Signaling at the primary cilium in development and disease

发育和疾病中初级纤毛的信号传导

基本信息

批准号：
8990974
负责人：
Saikat Mukhopadhyay
金额：
$ 27.65万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-01-01 至 2019-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8990974
关键词：
Address Affinity Chromatography Automobile Driving Biochemical Biological Biological Assay Cells Cilia Complex Cultured Cells Cyclic AMP Cyclic AMP-Dependent Protein Kinases Defect Development Disease Endocytosis Endocytosis Pathway Excision Feedback Flagella Foundations G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors Health Human Mass Spectrum Analysis Mediating Membrane Modeling Monitor Neural Tube Development Organelles Orphan Pathogenesis Pathology Pathway interactions Phosphorylation Process Protein Family Proteins Proteolytic Processing Proteomics RNA Interference Receptor Signaling Regulation Role SHH gene Sensory Signal Transduction Signaling Molecule Site Solid Sonic Hedgehog Pathway Specificity Subcellular structure Testing Vertebrates base ciliopathy desensitization genetic approach human disease insight knock-down mouse model receptor receptor function receptor internalization research study reverse genetics smoothened signaling pathway tool trafficking transcription factor

项目摘要

DESCRIPTION (provided by applicant): Almost every quiescent vertebrate cell has an organelle called the primary cilium. Cilia and flagella are among the earliest described subcellular structures; however, the primary cilium was long mistaken as vestigial. The primary cilia are now considered to function as cellular antennae for sensing a wide variety of signals, and defects in these organelles are increasingly being implicated in diverse developmental and degenerative human diseases called "ciliopathies". Although, mechanisms driving ciliary assembly have been studied, the principles underlying ciliary signaling are largely unknown. The main focus of this proposal is to investigate the spatio-temporal organization of signaling in the primary cilium, and the unique features that have resulted in its widespread use as a signaling compartment. The primary cilium is indispensable for sonic hedgehog (Shh) signaling during neural tube development by regulating the downstream bifunctional Gli transcription factors. The cAMP activated protein kinase A (PKA) triggers proteolytic processing that generates Gli repressors in a cilia-dependent manner; however, pathways that promote PKA activation, and the exact function of cilia is unclear. We recently identified an orphan ciliary G-protein-coupled receptor (GPCR), Gpr161 that acts as a negative regulator of Shh signals and Gli processing via cAMP signaling. Active Shh signaling also results in removal of this receptor from the primary cilia. Thus, Gpr161 is likely to activate PKA by increasing ciliary cAMP levels, while also being regulated by Shh signaling in a positive feedback circuit. Our discovery of Gpr161 allows us to address the following outstanding questions in ciliary signaling. First, how are signaling molecules dynamically compartmentalized in the primary cilia? Second, what are the phenotypic consequences of disrupting signaling in the primary cilia? Our ability to uncouple distinct aspects of Gpr161's function, such as signaling, ciliary localization and removal from cilia, and study endogenous pools in cilia help us mechanistically dissect signaling in the context of intact cilia. Here, we propose to identify regulatory mechanisms underlying Gpr161-mediated ciliary signaling using integrative approaches. First, we will identify factors determining specificity of ciliary localization of GPCRs utilizing proteomic approaches with membrane-targeted ciliary localization motifs. We will test the role of these potential candidates in ciliar trafficking of endogenous Gpr161 using knockdown assays. Second, we will define mechanisms involved in Shh-dependent removal of Gpr161 from the cilia by identifying regions of the receptor that are necessary for this process, and study the cross-talk between Shh signaling, Gpr161 activity, desensitization, and endocytosis pathways. Third, we will study the developmental consequences of disrupting dynamic regulation of Gpr161 in the cilia, and test the role of Gpr161-mediated ciliary cAMP signaling during compartmentalized PKA activation in the Shh pathway. These experiments will establish a solid foundation for studying ciliary regulation and compartmentalization of GPCRs in developmental pathways.

描述（由申请人提供）：几乎每个静止的脊椎动物细胞都有一个称为初级纤毛的细胞器。纤毛和鞭毛是最早被描述的亚细胞结构之一。然而，初级纤毛长期以来被误认为是退化的。现在认为初级纤毛具有感知多种信号的细胞天线的功能，并且这些细胞器的缺陷越来越多地与被称为“纤毛病”的多种发育和退行性人类疾病有关。尽管已经研究了驱动纤毛组装的机制，但纤毛信号传导的基本原理在很大程度上尚不清楚。该提案的主要重点是研究初级纤毛中信号传导的时空组织，以及导致其广泛用作信号传导室的独特特征。初级纤毛通过调节下游双功能 Gli 转录因子，对于神经管发育过程中的声波刺猬 (Shh) 信号传导是不可或缺的。 cAMP 激活的蛋白激酶 A (PKA) 触发蛋白水解过程，以纤毛依赖性方式产生 Gli 阻遏物；然而，促进 PKA 激活的途径以及纤毛的确切功能尚不清楚。我们最近发现了一种孤儿睫状 G 蛋白偶联受体 (GPCR) Gpr161，它通过 cAMP 信号传导充当 Shh 信号和 Gli 处理的负调节因子。活跃的 Shh 信号传导还会导致该受体从初级纤毛中去除。因此，Gpr161 可能通过增加纤毛 cAMP 水平来激活 PKA，同时也受到正反馈回路中的 Shh 信号传导的调节。 Gpr161 的发现使我们能够解决纤毛信号传导中的以下突出问题。首先，信号分子如何在初级纤毛中动态划分？其次，破坏初级纤毛信号传导会产生什么表型后果？我们能够解开 Gpr161 功能的不同方面，例如信号传导、纤毛定位和从纤毛中去除，并研究纤毛中的内源池，这有助于我们在上下文中机械地剖析信号传导完整的纤毛。在这里，我们建议使用综合方法来确定 Gpr161 介导的纤毛信号传导的潜在调节机制。首先，我们将利用具有膜靶向纤毛定位基序的蛋白质组学方法来确定决定 GPCR 纤毛定位特异性的因素。我们将使用敲低实验测试这些潜在候选者在内源性 Gpr161 纤毛运输中的作用。其次，我们将通过识别该过程所需的受体区域来定义依赖于Shh从纤毛中去除Gpr161的机制，并研究Shh信号传导、Gpr161活性、脱敏和内吞途径之间的串扰。第三，我们将研究破坏纤毛中 Gpr161 动态调节的发育后果，并测试 Gpr161 介导的纤毛 cAMP 信号在 Shh 通路中 PKA 激活过程中的作用。这些实验将为研究发育途径中 GPCR 的纤毛调节和区室化奠定坚实的基础。