Next generation G protein activity biosensors

意义：G蛋白偶联受体(GPCRs)是一个膜蛋白大家族， 启动细胞对一系列细胞外信号的反应，如中性递质、激素或 光子。因此，它们对许多生理过程及其频繁的失调都是至关重要的。 会导致人类疾病。这与美国食品和药物管理局批准的药物中30%的靶向是一致的 GPCRs。GPCRs的主要作用机制是通过激活异源三聚体G蛋白，因此 开发高保真和精确的G蛋白活性监测工具是阐明G蛋白活性模式的关键 许多神经递质、激素或药物的作用，以及发现新的治疗剂。我们的目标是 开发一种新型的基因编码光学传感器来监测异三聚体的激活 G蛋白直接、实时和在细胞内的内源性水平。如果成功，我们的项目将提供 高精度G蛋白活性生物传感器：(1)适用于监测内源G蛋白活性 与生理相关的实验系统，(2)易于转移到其他研究人员，以及(3)可扩展 以获得更高的吞吐量。因此，我们在这里建议开发的生物传感器将在该领域产生深远的影响 通过能够以前所未有的细节和准确性研究自然条件下的G蛋白活性。 背景：荧光或生物发光共振能量转移(FRET)的发展 或BRET)技术对GPCR领域的发展至关重要，因为它允许敏感和精确的 活细胞中G蛋白活性的测定。然而，基于RET的G蛋白测量技术 到目前为止开发的激活仍然存在阻碍进展的限制。其一是他们依赖于异类 G蛋白的过度表达，从而影响读数的保真度。此外，还要求 多个成分的同时过表达排除了它们在遗传基因系统中的使用 操纵并不容易。另一个可能的限制是他们测量Gα-Gβγ亚单位解离/ 重排，GPCRs激活G蛋白的一种近端但仍是间接的测量(由 核苷酸交换)。 AIMS简介：我们设想了基于Bret的生物传感器的设计，以监测内源性 通过检测4G蛋白中每一种G蛋白的Gα亚基的形成来检测G蛋白的活性 亚家族(Gi/o、Gs、Gq/11、G12/13)。我们的方法基于生物传感器的模块化设计，分两步进行 进程。在第一步(SA#1)中，我们将识别与活性Gα亚基特异结合的蛋白质模块 每个家庭，并验证他们可以报告活动时，纳入两个组成部分(捐赠者/接受者) 布雷特系统。在第二步(SA#2)中，生物传感器将被适应于一个单分子系统，该系统报告 在天然表达的G蛋白存在的情况下，分子内Bret的活性。

SIGNIFICANCE: G protein-coupled receptors (GPCRs) are a large family of membrane proteins that initiate cellular responses to a wide range of extracellular signals, like neutrotransmitters, hormones or photons. Consequently, they are critical for many physiological processes and their dysregulation frequently leads to human disease. This is in good agreement with the fact that >30% of FDA-approved drugs target GPCRs. The main mechanism of action of GPCRs is through the activation of heterotrimeric G proteins, so developing tools to monitor G protein activity with high fidelity and precision is crucial to elucidate the mode of action of many neurotransmitters, hormones or drugs, and to discover novel therapeutic agents. Our goal is to develop a new class of genetically-encoded optical sensors to monitor the activation of heterotrimeric G proteins directly, in real time and at the endogenous level in cells. If successful, our project will deliver high precision G protein activity biosensors that are (1) adaptable to monitor endogenous G protein activity in physiologically relevant experimental systems, (2) readily transferable to other investigators, and (3) scalable for higher throughput. Thus, the biosensors we propose to develop here will have a profound impact in the field by enabling the study of G protein activity under native conditions with unprecedented detail and accuracy. BACKGROUND: The development of fluorescence or bioluminescence resonance energy transfer (FRET or BRET) techniques has been crucial for the advance of the GPCR field by allowing the sensitive and precise measurement of G protein activity in living cells. However, RET-based techniques to measure G protein activation developed to date still have limitations that dampen progress. One is that they rely on the ectopic overexpression of G proteins, thereby compromising the fidelity of readouts. Also, the requirement for simultaneous overexpression of multiple components precludes their use in systems in which genetic manipulation is not easy. Another possible limitation is that they measure Gα-Gβγ subunit dissociation/ rearrangement, a proximal but still indirect measure of G protein activation by GPCRs (which is defined by nucleotide exchange). SYNOPSIS OF AIMS: We have envisioned the design of BRET-based biosensors to monitor endogenous G protein activity by detecting the formation of GTP-bound Gα subunits for each one of the 4 G protein subfamilies (Gi/o, Gs, Gq/11, G12/13). Our approach is based on the modular design of biosensors in a two-step process. In the first step (SA#1), we will identify protein modules that specifically bind to active Gα subunits of each family and validate that they can report activity when incorporated into a two-component (donor/acceptor) BRET system. In the second step (SA#2), the biosensors will be adapted to a unimolecular system that reports activity by intramolecular BRET in the presence of natively expressed G proteins.