Direct chemogenetic control of heterotrimeric G protein signaling

摘要 意义：G蛋白偶联受体（GPCR）启动细胞对许多不同刺激的反应，如 神经递质激素或光子它们对许多生理过程及其 失调经常导致人类疾病，这也与>30%的FDA- 批准的药物靶向GPCR。GPCR是神经和神经精神疾病的关键药理学靶点 基于它们作为代谢性神经递质受体的功能的疾病， 神经调节GPCR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， 异源三聚体G蛋白信号传导由于缺乏足够的实验研究而难以阐明。 在细胞环境中以高精度和特异性操纵其活性的工具。我们的目标是发展一个 直接激活异源三聚体G蛋白而不干扰GPCR或其他化学遗传工具的新类别 细胞过程一般来说，化学遗传学是指一种蛋白质被改造成与 与之前未被发现的化合物混合这里开发的工具将使调查人员能够 这一研究领域操纵和剖析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它生物过程的治疗性调节。 背景：在刺激后，GPCR促进GTP负载在异源三聚体G Gαβγ蛋白。反过来，Gα-GTP与效应蛋白结合以传播信号。背景下 神经传递，Gs的Gα蛋白（例如，Gαs）或Gq/11（例如，Gαq）家族主要是 神经刺激性的，由于它们分别增加cAMP或细胞内Ca 2+的能力。相反，Gα Gi/o家族的蛋白质（例如，Gαi）通过抑制cAMP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蛋白介导， 也就是说，腺苷酸环化酶代表Gαs和Gαi，磷脂酶C代表Gαq。Gβγ也有助于神经调节 通过调节离子通道。我们已经设想并部分验证了一种化学遗传学方法 实现G蛋白的直接和特异性激活，而不需要GPCR。 目标概要：在目标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。 细胞这些构建体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 目的2，我们将测试这些化学活化的G蛋白的性能，通过使用下游信号 读数直接依赖于细胞系和神经元原代培养物中的同源G蛋白效应物。 .

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. .