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感应各种信号的细胞天线，这些细胞器的缺陷越来越多地与各种发育和退行性人类疾病（称为“纤毛病”）有关。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。本研究的主要重点是研究初级纤毛信号的时空组织，以及导致其广泛用作信号室的独特特征。初级纤毛通过调节下游双功能Gli转录因子，在神经管发育过程中对sonic hedgehog （Shh）信号传递起着不可或缺的作用。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抑制因子；然而，促进PKA激活的途径以及纤毛的确切功能尚不清楚。我们最近发现了一个孤儿纤毛g蛋白偶联受体（GPCR） Gpr161，它通过cAMP信号作为Shh信号和Gli加工的负调节因子。活跃的Shh信号也会导致这种受体从初级纤毛中移除。因此，Gpr161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到Shh信号的调节。我们对Gpr161的发现使我们能够解决以下在纤毛信号传导中悬而未决的问题。首先，信号分子是如何在初级纤毛中动态划分的？其次，干扰初级纤毛信号传导的表型后果是什么？我们能够解开Gpr161功能的不同方面，如信号，纤毛定位和去除纤毛，并研究纤毛中的内源性池，这有助于我们在机械上解剖背景下的信号传导

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.