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）启动了对许多不同刺激的细胞反应，例如 神经递质，激素或照片。它们对于许多物理过程及其 失调经常导致人类疾病，这也与以下事实一致。 批准的药物靶向GPCR。 GPCR是神经和神经精神病学的关键药物靶标 疾病基于其作为代谢性神经递质接收者的功能，在 神经调节。 GPCR作用的主要机理是通过激活异三聚体G蛋白， 在4个家庭（GS，GI/O，GQ/11，G12/13）中广泛分配。但是，机制和后果 由于缺乏足够的实验 在细胞环境中以高精度和特异性来操纵其活动的工具。我们的目标是开发 新的化学生成工具直接激活异三聚体G蛋白而不会扰动GPCR或其他 细胞过程。通常，化学遗传学是指蛋白质相互作用的方法 具有先前未识别的化合物。这里要开发的工具将允许调查人员进入 通过操纵和剖析G蛋白激活的功能后果的研究领域 前所未有的精度，从而揭示了生理，病理学，病理学的基本机制 或神经递质反应和其他生物学过程的热调节。 背景：刺激后，GPCR促进了杂点Gα亚基上的GTP载荷 蛋白质（Gαβγ）。反过来，Gα-GTP与效应蛋白结合以传播信号传导。在 神经传递，GS的Gα蛋白（例如GαS）或GQ/11（例如GαQ）家族是主要的 神经刺激分别增加了cAMP或细胞内Ca2+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。这些效果是 通过直接结合到控制第二信使水平的效应蛋白的介导和调节 即，GαS和GαQ的GαS的腺苷酸周期或GαQ的磷脂酶C。 Gβγ也有助于神经调节 通过离子通道的调节。我们已经设想并部分验证了一种化学遗传方法 无需GPCR即可实现G蛋白的直接和特异性激活。 目标的提要：在AIM 1中，我们将确定工程GαI，GαS，GαQ或Gβγ所需的组件 被哺乳动物含有已知靶标或作用的化学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来评估。在 AIM 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 读数直接取决于细胞系和神经元原发性培养物中的同源G蛋白效应。 。