Biology of the ARL13B GTPase

描述（由申请人提供）：纤毛引起了人们的浓厚兴趣，因为它们在许多人类遗传疾病（统称为纤毛病，包括Joubert综合征（JS））和脊椎动物/小鼠发育中具有重要作用；对它们在细胞信号传导方面的核心作用的认识也在增长。原发的、不动的纤毛是基于微管的突起，几乎存在于每一个真核细胞中。我们关注小GTPase ARL13B，因为它的缺失会导致以下缺陷：(1)纤毛结构缺陷；(2)纤毛特异性蛋白的运动和定位缺陷；(3)纤毛内特异性蛋白的运动和定位缺陷；(4)ARL13突变的患者会出现Joubert综合征（JS），包括肾脏、眼部和脑部异常。该应用程序有四个具体目标，其中我们提出(1)定义ARL13B作为GTPase的生化特性并鉴定和表征新的效应物，(2)鉴定、纯化和表征ARL13B GTPase激活蛋白（GAPs），(3)确定下游ARL13B信号传导的复杂性水平并鉴定一种或多种效应物，(4)确定JS引起小鼠ARL13B突变的组织特异性后果。我们将利用两家pi的互补专业知识开发与疾病过程直接相关的纤毛ARL13B信号传导模型。我们将在小鼠胚胎成纤维细胞中建立一个新的基因缺失系统，并对ARL13B作为一种调节性GTPase进行详细的生化研究和建模，以建立ARL13B在细胞和动物中的作用的分子模型。总之，这些方法将使我们能够识别并严格测试ARL13B作用的下游效应物和GTPase激活蛋白（gap）在JS影响的通路发展中的作用。最后，通过定义JS致病ARL13B突变的体内组织特异性功能，我们将建立疾病模型，并确定组织特异性ARL13B功能和患者表型变异性的机制。总之，这些目标将为建立Arl13b作用模型提供坚实的实验基础，对纤毛功能、细胞信号传导、哺乳动物发育和人类疾病的许多方面具有明确的意义。

DESCRIPTION (provided by applicant): Cilia have sparked keen interest because of their proven importance in many human genetic diseases (collectively termed ciliopathies and including Joubert syndrome (JS)) and in vertebrate/mouse development; appreciation of their central role in aspects of cell signaling is growing, as well. Primary, immotile cilia are microtubule-based projections found on virtually every eukaryotic cell. We focus on the small GTPase, ARL13B, because its absence leads to defects in (1) cilia structure, (2) movement and localization of specific proteins to and (3) within cilia, and (4) patients with mutations in ARL13 have Joubert syndrome (JS), which involves renal, ocular, and brain anomalies. This application has four specific aims, in which we propose to (1) define the biochemical properties of ARL13B as a GTPase and identify and characterize novel effectors, (2) identify, purify, and characterize ARL13B GTPase activating proteins (GAPs), (3) determine the level of complexity in downstream ARL13B signaling and identify one or more effectors, (4) determine the tissue-specific consequences of JS causing ARL13B mutations in mice. We will use the complementary expertise of the two PIs to develop models for ARL13B signaling at cilia, with direct relevance to disease processes. We will pair a novel genetic deletion system in mouse embryo fibroblasts with detailed biochemical studies and modeling of ARL13B as a regulatory GTPase to develop molecular models for ARL13B's actions in cells and animals. Together, these approaches will allow us to identify and rigorously test downstream effectors and GTPase activating proteins (GAPs) of ARL13B actions in the development of the pathways affected by JS. Finally, by defining the in vivo, tissue specific function of the JS causative ARL13B mutations, we will both model the disease and identify the mechanism underlying tissue-specific Arl13b function and patient phenotypic variability. Together these aims will provide a solid experimental basis for establishing models of Arl13b actions, with clear implications for many aspects of cilia function, cell signaling, mammalian development, and human disease.