INVESTIGATION OF ROLES OF HEPARAN SULFATE PROTEOGLYCANS (HSPGS)

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 用荧光共聚焦显微镜研究硫酸乙酰肝素蛋白多糖(HSPGs)在骨形态发生蛋白-2(BMP-2)配体-受体组装和受体齐聚中的作用 硫酸乙酰肝素蛋白多糖(HSPGs)是细胞表面和细胞外基质中高度糖化和硫酸盐化的蛋白质。已知的线状和高度硫酸化的多糖HS链参与了几类多肽生长因子的信号传递，包括骨形态发生蛋白(BMPs)。众所周知，BMPs参与了原肠形成、神经发生、软骨形成、背腹模式形成和细胞凋亡等多种基本发育过程。目前尚不清楚HSPGs是如何调控BMP信号的。 在这里，我们将利用流式细胞术研究HS在BMP II型受体预组装和BMP2配体诱导的哺乳动物细胞表面受体寡聚中的作用。我们的初步生化数据为HS调节BMP作用的机制提供了新的见解，但不允许我们实时观察高阶受体寡聚。此外，HSPGs含有大量的多糖链，在实验过程中很难进行某些生化修饰。FCM将使我们能够研究HSPGs、BMP配体和两种类型的BMP受体之间可能的相互作用，而不受生化实验程序的限制。 以下是我们计划做的事情： 1.酶法去除HSPGs后检测II型BMP受体(BMPRII)的二聚化； 2.检测BMP配体刺激下BMPRII与GLYPICAN-1(哺乳动物细胞中HSPGs的主要形式)之间的可能联系； 3.检测BMP刺激和/或去除HSPGs时I型BMP受体(BMPRIA)和BMPRII的寡聚状态。 目前，我们有稳定表达BMPRII-EYFP(黄色荧光)的小鼠PC12细胞，并且已经成功地进行了测试。我们也有BMPRIA和Glypican1(都带有荧光蛋白标签)构建物可供使用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Full title: Investigation of roles of heparan sulfate proteoglycans (HSPGs) in bone morphogenetic protein-2 (BMP-2) ligand-receptor assembly and receptor oligomerization by fluorescence confocal microscopy Heparan sulfate proteoglycans (HSPGs) are highly glycated and sulfated proteins on cell surfaces and in the extracellular matrix. The linear and highly sulfated polysaccharide HS chains have been known to participate in signaling of several classes of polypeptide growth factors, including bone morphogenetic proteins (BMPs). It is known that BMPs involve in fundamental developmental processes as diverse as gastrulation, neurogenesis, chondrogenesis, dorsal-ventral patterning, and apoptosis. Exactly how HSPGs regulate BMP signaling remains unclear. Here we will investigate the roles of HS in BMP type II receptor pre-assembly, and BMP2 ligand-induced receptor oligomerization on cultured mammalian cell surface by employing FCM. Our preliminary biochemical data provided new insights into the mechanisms of HS regulating BMP actions, but did not allow us to look into higher-order receptor oligomerization in real time. Besides, HSPGs consist of huge amount of polysaccharide chains, and make it very difficult to apply some biochemical modification during experimental procedures. FCM will allow us to investigate the possible interactions among HSPGs, BMP ligand, and two types of BMP receptors without the limitations of biochemical experimental procedures. The following is what we plan to do: 1. Detecting type II BMP receptor (BMPRII) dimerization upon enzymatic removal of HSPGs; 2. Detecting possible association between BMPRII and glypican-1 (a predominant form of HSPGs in mammalian cells) upon BMP ligand stimulation; 3. Detecting oligomerization status of type IA BMP receptor (BMPRIA) and BMPRII upon BMP stimulation and/or removal of HSPGs. Currently we have mouse PC12 cells stably expressing BMPRII-EYFP (yellow fluorescence) and have successfully tested it before. We also have BMPRIA and glypican1 (both with fluorescence protein tags) constructs ready to use.