Alternative mechanisms of signaling via trimeric G proteins

通过三聚体 G 蛋白传递信号的替代机制

• 批准号: 10374905
• 负责人: Mikel Garcia-Marcos
• 金额: $ 47.87万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10374905
• 关键词: Achievement; Adenylate Cyclase; Affect; Area; Binding; Binding Proteins; Biological Assay; Biosensor; Brain; Brain region; Cell physiology; Cells; Chronic inflammatory pain; Coin; Communication; Complex; Coupled; Cyclic AMP; Cytoplasmic Protein; Data; Disease; Ensure; Epilepsy; Excitatory Synapse; Family; G Protein-Coupled Receptor Signaling; G-Protein Signaling Pathway; G-Protein-Coupled Receptors; GTP-Binding Protein Regulators; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; GTPase-Activating Proteins; Genetic; Goals; Grant; Guanine Nucleotide Dissociation Inhibitors; Guanosine Triphosphate; Heterotrimeric GTP-Binding Proteins; Human; Hydrolysis; Hyperalgesia; Impact Seizures; Impairment; In Vitro; Inhibitory Synapse; Ion Channel; Knockout Mice; Laboratories; Link; Maps; Mass Spectrum Analysis; Mechanics; Mediating; Mediator of activation protein; Molecular; Neurons; Neuropathy; Neurotransmitters; Nucleotides; Pain; Pattern; Peripheral Nervous System; Pharmacology; Physiological; Physiology; Population; Post-Translational Protein Processing; Predisposition; Process; Proteins; Public Health; RGS Proteins; Regulation; Reporting; Role; Seizures; Signal Transduction; Signaling Protein; Site; Slice; Stimulus; Synapses; Testing; Therapeutic; Wild Type Mouse; base; chronic pain; human disease; insight; member; mouse model; nervous system disorder; neuroregulation; neurotransmission; novel; prototype; receptor; response; spinal nerve posterior root; synaptic function

SIGNIFICANCE: Our long-term goal is to discover novel regulatory components of a core mechanism of cellular communication, i.e., signaling via heterotrimeric G proteins (henceforth trimeric G proteins), and to characterize the molecular mechanisms that underlie their involvement in human disease. Our emphasis is on cytoplasmic factors that regulate G protein signaling, which have been (and remain) understudied compared to G protein-coupled receptors (GPCRs), the main activating inputs for trimeric G proteins. During the last grant cycle, we made significant advances in this area by achieving all the originally proposed goals, thereby establishing a new paradigm of G protein regulation. We demonstrated the existence of a family of cytoplasmic activators of G proteins, dissected the structural basis for their molecular activity, and established the consequences of their dysregulation in specific cellular processes and human diseases. The specific goal of this renewal application is to characterize the prototype member of a potential new class of G protein regulators that has been linked to chronic pain and epilepsy. The achievement of our goals would provide deep mechanistic insights into a new paradigm of GPCR-G protein regulation that fine tunes inhibitory neuromodulation, and establish a new framework to devise therapeutic strategies for neurological disorders like chronic pain and epilepsy. BACKGROUND: In the course of a screen of proteins that bind to Gαi subunits of Gα-Gβγ timeric complexes, we identified a protein that regulates G proteins via a unique and novel mechanism. We have coined the term “paradoxical G protein regulator” (PGR) to convey that it upregulates the modulation of some G protein effectors while simultaneously downregulating the modulation of other G protein effectors. Loss of this “PGR” is known to alter GPCR signaling in neurons of the peripheral nervous system and causes chronic pain. It has also been linked to epilepsy. Despite its clear biomedical importance, the molecular mechanism by which this G protein regulator operates, and how it modulates neurotransmission in the brain are completely unknown. SYNOPSIS OF AIMS: Based on compelling preliminary data, we propose that the PGR modulates both Gαi- and Gβγ-dependent signaling without directly affecting the G protein enzymatic activity (i.e., nucleotide binding and/or hydrolysis), and that this novel mechanism fine tunes GPCR signaling in brain neurons to influence seizure susceptibility. In Aim#1 we will dissect how the PGR regulates G proteins at the molecular level by characterizing how it engages physically Gαi and the consequences of this physical engagement on G protein signaling to different effectors. In Aim#2 we will characterize how it regulates GPCR signaling and neurotransmission at the cellular level by using primary cultures of neurons and brain slices from wild-type and KO mice. In Aim#3 we will determine the PGR's role at the physiological level by establishing how it impacts seizure susceptibility using genetically modified mouse models.

意义：我们的长期目标是发现核心机制的新监管组成部分 细胞通讯，即通过异三聚体G蛋白(下称三聚体G蛋白)传递信号，以及 描述它们参与人类疾病的分子机制。我们的重点是 调节G蛋白信号的细胞质因子，一直(和仍然)研究不足，与 G蛋白偶联受体(GPCRs)，三聚体G蛋白的主要激活输入。在最后一次拨款期间 周期，我们在这方面取得了重大进展，实现了最初提出的所有目标，从而 建立G蛋白调控的新范式。我们证明了一个细胞质家族的存在 G蛋白的激活剂，剖析了其分子活性的结构基础，并建立了 它们在特定的细胞过程和人类疾病中的失调的后果。 此续订应用程序的特定目标是描述潜在新类的原型成员的特征 G蛋白调节剂与慢性疼痛和癫痫有关。我们目标的实现将会 提供对微调抑制的GPCR-G蛋白调控新范例的深刻机械洞察力 神经调节，并建立一个新的框架来设计神经疾病的治疗策略 比如慢性疼痛和癫痫。 背景：在筛选与Gα-Gα时间复合体的GβγI亚单位结合的蛋白质的过程中， 我们发现了一种通过一种独特而新颖的机制来调节G蛋白的蛋白质。我们创造了这个术语 “矛盾G蛋白调节剂”(PGR)表达它上调某些G蛋白的调节 同时下调其他G蛋白效应器的调节。失去这一“PGR”是 已知会改变周围神经系统神经元中的GPCR信号，并导致慢性疼痛。它有 也被认为与癫痫有关。尽管它在生物医学上具有明显的重要性，但它的分子机制 G蛋白调节器的作用，以及它如何调节大脑中的神经传递完全未知。 AIMS简介：基于令人信服的初步数据，我们认为PgR对G-αI- 和Gβγ依赖的信号传递，而不直接影响G蛋白的酶活性(即核苷酸结合 和/或水解)，并且这一新的机制微调大脑神经元中的GPCR信号以影响 癫痫敏感度。在目标1中，我们将剖析PGR如何通过以下方式在分子水平上调节G蛋白 表征它是如何与GαI物理接触的，以及这种物理接触对G蛋白的影响 向不同的效应器发送信号。在目标#2中，我们将描述它是如何调节gpr信号和 用原代培养的神经细胞和来自野生型和大鼠脑片的神经细胞水平的神经传递 Ko老鼠。在目标3中，我们将通过确定PGR如何影响生理水平来确定它在生理水平上的作用 利用转基因小鼠模型研究癫痫易感性。