Heparan sulfate proteoglycans in signaling and development

项目摘要 我们研究的长期目标是了解蜂窝通信的基本原理 由硫酸乙酰肝素蛋白多糖(HSPGs)介导。HSPG是一种特殊类型的碳水化合物修饰 作为各种生长因子的共同受体的蛋白质，包括骨形态发生蛋白， WNT/Wingless和Hedgehog。这些HSPG共受体在数量和强健的 控制信令输出。我们利用果蝇模型研究了HSPGs的体内功能。我们早些时候 研究已经确定了HSPGs在发育中的关键作用，即在形态发生信号和 梯度形成。此外，我们还证明了热休克蛋白G作为一种 普适因素。尽管蛋白多糖生物学已经取得了重大进展，但仍有几个主要问题 仍有待阐明。例如，热休克蛋白G的共同受体活性的分子机制是 人们对此知之甚少。不同的HS结构如何调节特定的信号和模式也是未知的 事件。现在至关重要的是开发一种跨学科的方法，这种方法可以直接将详细的HS联系起来 结构基序、配体结合、数量信号输出和发育/生理表型。 我们以前的研究表明，HSPGs与其他因素协同作用，发挥协同受体作用。 细胞表面的活性。为了揭示共受体功能的分子基础，我们使用蛋白质组学和 用遗传方法来确定这些失踪球员的身份。我们最近发现气管(WDP)，一种 含有四个富含亮氨酸重复序列的跨膜蛋白是一种新的HH负联合受体 发信号。我们还发现WDP是一种硫酸软骨素蛋白多糖(CSPG)。因此，HH途径是 由两类蛋白多糖辅助受体控制：HSPG(正调节因子)和WDP(CSPG，a 负调节器)。我们将阐明这种“双PG共同受体系统”如何在数量上实现 受控的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和形态发生，我们将建立 一种使用果蝇细胞的“体外”模型。尽管果蝇模型在体内的许多优点 研究表明，有关果蝇HS结构的信息有限。这主要是由于代谢困难造成的。 用果蝇动物体内放射性标记HS。为了填补这一空白，我们最近创作了一部小说 5种HS修饰酶突变的果蝇细胞系。我们使用这些细胞系的研究将提供一种 果蝇HS的详细结构信息与丰富的生物学知识的直接联系 在过去二十年中使用这种动物模型获得的表型信息。 总之，这一应用将通过以下方式推进我们的领域：(1)定义基本的分子机制 (2)全面了解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.