Novel signaling pathways in the ciliary inversin compartment

项目总结 初级纤毛功能对正常左右不对称的发展至关重要，肾脏、心脏、中枢 神经系统和许多其他器官。目前的数据表明纤毛起信号转导的作用。 细胞器，区分受体、适配器和下游的多种转导机制 信号通路。然而，基本纤毛信号的身份以及信号是如何由 纤毛仍不清楚。倒置蛋白、NEK8、ANKS6和肾囊藻蛋白-3四种纤毛蛋白的复合体 构成所谓的睫状肌内翻室(IC)，位于睫状干的近端。 我们和其他人发现，这种IC是脊椎动物纤毛功能所必需的，INVS，NEK8， ANKS6和NPHP3均可导致包括L-/R-不对称扰动、先天性 心脏缺陷和多囊肾。这些缺陷表现为PKD2的完全敲除，PKD2是两个基因之一 常染色体显性遗传性多囊肾病的主要基因，提示ADPKD-1下游的信号转导 IC基因产物也沿着同样的途径出现。我们发现IC蛋白也控制纤毛长度和 鞭毛内转运(IFT)速度，需要cAMP信号的过程。没想到，我们发现 肾囊藻毒素-3是一种新的GTP酶，提示IC信号和cAMP信号之间存在因果联系。在这 GRANT建议，我们的目的是研究肾囊藻毒素-3 GTP酶机制的功能意义， 确定酶的催化参数，并询问IC之间的分子连接 成分和上下游信号因子之间的相互作用。 我们假设，肾囊藻蛋白-3的作用类似于刺激性异三聚体G蛋白α亚基， 沿着GTP-水解循环，关闭和开启其活性状态。在目标1中，我们将研究核苷酸 纯化的重组肾囊蛋白-3的亲和力和转化率，以及影响Pcy的等位基因 催化部位，这会导致小鼠多囊肾病；与GαS相比，我们将测试 纤毛G蛋白偶联受体可利用肾囊蛋白-3作为信号转接子，而腺苷是否 环化酶可能被肾囊素-3刺激而产生cAMP。在目标2中，我们研究分子 IC组件层次结构的详细信息。我们试图确定哪些蛋白质结构域是需要招募的 将肾囊藻毒素-3正确定位，以维持整个复合体的完整性，如果致病 突变会破坏结构的稳定。我们将最终测量纤毛cAMP水平，作为IC的函数 操作，并测试单个IC蛋白的缺失或突变是否会改变纤毛cAMP水平。 这些研究将纤毛病基因功能与cAMP信号联系起来，从而解决了 纤毛生物学对囊性肾病发病机制的重要意义。结果将会有所帮助 识别IC中可能导致新疗法设计的可用药酶靶点。

Project summary Primary cilia function is essential for the development of normal left-right asymmetry, kidneys, heart, central nervous system and many other organs. Current data indicate that the cilium functions as a signal transduction organelle, compartmentalizing receptor-, adaptor- and downstream transduction mechanisms for multiple signaling pathways. However, the identity of essential cilia signals and how signals are transduced by the cilium remains unclear. A complex of four ciliary proteins, inversin, NEK8, ANKS6 and nephrocystin-3 constitutes the so-called ciliary inversin compartment (IC), localized in the proximal portion of the ciliary shaft. We and others found that the IC is required for cilium function in vertebrates, with mutations in INVS, NEK8, ANKS6 and NPHP3 all leading to ciliopathy syndromes that include L-/R-asymmetry perturbation, congenital heart defects and polycystic kidneys. These defects phenocopy the full knockout of PKD2, one of the two major autosomal-dominant polycystic kidney disease genes, suggesting that signaling downstream of ADPKD- and IC-gene products occurs along the same pathway. We found that IC proteins also control ciliary length and intraflagellar transport (IFT) velocity, processes that require cAMP signaling. Unexpectedly, we find that nephrocystin-3 acts as a novel GTPase, suggesting a causal link between IC- and cAMP-signaling. In this grant proposal, we aim to investigate the functional significance of the nephrocystin-3 GTPase mechanism, defining the catalytic parameters of the enzyme, and interrogating molecular connections among the IC constituents and to upstream- and downstream signaling factors. We hypothesize that nephrocystin-3 acts analogous to a stimulatory heterotrimeric G-protein α-subunit, switching its activity state off and on, along a GTP-hydrolysis cycle. In Aim #1, we will investigate nucleotide affinitiy and -turnover of the purified recombinant nephrocystin-3, along with the pcy allele that affects the catalytic site, and that gives rise to polycystic kidney disease in mice; compared to GαS. We will test, whether ciliary G-protein coupled receptors may utilize nephrocystin-3 as a signal adaptor, and whether adenylyl cyclases may be stimulated by nephrocystin-3, to generate cAMP. In Aim #2, we investigate the molecular details of the IC assembly hierarchy. We seek to identify what protein domains are necessary to recruit nephrocystin-3 to its proper localization, to maintain the integrity of the whole complex, and if disease-causing mutations destabilize the structure. We will ultimately measure ciliary cAMP levels, as a function of IC manipulations, and test whether deletion or mutation of individual IC proteins change ciliary cAMP levels. These studies connect ciliopathy gene function to cAMP signaling, thereby addressing outstanding issues in ciliary biology with important implications for cystic kidney disease pathogenesis. The results will help identifying druggable enzymatic targets within the IC that may lead to the design of novel therapies.