EXTRACELLULAR MATRIX AND CELL ATTACHMENT PROTEINS

细胞外基质和细胞附着蛋白

• 批准号: 2092409
• 负责人: HAROLD P ERICKSON
• 金额: $ 25.13万
• 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 1997-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2092409
• 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.

继续研究将主要集中于Tenascin-巨人 肿瘤细胞外基质中的六边形分子，愈合 伤口和特定的胚胎组织。我们已经在做转基因 那些过表达tenascin的老鼠。我们建议让其他人 六边形组件（单刺小鼠）和带有小鼠的缺陷 Tenascin分泌的耗竭。我们将确定 胚胎上的过表达，不渗透或六边形缺陷 发育，肿瘤和伤口愈合。第二个项目是搜索 果蝇和秀丽隐杆线虫中的Tenascin基因。这些物种具有很高的 开发了遗传图和大量突变体，提供了强大的 了解替纳斯汀的功能的新方法。我们将继续 我们对Tenascin细胞生物学和生物化学的研究，以识别 并表征了不同域的细胞表面受体 Tenascin。该项目的关键工具是细菌库 我们最近开发的表达蛋白，提供大量 六边形臂的定义段数量。我们将使用一个 类似的方法来鉴定与替纳斯蛋白结合的ECM分子。其他 持续的项目是表征我们的一种新形式的层粘连蛋白 从细胞培养中纯化替纳丝蛋白期间确定 上清液。这种新蛋白质似乎具有一种变体链，一个独特的 组织分布，细胞粘附活性与 任何已知的层粘连蛋白。最后，我们建议扩大我们的协作 关于Tenascin X射线晶体学的项目。我们的方法是 使用我们的PCR方法一次结晶一个域 精确定义的细菌表达蛋白。两个FN-III域 到目前 给出了出色的晶体和高分辨率X射线衍射 研究正在进行中。现在提出了其他域名 研究。整个六边形的原子分辨率结构是 可行的目标。