CELL ADHESION FUNCTIONS & CLONING OF VLA PROTEINS

细胞粘附功能

• 批准号: 3295658
• 负责人: MARTIN E HEMLER
• 金额: $ 14.91万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-08-01 至 1992-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3295658
• 关键词: cell adhesion; extracellular matrix; fibronectins; glycoproteins; laminin; liposomes; membrane proteins; molecular cloning; monoclonal antibody; nucleic acid probes; receptor

The long term goals of this research are to 1) characterize each member of the VLA family of five heterodimers with regard to cell matrix adhesion functions and 2) obtain genetic sequence information essential for the long term understanding of VLA proteins. Already it is known that antisera to the VLA Beta subunit (common to all VLA structures) blocks cell adhesion to fibronectin and laminan, and two VLA structures (VLA-3 nd VLA- 5) resemble known receptors for fibronectin. The availability of both a panel of monoclonal antibodies recognizing different VLA subunits, and an abundant supply of purified VLA proteins now makes it feasible to carry out the following specific aims. (1) The matrix-adherence capabilities for each VLA heterodimer will be determined against a panel of cell matric components (e.g. fibronectin, laminan, collagens). Native VLA structures on whole cells, and purified VLA in liposomes will be tested for adherence, and results will be confirmed using specific anti-VLA antibodies in blocking and cell surface modulation studies. (2) To determine the importance of arg-gly-asp (R-G-D) recognition in VLA- mediated adhesion, a) the inhibitory capacities or R-G-D- containing peptides will be tested, b) anti-alpha and anti-beta sera as well as alpha and/or beta liposomes will be used to evaluate the relative contributions of VLA subunits to R-G-D recognition, and c) peptides containing the R-G-D sequence will be specifically crosslinked to VLA proteins to identify the alpha or beta subunit(s) involved in R-G-D recognition. (3) To obtain basic structural information, genes for VLA subunits will be cloned and sequenced. Antisera to purified alpha subunits will be used to screen lamda gt11 cDNA libraries, and if necessary, probes based on known protein sequence will be used to screen lambda gt10 cDNA libraries. This information will a) allow detailed comparisons of VLA subunit structures and b) allow further comparisons within a broader "super family" of more distantly related cell surface recognition structures such as LFA-1 and Mac-1 proteins. Specific cell adhesion to extracellular matrix proteins is a phenomenon of fundamental importance to tissue organization, cell migration, embryogenesis, neoplastic transformation and tumor cell metastasis. Thus structural information from detailed biochemical analyses of the VLA protein family now can provide a significant framework for organizing, comparing, and understanding the many diverse cell surface glycoproteins which have been implicated in cell matrix adhesion.

这项研究的长期目标是1)描述每一种 VLA家族的五个杂二聚体中的一员 细胞基质黏附函数和2)获得遗传序列 对长期了解VLA至关重要的信息 蛋白质。已知VLA Beta的抗血清 亚基(对所有VLA结构通用)阻止细胞与 纤维连接蛋白和层粘连蛋白，以及两种VLA结构(VLA-3和VLA-3)。 5)与已知的纤维连接蛋白受体相似。是否可以使用 都是一组识别不同VLA的单抗 亚基，以及现在大量纯化的VLA蛋白 使实现以下具体目标成为可能。(1) 每个VLA异二聚体的基质附着能力将是 根据一组细胞矩阵成分(例如 纤维连接蛋白、层粘连蛋白、胶原蛋白)。整体上自然的VLA结构 细胞和脂质体中纯化的VLA将进行粘附性测试， 结果将使用特定的抗VLA抗体在 阻断和细胞表面调制研究。(二)确定 精氨酸-甘氨酸-天冬氨酸(R-G-D)识别在VLA中的重要性 中介黏附，a)抑制能力或R-G-D- 将检测含有多肽，b)抗-α和抗-β血清 以及阿尔法和/或贝塔脂质体将用于评估 VLA亚基对R-G-D识别的相对贡献，以及 C)含有R-G-D序列的多肽将被特异性地 与VLA蛋白交联以识别α或β 参与R-G-D识别的亚单位(S)。(3)取得基本的 结构信息，VLA亚基的基因将被克隆和 已排序。纯化的阿尔法亚基的抗血清将用于 筛选Lamda gt11 cdna文库，如有必要，还可基于探针 已知的蛋白质序列将用于筛选lambda gt10 文库的构建。此信息将a)允许详细说明 VLA亚基结构和b)的比较允许进一步 在一个更广泛的“超级家庭”中进行比较 相关的细胞表面识别结构，如LFA-1和 Mac-1蛋白。 细胞对细胞外基质蛋白的特异性黏附是一种 对组织组织至关重要的现象， 细胞迁移、胚胎发生、肿瘤转化和 肿瘤细胞转移。因此，来自详细信息的结构信息 对VLA蛋白家族的生化分析现在可以提供 组织、比较和管理的重要框架 了解多种不同的细胞表面糖蛋白 与细胞基质黏附有关。