Direct chemogenetic control of heterotrimeric G protein signaling

摘要 意义：G蛋白偶联受体(GPCRs)启动细胞对许多不同刺激的反应，如 神经递质、荷尔蒙或光子。它们对许多生理过程和它们的 监管失调经常会导致人类疾病，这也与这样一个事实相一致：美国食品和药物管理局30%- 批准的药物针对GPCRs。GPCRs是神经病学和神经精神病学的关键药理靶点 以代谢性神经递质受体功能为基础的疾病在 神经调节。GPCRs的主要作用机制是通过激活异源三聚体G蛋白， 它们大致分为4个科(Gs、Gi/o、Gq/11、G12/13)。然而，这一机制和后果 由于缺乏足够的实验研究，异三聚体G蛋白信号转导机制一直难以阐明 在细胞环境中以高精确度和特异度操纵其活动的工具。我们的目标是开发一种 一种新的化学发生工具，可直接激活异源三聚体G蛋白，而不干扰GPCRs或其他 细胞过程。一般说来，化学遗传学指的是一种使蛋白质相互作用的方法。 以前未被识别的化合物。将在这里开发的工具将允许调查人员进入 这一领域的研究旨在操纵和剖析G蛋白激活的功能后果 前所未有的精确度，从而揭示了生理、病理、 或神经递质反应和其他生物过程的治疗调节。 背景：在刺激下，GPCRs促进异源三聚体G的Gα亚单位上的GTP负荷 蛋白质(Gαβγ)。反过来，G-α-GTP结合到效应蛋白上来传播信号。在…的背景下 神经传递，G家族的Gα蛋白(如GαS)或GQ/11(如GαQ)家族主要是 它们分别通过增加cAMP或细胞内钙离子的能力发挥神经刺激作用。相比之下，Gα Gi/o家族的蛋白(如GαI)通过抑制cAMP而引起神经抑制。这些影响是 通过直接结合和调节控制第二信使水平的效应器蛋白来调节- 也就是说，GαS和GαI的腺酰环化酶，或GαQ的磷脂酶C。Gβγ也参与神经调节 通过对离子通道的调节。我们已经设想并部分验证了一种化学发生方法 在不需要GPCRs的情况下实现G蛋白的直接和特异性激活。 AIMS简介：在AIMS 1中，我们将确定设计GαI、GαS、GαQ或Gβγ所需的组件 被化合物激活的蛋白质，这些化合物在哺乳动物中没有已知的靶点或作用。 细胞。这些构建物将通过使用直接检测活性G蛋白的光学生物传感器进行评估。在……里面 目的2，我们将通过使用下游信号来测试这些化学激活的G蛋白的性能 在细胞系和神经元原代培养中，读数直接依赖于同源G蛋白效应器。 。

ABSTRACT SIGNIFICANCE: G protein-coupled receptors (GPCRs) initiate cellular responses to many different stimuli, like neurotransmitters, hormones or photons. They are critical for many physiological processes and their dysregulation frequently leads to human disease, which is also in agreement with the fact that >30% of FDA- approved drugs target GPCRs. GPCRs are key pharmacological targets in neurological and neuropsychiatric diseases based on their function as metabotropic neurotransmitter receptors with a prominent role in neuromodulation. The main mechanism of action of GPCRs is through activation of heterotrimeric G proteins, which are broadly divided in 4 families (Gs, Gi/o, Gq/11, G12/13). However, the mechanisms and consequences of heterotrimeric G protein signaling have been difficult to elucidate because of the lack of adequate experimental tools to manipulate their activity with high precision and specificity in a cellular context. Our goal is to develop a new class of chemogenetic tool to directly activate heterotrimeric G proteins without perturbing GPCRs or other cellular processes. Chemogenetics, in general, refers to a method by which a protein is engineered to interact with previously unrecognized chemical compounds. The tools to be developed here will allow investigators in this field of research to manipulate and dissect the functional consequences of G protein activation with unprecedented precision, thereby revealing fundamental mechanisms that underlie physiological, pathological, or therapeutic modulation of neurotransmitter responses and other biological processes. BACKGROUND: Upon stimulation, GPCRs promote GTP loading on the Gα-subunit of heterotrimeric G proteins (Gαβγ). In turn, Gα-GTP binds to effector proteins to propagate signaling. In the context of neurotransmission, Gα proteins of the Gs (e.g., Gαs) or the Gq/11 (e.g., Gαq) family are primarily neurostimulatory by virtue of their ability to increase cAMP or intracellular Ca2+, respectively. In contrast, Gα proteins of the Gi/o family (e.g., Gαi) cause neuroinhibition via suppression of cAMP. These effects are mediated through direct binding to and modulation of effector proteins that control second messenger levels— i.e., adenylyl cyclases for Gαs and Gαi, or phospholipases C for Gαq. Gβγ also contributes to neuromodulation through the regulation of ion channels. We have envisioned and partially validated a chemogenetic approach to achieve the direct and specific activation of G proteins without the need of GPCRs. SYNOPSIS OF AIMS: In Aim 1, we will identify the components required to engineer Gαi, Gαs, Gαq, or Gβγ proteins that are activated by chemical compounds that do not have known targets or effects in mammalian cells. These constructs will be evaluated by using optical biosensors that directly detect active G proteins. In Aim 2, we will test the performance of these chemically-activated G proteins by using downstream signaling readouts directly dependent on cognate G protein effectors in cell lines and in neuronal primary cultures. .