CLIC function in GPCR-mediated Rho/Rac signaling

CLIC 在 GPCR 介导的 Rho/Rac 信号传导中的功能

• 批准号: 10552564
• 负责人: Jan K. Kitajewski
• 金额: $ 39.73万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10552564
• 关键词: Address; Adrenergic Agents; Agonist; Amino Acids; Binding; Biological Assay; Biosensor; Blood; C-terminal; CLIC4 gene; Caenorhabditis elegans; Cardiovascular system; Cell membrane; Cells; Chimera organism; Chloride Channels; Chlorides; Clinical Trials; Complex; Data; Defect; Development; Disease; Embryo; Endothelial Cells; Endothelium; Engineering; FDA approved; Family; Fibroblasts; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits, Gs; Genes; Genetic; Glutathione S-Transferase; Guanosine Triphosphate Phosphohydrolases; Heterotrimeric GTP-Binding Proteins; Human; Human Biology; Ion Channel; Knowledge; Label; Link; Lymphatic Endothelial Cells; Mammalian Cell; Measures; Mediating; Membrane; Modeling; Molecular; Mus; Mutant Strains Mice; Mutation; N-terminal; Nucleic Acid Regulatory Sequences; Orthologous Gene; Pathway interactions; Pericytes; Pharmaceutical Preparations; Phenotype; Physiological; Physiology; Protein Family; Protein Tyrosine Kinase; Proteins; Proteomics; Regulation; Role; SH3-Binding Motif; Signal Pathway; Signal Transduction; Specificity; Sphingosine-1-Phosphate Receptor; Structure; Surveys; System; Testing; Thrombin; Tube; Variant; Work; angiogenesis; cell behavior; cell type; clinically significant; functional restoration; gene conservation; in vivo; mutant; myristoylation; novel; protein complex; response; rho; rho GTP-Binding Proteins; sphingosine 1-phosphate; tool

G-protein-coupled receptor (GPCR)-mediated regulation of the GTPases Rho and Rac is a conserved signaling module required for vital cell behaviors. However, the mechanisms that connect GPCRs, and their heterotrimeric G-protein (Gα/β/γ) partners, to Rho/Rac are not fully delineated. Our studies in C. elegans and human endothelial cells have revealed a new conserved player required for Rho/Rac signaling: the Chloride Intracellular Channel (CLIC) family of proteins. Our data shows that CLICs are required in two important endothelial GPCR pathways (S1P/S1P Receptor and thrombin/PAR) that function through Gα12/13 and Gαi to activate Rho and Rac. This CLIC function is evolutionarily conserved, because we found that in C. elegans the CLIC ortholog exc-4 genetically interacts with the Gα12 ortholog gpa-12 and with the Rac orthologs ced-10 and mig-2. The molecular function of CLICs has long remained a mystery. Based on sequence and structural similarities, they have been proposed to function as chloride channels and/or as glutathione S-transferases (GST). Previous work has shown that EXC-4 membrane localization is mediated by an N-terminal domain and this localization is critical for function. We have now found that CLIC membrane localization is also critical for its role in GPCR-mediated Rho/Rac activation in endothelial cells, and that replacement of the membrane-targeting N-terminus with a myristoylation signal is sufficient to restore this function. Since the channel and GST activities of CLICs require an intact N- terminus we have discovered a novel activity for CLICs. We hypothesize that CLICs are membrane-localized regulators of Rho and Rac that respond to GPCR-Gα signaling. In Aim 1 we will determine how CLICs couple GPCR-Gα (Gα12/13 and Gαi) signaling to Rho and Rac. We will survey the requirement for CLICs in different cell and signaling contexts to define key GPCR-Gα combinations that utilize CLICs to regulate of Rho and Rac. We will use cutting-edge bio-sensors and genetic tools to measure and modulate signaling to determine which step in the GPCR-Gαβγ-Rho/Rac cascade requires CLICs. Finally, we will test whether CLICs physically interact with Rho/Rac to modulate signaling. In Aim 2 we will define the determinants by which CLICs regulate Rho/Rac in human cells and in C. elegans by performing structure-function analyses, focused on the EXC-4/CLIC C- terminus. Critical domains defined in this Aim will be tested for their ability to interact with Rho/Rac (as defined in Aim 1). In Aim 3 we will carry out unbiased genetic and proteomic screens in C. elegans to find conserved players that genetically and physically interact with EXC-4/CLIC to further elucidate how CLICs regulate Rho/Rac signaling. We will then test whether human orthologs of genes identified in these screens influence Rho/Rac signaling in mammalian cells. By defining new mechanisms of action for CLICs in GPCR-Gαβγ-Rho/Rac signaling we will significantly increase our knowledge of how GPCRs influence human biology and uncover new ways of targeting these pathways.

G蛋白偶联受体(GPCR)介导的GTP酶Rho和Rac是一种保守信号 重要细胞行为所需的模块。然而，连接GPCRs及其异三聚体的机制 G-蛋白(G-α/β/γ)伙伴，到Rho/Rac还没有完全被描述。我们对线虫和人内皮细胞的研究 细胞已经揭示了Rho/Rac信号所需的一个新的保守角色：氯离子细胞内通道 (CLIC)蛋白质家族。我们的数据显示，在两条重要的内皮GPCR通路中需要CLICS (S1P/S1P受体和凝血酶/PAR)，通过Gα12/13和GαI激活Rho和Rac。这 CLIC功能在进化上是保守的，因为我们发现在线虫中，CLIC直系同源基因exc-4 基因上与GPA-12的Gα12同源基因以及RACC10和MIG-2的同源基因相互作用。分子 陈词滥调的作用长期以来一直是个谜。基于序列和结构的相似性，它们一直是 建议作为氯离子通道和/或谷胱甘肽S转移酶(GST)发挥作用。以前的工作已经表明 Exc-4膜的定位是由N-末端结构域介导的，这种定位对功能至关重要。 我们现在发现CLIC膜的定位对其在GPCR介导的Rho/RAC中的作用也是至关重要的 在内皮细胞中的激活，以及以肉豆蔻酰化取代膜靶向N-末端 信号足以恢复这一功能。由于CLICS的通道和GST活动需要完整的N- 最后，我们发现了一种新的古典文学活动。我们假设古典文学是局限于膜的 Rho和Rac的调节因子，对gpr-Gα信号作出反应。在目标1中，我们将确定陈词滥调是如何结合的 GPCRGα(Gα12/13和GαI)向Rho和Rac发送信号。我们将调查不同单元中对Clics的需求 和信令上下文来定义关键的GPCRGα组合，这些组合利用CLICS来调节Rho和Rac。我们 将使用尖端生物传感器和遗传工具来测量和调制信号，以确定哪一步 在GPCRG中，αβγ-RHO/RAC级联需要经典。最后，我们将测试经典之作是否与 RHO/RAC来调制信令。在目标2中，我们将定义CLICS调节RHO/RAC的决定因素 通过对人体细胞和线虫进行结构-功能分析，重点研究了exc-4/clic C- 终点站。将测试在此目标中定义的关键域与RHO/RAC(如所定义的)交互的能力 目标1)。在目标3中，我们将在线虫中进行无偏见的遗传和蛋白质组筛选，以找到保守的 基因和物理上与exc-4/clic相互作用的玩家，以进一步阐明Clics如何调控Rho/Rac 发信号。然后，我们将测试在这些屏幕上识别的基因的人类同源基因是否会影响Rho/RAC 哺乳动物细胞中的信号。通过定义CLICS在GPCRGαβγ-RHO/RAC信号中的新作用机制 我们将显著增加我们对GPCRs如何影响人类生物学的知识，并发现新的 瞄准这些小路。