Heparan sulfate proteoglycans in signaling and development

项目摘要 我们研究的长期目标是了解细胞通讯的基本原理 由硫酸乙酰肝素蛋白聚糖（HSPG）介导。HSPG是一种特殊类型的碳水化合物修饰的 作为各种生长因子的共受体的蛋白质，包括骨形态发生蛋白， Wnt/Wingless和Hedgehog。这些HSPG共受体在定量和稳健方面发挥着关键作用 控制信号输出。我们使用果蝇模型研究HSPGs的体内功能。我们早先 研究已经确定了HSPG在发育中的关键作用，即在形态发生素信号传导中， 梯度形成此外，我们已经证明HSPG作为一种调节因子调节干细胞生态位。 普遍因素虽然蛋白聚糖生物学已经取得了重大进展，但仍存在一些主要问题， 仍有待阐明。例如，HSPG的共受体活性的分子机制是 不太了解。不同的HS结构如何调节特定的信号传导和模式也是未知的 事件现在必须发展一种跨学科的方法，这种方法可以直接将详细的HS 结构基序、配体结合、定量信号输出和发育/生理表型。 我们前期的研究表明，HSPGs与其他因素协同作用，发挥共受体的功能， 细胞表面的活性。为了揭示辅助受体功能的分子基础，我们使用蛋白质组学和 通过遗传学方法来识别这些缺失的参与者。我们最近发现，气管（Wdp），一个 一种含有四个富含亮氨酸重复序列的跨膜蛋白，作为Hh的一种新型负性共受体 信号我们还表明，Wdp是硫酸软骨素蛋白聚糖（CSPG）。因此，Hh途径是 由两类蛋白聚糖共受体控制：HSPG（正调节剂）和Wdp（CSPG， 负调节器）。我们将阐明这样一个“双PG共受体系统”如何实现定量 控制信号输出和精确的图案形成。 为了了解HS结构的变化如何影响信号传导和形态发生，我们将建立 使用果蝇细胞的“体外”模型。尽管果蝇模型有许多优点， 研究，果蝇HS结构的信息是有限的。这主要是由于代谢的困难 使用果蝇动物在体内放射性标记HS。为了填补这一空白，我们最近开发了一种新的 果蝇细胞系突变的五个HS修饰酶。我们使用这些细胞系的研究将提供 果蝇HS的详细结构信息与丰富的生物学知识之间的直接联系 表型信息在过去二十年中使用该动物模型获得。 总之，该应用将通过以下方式推进我们的领域：（1）定义基本分子机制 （2）全面认识HS的结构与功能关系。

Project Summary The long-term goal of our research is to understand fundamental principles of cell communications mediated by heparan sulfate proteoglycans (HSPGs). HSPGs are a special type of carbohydrate-modified proteins that serve as co-receptors for various growth factors, including bone morphogenetic proteins, Wnt/Wingless, and Hedgehog. These HSPG co-receptors play critical roles in quantitative and robust control of signaling output. We study in vivo functions of HSPGs using the Drosophila model. Our earlier research has established critical roles of HSPGs in development, namely in morphogen signaling and gradient formation. In addition, we have demonstrated that HSPGs regulate the stem cell niche as a universal factor. Although proteoglycan biology has made significant progress, several major questions still remain to be elucidated. For example, the molecular mechanisms of co-receptor activities of HSPGs are poorly understood. It is also unknown how distinct HS structures regulate specific signaling and patterning events. It is now critical to develop an interdisciplinary approach, which can directly link detailed HS structural motifs, ligand binding, quantitative signaling output, and developmental/physiological phenotypes. Our previous studies suggested that HSPGs cooperate with other factors to exert co-receptor activity on the cell surface. To reveal the molecular basis for co-receptor function, we use proteomic and genetic approaches to identify these missing players. We recently found that Windpipe (Wdp), a transmembrane protein containing four leucine-rich repeats, acts as a novel negative co-receptor for Hh signaling. We also showed that Wdp is a chondroitin sulfate proteoglycan (CSPG). Thus, the Hh pathway is controlled by two classes of proteoglycan co-receptors: HSPGs (positive regulators) and Wdp (CSPG, a negative regulator). We will elucidate how such a "dual PG co-receptor system" achieves quantitatively controlled signaling output and precise pattern formation. To understand how a change in HS structure affects signaling and morphogenesis, we will establish an "in vitro" model using Drosophila cells. Despite the many strengths of the Drosophila model for in vivo studies, information on Drosophila HS structure is limited. This is mainly due to the difficulty of metabolic radio-labeling of HS in vivo using Drosophila animals. To fill this gap, we have recently generated novel Drosophila cell lines mutant for five HS modifying enzymes. Our studies using these cell lines will provide a direct link between detailed structural information of Drosophila HS and a wealth of knowledge on biological phenotypic information obtained over the last two decades using this animal model. Together, this application will advance our field by: (1) defining fundamental molecular mechanisms of co-receptor function and (2) comprehensive understanding of the structure-function relationship of HS.