EXTRACELLULAR MATRIX AND CELL ATTACHMENT PROTEINS

细胞外基质和细胞附着蛋白

• 批准号: 2007704
• 负责人: HAROLD P ERICKSON
• 金额: $ 29.45万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-05-01 至 2000-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2007704
• 关键词: Caenorhabditis elegans; Drosophilidae; X ray crystallography; affinity chromatography; binding proteins; cell adhesion molecules; embryogenesis; enzyme linked immunosorbent assay; extracellular matrix; gene expression; genetic library; genetically modified animals; immunochemistry; laboratory mouse; laminin; membrane proteins; neoplastic growth; northern blottings; polymerase chain reaction; protein structure function; tissue /cell culture; western blottings; wound healing

Continued studies will focus primarily on tenascin - the giant hexabrachion molecule in the extracellular matrix of tumors, healing wounds and specific embryonic tissues. We are already making transgenic mice that overexpress tenascin. We propose to make others that will be defective in hexabrachion assembly (monobrachion mice), and mice with depletion in tenascin secretion. We will determine the effects of overexpression, underexpression or defective hexabrachions on embryonic development, tumors and wound healing. A second project is a search for the tenascin gene in Drosophila and C. elegans. These species have highly developed genetic maps and large numbers of mutants, offering a powerful new approach to understanding the functions of tenascin. We will continue our studies of the cell biology and biochemistry of tenascin, to identify and characterize cell surface receptors for different domains of tenascin. A key tool for this project is the library of bacterial expression proteins that we have recently developed, providing large quantities of defined segments of the hexabrachion arm. We will use a similar approach to identify ECM molecules that bind to tenascin. Another continuing project is to characterize a new form of laminin that we identified during the purification of tenascin from cell culture supernatant. This new protein appears to have a variant A chain, a unique tissue distribution, and a cell adhesion activity quite different from any known laminin. Finally, we propose to expand our collaborative projects on the x-ray crystallography of tenascin. Our approach is to crystallize one domain at a time, using our PCR approach to make precisely defined bacterial expression proteins. The two FN-III domains that we have tried so far, the RGD domains from tenascin and fibronectin, have given excellent crystals, and high resolution x-ray diffraction studies are in progress. Additional domains are now proposed for future studies. An atomic resolution structure of the entire hexabrachion is a feasible goal.

继续的研究将主要集中在Tenascin-巨人 六臂细胞分子在肿瘤细胞外基质中的作用 伤口和特定的胚胎组织。我们已经在制造转基因产品了 过度表达Tenascin的小鼠。我们建议让其他人成为 六臂臂组装缺陷(单臂臂小鼠)，小鼠 Tenascin分泌耗尽。我们将确定 胚胎上的高表达、低表达或缺陷六臂 发展、肿瘤和伤口愈合。第二个项目是搜索 果蝇和线虫中的tenascin基因。这些物种有很高的 开发了遗传图谱和大量突变体，提供了强大的 理解Tenascin功能的新途径。我们将继续 我们对Tenascin的细胞生物学和生化研究，以鉴定 并对不同结构域的细胞表面受体进行表征 Tenascin。这个项目的一个关键工具是细菌库 我们最近开发的表达蛋白，提供了大量的 六臂臂的定义节段的数量。我们将使用 识别与Tenascin结合的ECM分子的类似方法。另一个 持续的项目是表征一种新形式的层粘连蛋白，我们 细胞培养提纯Tenascin过程中的鉴定 上清液。这种新的蛋白质似乎有一个变异的A链，一个独特的 组织分布，与细胞黏附活性大相径庭 任何已知的层粘连蛋白。最后，我们建议扩大我们的协作 Tenascin的x射线结晶学研究项目。我们的方法是 一次结晶一个结构域，使用我们的PCR方法来制作 精确定义的细菌表达蛋白。两个FN-III结构域 我们到目前为止已经尝试过了，来自Tenascin和FN的RGD结构域， 已经给出了优秀的晶体和高分辨率的x射线衍射仪 研究正在进行中。现在建议为未来提供更多域名 学习。整个六臂粒子的原子分辨率结构是 可行的目标。