Direct chemogenetic control of heterotrimeric G protein signaling

异源三聚体 G 蛋白信号传导的直接化学遗传学控制

• 批准号: 10590217
• 负责人: Mikel Garcia-Marcos
• 金额: $ 45.38万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10590217
• 关键词: Adenylate Cyclase; Adopted; Affinity; Agreement; Antiviral Agents; Binding; Biological Assay; Biological Process; Bioluminescence; Biosensor; Cell Line; Cell Surface Receptors; Cell physiology; Cells; Chemicals; Clinical; Communication; Communities; Cyclic AMP; Disease; Drug Targeting; Energy Transfer; Engineering; Etiology; Event; FDA approved; Family; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP Binding; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Genetic; Goals; Guanosine Triphosphate; Hepatitis C; Heterotrimeric G Protein Subunit; Heterotrimeric GTP-Binding Proteins; Hormones; Ion Channel; Left; Length; Ligands; Location; Mammalian Cell; Measures; Mediating; Methods; Mus; Names; Neurons; Neurosciences; Neurotransmitter Receptor; Neurotransmitters; Optics; Outcome; Output; Pathologic; Peptides; Performance; Pharmaceutical Preparations; Pharmacology; Phospholipase C; Photons; Physiological; Physiological Processes; Proteins; Receptor Activation; Regulation; Research; Research Personnel; Role; Second Messenger Systems; Signal Transduction; Signaling Protein; Specificity; Stimulus; Technology; Therapeutic; Transducers; base; design; experimental study; human disease; improved; nervous system disorder; neuropsychiatric disorder; neuroregulation; neurotransmission; new technology; novel; performance tests; protein activation; receptor; receptor-mediated signaling; response; subcellular targeting; tool

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. .

抽象的 意义：G 蛋白偶联受体 (GPCR) 启动细胞对许多不同刺激的反应，例如 神经递质、激素或光子。它们对于许多生理过程及其作用至关重要 失调经常导致人类疾病，这也与 > 30% 的 FDA- 已批准的药物以 GPCR 为靶点。 GPCR 是神经病学和神经精神病学的关键药理学靶点 基于其作为代谢型神经递质受体的功能的疾病，在 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 个家族（Gs、Gi/o、Gq/11、G12/13）。然而，其机制和后果 由于缺乏足够的实验数据，异三聚体 G 蛋白信号传导一直难以阐明。 在细胞环境中以高精度和特异性操纵其活动的工具。我们的目标是开发一个 新型化学遗传学工具，可直接激活异源三聚体 G 蛋白，而不干扰 GPCR 或其他 细胞过程。一般来说，化学遗传学是指一种将蛋白质改造为相互作用的方法 与以前未被识别的化合物。这里开发的工具将使研究人员能够 这一研究领域旨在操纵和剖析 G 蛋白激活的功能后果 前所未有的精度，从而揭示生理、病理、 或神经递质反应和其他生物过程的治疗调节。 背景：刺激后，GPCR 会促进异源三聚体 G 的 Gα 亚基上的 GTP 负载 蛋白质（Gαβγ）。反过来，Gα-GTP 与效应蛋白结合以传播信号传导。在这样的背景下 在神经传递中，Gs（例如 Gαs）或 Gq/11（例如 Gαq）家族的 Gα 蛋白主要是 由于它们分别增加 cAMP 或细胞内 Ca2+ 的能力而具有神经刺激作用。相比之下，Gα Gi/o 家族蛋白（例如 Gαi）通过抑制 cAMP 引起神经抑制。这些效果是 通过直接结合和调节控制第二信使水平的效应蛋白来介导—— 即 Gαs 和 Gαi 的腺苷酸环化酶，或 Gαq 的磷脂酶 C。 Gβγ 也有助于神经调节 通过离子通道的调节。我们设想并部分验证了化学遗传学方法 无需GPCR即可实现G蛋白的直接特异性激活。 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 由在哺乳动物中没有已知靶标或作用的化合物激活的蛋白质 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 目标 2，我们将通过使用下游信号传导来测试这些化学激活的 G 蛋白的性能 读数直接依赖于细胞系和神经元原代培养物中的同源 G 蛋白效应器。 。