BIOLOGY OF THE CELL ADHESION MOLECULE CD31

CD 31是最近描述的一种跨膜蛋白，其属于 细胞表面粘附分子免疫球蛋白基因家族。 CD31 表达限于血管内皮、白细胞和血小板。 在血管内皮细胞中，CD 31定位于细胞间 交叉点 我们已经获得了几条数据线，表明CD 31可能 在内皮的急性炎症反应中起作用， 免疫应答中发生的初始粘附相互作用：1. 血小板或内皮细胞的刺激与急性炎症 激动剂如组胺的凝血酶或T细胞活化， 植物血凝素导致CD 31立即磷酸化， 蛋白激酶C依赖性机制。 2. T淋巴细胞活化后， CD 31表达下调。 3.一种CD 31单克隆抗体LYP 21， 抑制同种异体混合淋巴细胞中的T淋巴细胞活化 反应 这个项目的目标建立在我们早期对CD 31的工作基础上。 目的#1：CD 31的表征和功能结果 磷酸化 细胞凋亡后CD 31磷酸化的化学计量学 激活将被确定。 CD 31氨基酸是 将通过掺入 在血小板活化期间放射性标记的磷酸盐，随后是酸 水解和氨基酸层析。 的功能性后果 CD 31磷酸化将通过利用同型粘附试验测定。 模型 CD 31蛋白将被纯化并包被在塑料上， 磷酸化和非磷酸化形式。 转染CD 31的COS细胞 将测试cDNA在静息和PKC-β条件下与纯化的CD 31的结合。 a）CD 31结合是否是同型的或 异型和B）磷酸化对CD 31结合的影响 亲和力 目的#2：确定CD 31粘附结构域。 CD 31单克隆抗体 抗体LYP 21干扰同种异体混合淋巴细胞反应， 这表明该表位可能是一个功能上重要的结构域， CD 31分子。 根据我们的克隆数据，我们初步定位了 可能的LYP 21结构域与该分子的23个氨基酸区域。 的 相关分子ICAM-1的粘附表位定位到两个 大多数远端结构域使得CD 31粘附功能也在 至少部分由结构域1和2中的表位介导。 合成CD 31 基于LYP 21表位的肽和来自第一和第二表位的序列 预测为亲水的、暴露的和可用于结合的结构域 将合成相互作用并测试对CD 31结合的影响。 目的#3：CD 31的生物学功能。 从以下方面获得的知识和试剂 上述目标将在两个模型系统中使用，以测试生物 CD 31的功能 内皮通透性，内皮-白细胞粘附 和白细胞的迁移将使用培养的EC进行研究， 由多孔膜分隔的双室系统。 CD 31在T细胞中的作用 将使用同种异体混合淋巴细胞研究细胞粘附 反应 CD 31的其他功能可能会成为 随着对这种分子了解的加深， 详细说明 CD 31的生物学，就其结构-功能关系而言， 表达的调控可能会在我们理解 在T细胞的初始阶段发生的分子事件 激活和宿主对急性炎症刺激的反应。 超出 研究这种细胞粘附现象的固有科学兴趣， 对这些基本过程的更详细的了解， 解决具有重大临床意义的问题的潜力，包括 病毒性疾病、急性和慢性炎性疾病的治疗， 产生免疫所需的粘附现象的解剖 反应和恶性细胞的血行转移。

CD31 is a recently described trans-membrane protein which belongs to the immunoglobulin gene family of cell surface adhesion molecules. CD31 expression is restricted to vascular endothelium, leukocytes and platelets. In vascular endothelial cells, CD31 is localized to intercellular junctions. We have obtained several lines of data to suggest that CD31 may play a role in the acute inflammatory response of endothelium and in initial adhesion interactions occurring in the immune response: 1. Stimulation of platelets or endothelial ells with acute inflammatory agonists such as thrombin of histamine or T cell activation with phytohemagglutinin results in immediate phosphorylation of CD31 by a protein kinase C-dependent mechanism. 2. After T lymphocyte activation, CD31 expression is down-regulated. 3. A CD31 monoclonal antibody, LYP21, inhibits T lymphocyte activation in the allogeneic mixed lymphocyte reaction. The goals of this project build upon our earlier work with CD31. Aim #1: Characterization and functional consequences of CD31 phosphorylation. The stoichiometry of CD31 phosphorylation after cell activation will be determined. The CD31 amino acids which are phosphorylated with cell activation will be determined by incorporation of radiolabeled phosphate during platelet activation, followed by acid hydrolysis and amino acid chromatography. The functional consequences of CD31 phosphorylation will be determined by utilizing a homotypic adhesion model. CD31 protein will be purified and coated onto plastic in phosphorylated and unphosphorylated forms. COS cells transfected with CD31 cDNA will be tested for binding to purified CD31 under resting and PKC- activated conditions to determine: a) whether CD31 binding is homotypic or heterotypic and b) the effects of phosphorylation upon CD31 binding affinity. Aim #2: Determination of CD31 adhesion domains. The CD31 monoclonal antibody LYP21 interferes with the allogeneic mixed lymphocyte reaction, suggesting that this epitope may be a functionally important domain on the CD31 molecule. Based on our cloning data, we have tentatively localized the probable LYP21 domain to a 23 amino acid region of the molecule. The localization of adhesion epitopes of the related molecule ICAM-1 to the two most distal domains makes it logical that CD31 adhesive function is also at least in part mediated by epitopes in domains 1 and 2. Synthetic CD31 peptides based on the LYP21 epitope and sequences from the first and second domains predicted to be hydrophilic, exposed and available for binding interactions will be synthesized and tested for effects on CD31 binding. Aim #3: Biological function of CD31. Knowledge and reagents gained from the above aims will be used in two model systems to test the biological function of CD31. Endothelial permeability, endothelial-leukocyte adhesion and leukocyte trans-migration will be studied using cultured EC grown in a two chamber system separated by a porous membrane. The role of CD31 in T cell adhesion will be studied using the allogeneic mixed lymphocyte reaction. It is likely that other function roles of CD31 will become apparent as more is learned about this molecule. The detailed elucidation of the biology of CD31, in terms of its structure-function relationship and regulation of expression may shed important insights in our understanding of the molecular events which occur in the initial stages of T cell activation and the host response to acute inflammatory stimuli. Beyond the inherent scientific interest of studying such cellular adhesion phenomena, a more detailed understanding of these fundamental processes has the potential for addressing problems of great clinical importance, including the therapy of viral diseases, acute and chronic inflammatory diseases, dissection of adhesion phenomena required for generation of the immune response and hematogenous metastasis of malignant cells.