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病(统称为纤毛疾病和包括Joubert综合征(JS))和脊椎动物/小鼠发育中都被证明是重要的；对它们在细胞信号方面的核心作用的认识也在不断增长。原生的、静止的纤毛是几乎在每个真核细胞上发现的以微管为基础的突起。我们关注小的GTP酶Arl13b，因为它的缺失导致(1)纤毛结构的缺陷，(2)特定蛋白在纤毛中的移动和定位，以及(4)ARL13突变的患者患有Joubert综合征(JS)，涉及肾、眼和脑的异常。这项应用有四个特定的目标，其中我们建议(1)将Arl13b的生化特性定义为GTP酶，并鉴定和表征新的效应物，(2)鉴定、纯化和鉴定Arl13b GTP酶激活蛋白(GAP)，(3)确定Ar13b下游信号的复杂程度并确定一个或多个效应物，(4)确定JS导致小鼠Ar13b突变的组织特异性后果。我们将利用这两个PI的互补专业知识来开发纤毛中Arl13b信号的模型，与疾病过程直接相关。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，结合详细的生化研究和对Arl13b作为调节GTP酶的建模，以开发Ar13b在细胞和动物中作用的分子模型。总之，这些方法将使我们能够识别并严格测试Arl13b在JS影响的通路发展中发挥作用的下游效应器和GTP酶激活蛋白(GAP)。最后，通过定义JS导致Arl13b突变的体内、组织特异性功能，我们将对疾病进行建模，并确定组织特异性Arl13b功能和患者表型变异的机制。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、哺乳动物发育和人类疾病的许多方面都有明确的影响。

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.