Signaling at the primary cilium in development and disease

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;然而，初级纤毛长期被误认为是退化的。初级纤毛现在被认为是作为细胞的触角，用于感知各种各样的信号，这些细胞器中的缺陷越来越多地被牵连在不同的发育和退行性人类疾病称为“ciliopathies”。虽然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是未知的。本提案的主要重点是调查的时空组织的信号在初级纤毛，和独特的功能，导致其广泛使用的信号室。 初级纤毛通过调节下游的双功能Gli转录因子，在神经管发育过程中的音刺猬（Shh）信号转导中不可或缺。cAMP激活的蛋白激酶A（PKA）触发蛋白水解过程，以纤毛依赖的方式产生Gli阻遏物;然而，促进PKA激活的途径和纤毛的确切功能尚不清楚。我们最近发现了一个孤儿睫状体G蛋白偶联受体（GPCR），Gpr 161，作为一个负调节Shh信号和Gli处理通过cAMP信号。活性Shh信号传导也导致从初级纤毛去除该受体。因此，Gpr 161可能通过增加纤毛cAMP水平来激活PKA，同时也在正反馈回路中受Shh信号转导调节。Gpr 161的发现使我们能够解决纤毛信号传导中的以下悬而未决的问题。首先，信号分子是如何在初级纤毛中动态划分的？第二，破坏初级纤毛中的信号传导的表型后果是什么？我们能够将Gpr 161功能的不同方面分开，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性池，这有助于我们在背景下机械地剖析信号传导 完整的纤毛在这里，我们建议使用综合的方法来确定潜在的Gpr 161介导的纤毛信号的调节机制。首先，我们将确定的因素，利用蛋白质组学方法与膜靶向纤毛定位图案的GPCR纤毛定位的特异性。我们将测试这些潜在的候选人的作用，在纤毛运输的内源性Gpr 161使用敲低测定。第二，我们将定义参与Shh依赖性去除Gpr 161从纤毛识别区域的受体是必要的，这一过程，并研究Shh信号，Gpr 161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Gpr 161的动态调节的发育后果，并测试Gpr 161介导的纤毛cAMP信号传导在Shh通路中的PKA激活过程中的作用。这些实验将为研究纤毛调控和GPCR在发育途径中的区室化奠定坚实的基础。

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.