INVESTIGATION OF ROLES OF HEPARAN SULFATE PROTEOGLYCANS (HSPGS)

硫酸乙酰肝素蛋白聚糖 (HSPGS) 作用的研究

• 批准号: 7724054
• 负责人: Arthur D Lander
• 金额: $ 0.77万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7724054
• 关键词: Apoptosis; BMP2 gene; Biochemical; Bone Morphogenetic Proteins; Cell surface; Chondrogenesis; Class; Computer Retrieval of Information on Scientific Projects Database; Confocal Microscopy; Data; Developmental Process; Dimerization; Dorsal; Excision; Extracellular Matrix; Fluorescence; Funding; Glypican; Grant; Growth Factor; Heparan Sulfate Proteoglycan; Inorganic Sulfates; Institution; Investigation; Ligands; Mammalian Cell; Modification; Mus; PC12 Cells; Pattern; Polysaccharides; Procedures; Proteins; Research; Research Personnel; Resources; Role; Signal Transduction; Source; Testing; Time; Title; United States National Institutes of Health; Unspecified or Sulfate Ion Sulfates; bone morphogenetic protein 2; bone morphogenetic protein receptor type II; bone morphogenetic protein receptors; insight; neurogenesis; polypeptide; receptor; type IA bone morphogenetic protein receptor

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 全标题：通过荧光共聚焦显微镜研究硫酸乙酰肝素蛋白聚糖 (HSPG) 在骨形态发生蛋白-2 (BMP-2) 配体-受体组装和受体寡聚化中的作用 硫酸乙酰肝素蛋白聚糖 (HSPG) 是细胞表面和细胞外基质中高度糖化和硫酸化的蛋白质。已知线性且高度硫酸化的多糖 HS 链参与几类多肽生长因子的信号传导，包括骨形态发生蛋白 (BMP)。众所周知，BMP 参与多种基本发育过程，如原肠胚形成、神经发生、软骨形成、背腹模式形成和细胞凋亡。 HSPG 究竟如何调节 BMP 信号传导仍不清楚。 在这里，我们将利用 FCM 研究 HS 在 BMP II 型受体预组装以及 BMP2 配体诱导的培养哺乳动物细胞表面受体寡聚化中的作用。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，但不允许我们实时研究高阶受体寡聚化。此外，HSPG由大量的多糖链组成，使得在实验过程中很难进行一些生化修饰。 FCM 将使我们能够研究 HSPG、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. 检测酶促去除 HSPG 后的 II 型 BMP 受体 (BMPRII) 二聚化； 2.检测BMP配体刺激后BMPRII和glypican-1（哺乳动物细胞中HSPG的主要形式）之间可能的关联； 3.在BMP刺激和/或去除HSPG后检测IA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，并且之前已经成功测试过。我们还有 BMPRIA 和 glypican1（均带有荧光蛋白标签）构建体可供使用。