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/小鼠发育中的重要性已得到证实;对它们在细胞信号传导方面的核心作用的认识也在增长。初级的，不动的纤毛是基于微管的突起，几乎在每一个真核细胞上都有发现。我们专注于小GTdR，ARL 13 B，因为它的缺失导致以下缺陷：（1）纤毛结构，（2）特定蛋白质的移动和定位以及（3）纤毛内，（4）ARL 13突变的患者患有Joubert综合征（JS），涉及肾脏，眼部和大脑异常。 本申请具有四个具体目的，其中我们提出（1）将ARL 13 B的生物化学性质定义为GTP酶并鉴定和表征新的效应物，（2）鉴定、纯化和表征ARL 13 B GTP酶活化蛋白（GAP），（3）确定下游ARL 13 B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ARL 13 B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与疾病过程直接相关的纤毛ARL 13 B信号转导模型。我们将在小鼠胚胎成纤维细胞中配对一种新的遗传缺失系统，并将ARL 13 B作为一种调节性GTdR进行详细的生化研究和建模，以开发ARL 13 B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和严格测试ARL 13 B作用的下游效应子和GTdR激活蛋白（GAP），以发展受JS影响的途径。最后，通过定义JS致病性ARL 13 B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ARL 13 B功能和患者表型变异性的机制。 这些目标将为建立Arl 13 b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.