/s) of Gq activation. In addition, we found that the guanine nucleotide exchange factor p63RhoGEF has a profound effect on the number of Gq proteins that become active upon stimulation of endogenous histamine H1 receptors. The sensor was also used to measure ligand-independent activation of the histamine H1 receptor (H1R) upon addition of a hypotonic stimulus. Conclusions: Our observations reveal that the application of a truncated mTurquoise as donor and a YFP-tagged Gg2 as acceptor in FRET-based Gq activity sensors substantially improves their dynamic range. This optimization enables the real-time single cell quantification of Gq signalling dynamics, the influence of accessory proteins and allows future drug screening applications by virtue of its sensitivity. Background Heterotrimeric G proteins are composed of Ga subunits and Gbg dimers, and can be activated by G-proteincoupled receptors (GPCRs). Upon binding of an agonist, the receptor changes its conformation, and acts as a guanine nucleotide exchange factor (GEF). By inducing the exchange of guanine diphosphate (GDP) for guanine triphosphate (GTP) in the Ga subunit, the G protein becomes active [1]. Gbg interacts with Ga through two interfaces: the switch region and the region formed by the N terminus of the Ga subunit. Binding of GTP upon receptor activation disrupts the switch interface, which is thought to trigger the dissociation of Gbg from Ga [2]. The family of Ga subunits consists of four classes; the subunits Gaq, Ga11, Ga14 and Ga16 belong to the Gq class. The principal target of the Gq class is phospholipase (PL)Cb [3]. Recently, RhoGEF proteins such as leukemia-associated Rho-guanine nucleotide exchange factor (LARG) and p63RhoGEF have been shown to directly interact with and to be regulated by Gaq, suggesting that they can link Gaqcoupled receptors to the activation of the small G protein RhoA [4-6]. Studying Gaq is important, because this protein is implicated in the development of myocardial hypertrophy after mechanical stress of the heart [7,8]. Because this is one of the triggers of cardiac failure, a leading cause of death in the western world, drugs to inhibit Gaq are much in demand [9]. Proteins belonging to the Gq class are also involved in the modulation of synaptic transmission [10,11], cell growth, platelet aggregation [12], glucose secretion, actin cytoskeletal rearrangements, hematopoietic cell differentiation, leukocyte activation and contraction of smooth muscle, emphasizing their importance in human physiology [13]. * Correspondence: Th.W.J.Gadella@uva.nl Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands Full list of author information is available at the end of the article Adjobo-Hermans et al. BMC Biology 2011, 9:32 http://www.biomedcentral.com/1741-7007/9/32 © 2011 Adjobo-Hermans et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Recently, several fluorescence resonance energy transfer (FRET) sensors have been developed to monitor the activation state of specific heterotrimeric G proteins in living cells upon GPCR activation [14-21]. In this paper, we report on the development of a highly sensitive sensor based on functional mTurquoise-tagged Gaq and yellow fluorescent protein (YFP)-tagged Gg2, which allows for monitoring of the location and G protein activation state of Gq in living cells and of the kinetics of the process. In addition, we describe the effects of ligand-dependent and ligand-independent stimulation of endogenous and overexpressed receptors, while concurrently monitoring the influence of effectors on the behaviour of the sensor. We opted for dual emission ratiometric FRET measurements supplemented with FRET-fluorescence lifetime imaging microscopy (FLIM) measurements to monitor the kinetics of Gq activation and FRET efficiencies, respectively.