BIOLOGY OF THE CELL ADHESION MOLECULE CD31

CD31是新近发现的一种跨膜蛋白，属于 免疫球蛋白基因家族的细胞表面黏附分子。CD31 表达仅限于血管内皮细胞、白细胞和血小板。 在血管内皮细胞中，CD31定位于细胞间 交汇点。我们已经获得了几行数据，表明CD31可能 在内皮和血管内皮细胞的急性炎症反应中发挥作用 在免疫反应中发生的初始黏附相互作用：1。 急性炎症对血小板或内皮细胞的刺激作用 组胺的凝血酶或T细胞激活等激动剂 植物血凝素导致CD31的即刻磷酸化 蛋白激酶C依赖的机制。2.T淋巴细胞活化后， CD31表达下调。3.CD31单抗LYP21， 抑制同种异体混合淋巴细胞中T淋巴细胞的活化 反应。这个项目的目标建立在我们早期对CD31的工作的基础上。 目标1：CD31的特性和功能后果 磷酸化。细胞后CD31磷酸化的化学计量学 激活将被确定。CD31氨基酸是 被细胞激活的磷酸化将通过掺入 在血小板激活过程中放射性标记的磷酸盐，随后是酸 水解层析和氨基酸层析。的功能后果 CD31的磷酸化将通过利用同型黏附来确定 模特。CD31蛋白将被提纯并涂覆在塑料上 磷酸化和非磷酸化形式。CD31基因转染的COS细胞 静息状态下检测CD31与纯化的CD31结合情况及PKC- 激活条件以确定：a)CD31结合是同型还是 异型和b)磷酸化对CD31结合的影响 亲和力。 目的#2：CD31粘附域的测定。CD31单抗 抗体LYP21干扰异基因混合淋巴细胞反应， 这表明这个表位可能是一个重要的功能域。 CD31分子。基于我们的克隆数据，我们已经初步定位了 可能的LYP21结构域到分子的23个氨基酸区域。这个 相关分子ICAM-1对二者黏附表位的定位 大多数远端结构域使得CD31的黏附功能也在 部分由结构域1和2的表位介导。合成CD31 基于LYP21表位的多肽及其第一和第二序列 预测的结构域是亲水性的、暴露的和可结合的 将合成相互作用并测试其对CD31结合的影响。 目的#3：CD31的生物学功能。从以下方面获得知识和试剂 上述目标将在两个模型系统中用于测试生物 CD31的功能。内皮通透性、内皮-白细胞黏附 并将使用培养的EC来研究白细胞的迁移 两室系统由多孔膜隔开。CD31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黏附。 反应。CD31的其他功能角色很可能会变成 随着人们对这种分子有了更多的了解，这是显而易见的。详细的说明 从CD31的结构-功能关系和生物学的角度 对表达的调控可能会给我们的理解带来重要的启示 发生在T细胞初始阶段的分子事件 激活和宿主对急性炎症刺激的反应。超越了 研究这种细胞黏附现象的内在科学兴趣， 要更详细地了解这些基本过程，请参阅 解决具有重大临床重要性的问题的潜力，包括 病毒性疾病、急、慢性炎症性疾病的治疗， 对产生免疫所需的粘连现象的剖析 恶性细胞的反应和血行转移。

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.