ENDOCAM/PECAM--A NOVEL VASCULAR CELL ADHESION MOLECULE

PECAM-1(CD31，Endocam)是免疫球蛋白超家族的成员 存在于内皮细胞、白细胞和血小板上，与 血管生成、白细胞迁移和白细胞激活。目标是 这一建议的目的是研究PECAM-1功能的分子基础。 初步数据表明，PECAM-I具有黏附分子的功能 能够调节异嗜性(PECAM-到未知的反- 受体)或亲水性(PECAM到PECAM)结合取决于 细胞质结构域的结构。多个可选拼接 最近发现了小鼠PECAM-1的异构体，并似乎 胞质结构域中外显子14的存在调节配基 专一性。为了了解这种独特性质的分子基础， 为了确定选择性剪接异构体的生物学意义， 提出了以下具体目标： 1.精确定位胞质结构域的区域 调节PECAM-1结合同嗜性或异嗜性的能力 并定义细胞质结构域的机制 调节PECAM-1功能。这将通过建设和 带有缺失和点突变的PECAM-1突变体的特性 根据自然产生的异构体的结果， 更精确地本地化监管区域(S) 细胞质结构域，探索细胞质 结构域调控PECAM-1的功能和嵌合结构的使用 含有PECAM-1胞浆结构域的各种异构体 通过腺病毒载体转导内皮细胞。 2.确定负责细胞外域的区域 亲异或亲同的结合。这将通过映射来实现 一系列具有生物活性的小鼠特异性抗PECAM-1表位的研究 抗体，含有特异点的PECAM-1构建体的评价 通过表位映射确定的关键区域的突变，以及通过检测 模拟可能的功能区域的多肽的能力 阻断同嗜性和异嗜性PECAM-1的胞外区- 中介聚合。 3.确定多个PECAM-1的生物学意义 异构体。这将通过评估模拟的多肽来实现 胞外结构域的假定功能区域 阻止人类白细胞通过内皮细胞的迁移 单层，测试具有明确体外功能的多肽的能力 阻断刺激下白细胞向硫代乙醇酸盐的迁移 小鼠体内炎症模型中的腹膜，并通过 小鼠MUPECAM-L体内外表达状况的测定 异构体。 这些研究将使我们更好地了解 细胞间黏附和结构/功能的机制 因此，PECAM-I和的关系最终可能在设计中具有价值 炎症性和恶性疾病的治疗。

PECAM-1 (CD31, endoCAM) is a member of the immunoglobulin superfamily present on endothelial cells, leukocytes and platelets implicated in angiogenesis, leukocyte transmigration, and leukocyte activation. The goal of this proposal is to study the molecular basis of PECAM-1 function. Preliminary data indicate that PECAM-I functions as an adhesion molecule capable of mediating either heterophilic (PECAM-to unknown counter- receptor) or homophilic (PECAM to PECAM) binding depending on the structure of the cytoplasmic domain. Multiple alternatively spliced isoforms of murine PECAM-1 have recently been identified and it appears that presence of exon 14 within the cytoplasmic domain regulates ligand specificity. To understand the molecular basis of this unique property and to define the biological significance of alternatively spliced isoforms, the following specific aims are proposed: 1. To precisely localize the regions of the cytoplasmic domain that regulate the ability of PECAM-1 to bind in a homophilic or heterophilic fashion and to define the mechanisms by which the cytoplasmic domain regulates PECAM-1 function. This will be accomplished by construction and characterization of PECAM-1 mutants with deletions and point mutations based on the results of the naturally occurring isoforms that will allow more precise localization of the "regulatory" region(s) with the cytoplasmic domain, exploration of mechanisms by which the cytoplasmic domain regulates PECAM-1 function, and use of chimeric constructs containing various isoforms of the cytoplasmic domain of PECAM-1 transferred by adenoviral vectors to endothelial cells. 2. To define the regions of the extracellular domain responsible for heterophilic or homophilic binding. This will be accomplished by mapping the epitopes of a series of bioactive murine specific anti -PECAM-1 antibodies, evaluation of PECAM-1 constructs containing specific point mutations in key regions identified by epitope mapping, and by testing the ability of peptides that mimic the putative functional regions of the extracellular domain to block homophilic and heterophilic PECAM-1- mediated aggregation. 3. To determine the biological significance of the multiple PECAM-1 isoforms. This will be accomplished by evaluating peptides that mimic the putative functional regions of the extracellular domain for their ability to block human leukocyte transmigration through an endothelial cell monolayer, testing the ability of peptides with defined in vitro functions to block leukocyte transmigration into thioglycollate stimulated peritoneum in an in vivo model of murine inflammation, and by determination of in vitro and in vivo expression status of muPECAM-l isoforms. These studies will provide both a better understanding of the basic mechanisms of cell-cell adhesion and of the structure/function relationships of PECAM-I and may thus ultimately be of value in designing therapies for inflammatory and malignant diseases.