Signaling at the primary cilium in development and disease

描述(申请人提供)：几乎每个静止的脊椎动物细胞都有一个叫做初级纤毛的细胞器。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。初级纤毛现在被认为是感知各种信号的细胞触角，这些细胞器中的缺陷越来越多地与各种发育和退化的人类疾病有关，这些疾病称为纤毛病。虽然，驱动纤毛组装的机制已经被研究，但纤毛信号的基本原理在很大程度上是未知的。这一建议的主要焦点是研究初级纤毛中信号的时空组织，以及导致其作为信号间隔被广泛使用的独特特征。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对Sonic Hedgehog(Shh)信号起着不可或缺的作用。CAMP激活的蛋白激酶A(PKA)以纤毛依赖的方式触发蛋白分解过程，产生Gli抑制物；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纤毛G蛋白偶联受体GPR161，它通过cAMP信号作为Shh信号和Gli处理的负调节因子。活跃的Shh信号也导致这种受体从初级纤毛上移除。因此，GPR161可能通过增加纤毛cAMP水平来激活PKA，同时也受到正反馈电路中Shh信号的调节。我们发现的GPR161使我们能够解决纤毛信号中的下列悬而未决的问题。首先，初级纤毛中的信号分子是如何动态划分的？第二，初级纤毛中信号中断的表型后果是什么？我们能够分离GPR161的S功能的不同方面，如信号、纤毛定位和纤毛去除，并研究纤毛的内生池，帮助我们机械地剖析上下文中的信号 完整的纤毛。在这里，我们建议使用综合方法来确定GPR161介导的纤毛信号的调控机制。首先，我们将利用膜定位纤毛定位模体的蛋白质组学方法来确定决定GPCRs纤毛定位特异性的因素。我们将使用基因敲除试验来测试这些潜在的候选基因在内源性GPR161纤毛运输中的作用。其次，我们将通过确定受体中参与这一过程的区域来确定依赖Shh从纤毛中移除GPR161的机制，并研究Shh信号、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区段激活过程中的作用。这些实验将为研究纤毛调节和GPCRs在发育途径中的区划奠定坚实的基础。

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.