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.

继续的研究将主要集中在腱生蛋白-巨人 肿瘤细胞外基质中的六臂分子，愈合 伤口和特定的胚胎组织。我们已经在制造转基因的 过度表达腱生蛋白的小鼠。我们建议让其他人 六臂体装配缺陷（单臂体小鼠）， 腱生蛋白分泌的耗竭。我们将确定 胚胎发育过程中的过表达、低表达或缺陷性六臂体 发育、肿瘤和伤口愈合。第二个项目是寻找 果蝇和C.优雅的。这些物种具有很高的 开发了基因图谱和大量的突变体，提供了一个强大的 这是一种了解tenascin功能的新方法。我们将继续 我们对腱生蛋白的细胞生物学和生物化学的研究， 并表征不同结构域的细胞表面受体， 腱生蛋白该项目的一个关键工具是细菌文库， 我们最近开发的表达蛋白， 数量的定义段的六臂。我们将使用一个 类似的方法来鉴定与腱生蛋白结合的ECM分子。另一 一个持续的项目是描述一种新形式的层粘连蛋白， 在从细胞培养物中纯化腱生蛋白的过程中鉴定 上清液。这种新蛋白质似乎有一个独特的A链变体 组织分布和细胞粘附活性与 任何已知的层粘连蛋白最后，我们建议扩大我们的合作， 腱生蛋白的X射线晶体学项目。我们的做法是 每次结晶一个结构域，使用我们的PCR方法， 精确定义的细菌表达蛋白。两个FN-III结构域 到目前为止，我们已经尝试过，来自腱生蛋白和纤连蛋白的RGD结构域， 具有优异的晶体和高分辨率的X射线衍射 研究正在进行中。现在提出了未来的其他领域 问题研究整个hexabrachion的原子分辨率结构是 可行的目标。