CELL ADHESION FUNCTIONS & CLONING OF VLA PROTEINS

本研究的长期目标是：1）描述每种 VLA家族的五个异源二聚体的成员， 细胞基质粘附功能; 2）获得基因序列 长期了解VLA所必需的信息 proteins. 已经知道VLA β的抗血清 亚基（所有VLA结构共有）阻断细胞粘附， 纤连蛋白和层粘连蛋白，以及两种VLA结构（VLA-3 nd VLA- 5)类似于已知的纤连蛋白受体。 的可用性 一组识别不同VLA的单克隆抗体 亚基，和丰富的供应，纯化的VLA蛋白，现在 使其能够实现以下具体目标。 (1)的 每个VLA异二聚体的基质粘附能力将是 根据一组细胞基质成分（例如， 纤连蛋白、层粘连蛋白、胶原蛋白）。 天然VLA结构整体 细胞，和脂质体中纯化的VLA将测试粘附性， 并将使用特异性抗VLA抗体确认结果， 阻断和细胞表面调节研究。 (2)以确定 arg-gly-asp（R-G-D）识别在VLA中的重要性， a）抑制能力或R-G-D- 将检测含有肽的血清，B）抗α和抗β血清 以及α和/或β脂质体将用于评价 VLA亚基对R-G-D识别的相对贡献，以及 c）含有R-G-D序列的肽将被特异性地 与VLA蛋白质交联以识别α或β 参与R-G-D识别的亚基。 (3)获得基本 结构信息，VLA亚基的基因将被克隆， 测序 纯化的α亚单位的抗血清将用于 筛选λ gt 11 cDNA文库，如果需要， 将使用已知蛋白质序列来筛选λ gt 10 cDNA文库。 该信息将：（a）详细 VLA亚基结构的比较和B）允许进一步 在更广泛的“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。 细胞与细胞外基质蛋白的特异性粘附是一种新的细胞粘附机制。 对组织结构至关重要的现象， 细胞迁移、胚胎发生、肿瘤转化和 肿瘤细胞转移 因此，从详细的结构信息 VLA蛋白家族的生化分析现在可以提供一个 组织、比较和 了解许多不同的细胞表面糖蛋白， 与细胞基质粘附有关。

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.