ENDOCAM/PECAM--A NOVEL VASCULAR CELL ADHESION MOLECULE

ENDOCAM/PECAM--一种新型血管细胞粘附分子

• 批准号: 2222803
• 负责人: Steven Mark Albelda
• 金额: $ 25.47万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2222803
• 关键词: cell cell interaction; cell migration; epitope mapping; flow cytometry; human tissue; immunoglobulins; inflammation; laboratory mouse; leukocyte adhesion molecules; mutant; platelets; point mutation; protein isoforms; protein structure function; transfection; vascular endothelium

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.

PECAM-1（CD 31，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， 血管生成、白细胞迁移和白细胞活化。目标 该方案的主要目的是研究PECAM-1功能的分子基础。 初步数据表明PECAM-1作为粘附分子发挥作用 能够介导嗜异性（PECAM-至未知的反- 受体）或嗜同性（PECAM对PECAM）结合，这取决于 胞质结构域的结构。多重选择性剪接 最近已经鉴定了鼠PECAM-1的同种型， 胞质结构域中外显子14的存在调节配体 的特异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同种型的生物学意义， 建议的具体目标如下： 1.为了精确定位细胞质区域， 调节PECAM-1在嗜同性或嗜异性细胞中结合的能力 的方式和定义的机制，细胞质域 调节PECAM-1功能。这将通过建设和 具有缺失和点突变的PECAM-1突变体的表征 基于天然存在的异构体的结果， 更精确地定位“调节”区域， 细胞质结构域，探索细胞质结构域的机制， 结构域调节PECAM-1功能和嵌合构建体的用途 含有PECAM-1胞质结构域的各种同种型 通过腺病毒载体转移到内皮细胞。 2.为了确定细胞外结构域的区域， 嗜异性或嗜同性结合。这将通过映射 一系列具有生物活性的鼠特异性抗PECAM-1抗体的表位 抗体，含有特异性点的PECAM-1构建体的评价 通过表位作图和检测 肽的能力，模拟推定的功能区的 细胞外结构域阻断嗜同性和嗜异性PECAM-1- 介导的聚集。 3.为了确定多个PECAM-1的生物学意义， 同种型。 这将通过评估模拟多肽的肽来实现。 细胞外结构域的推定功能区， 阻断人类白细胞通过内皮细胞的迁移 单层，测试具有确定体外功能的肽的能力 阻止白细胞迁移到巯基乙酸盐刺激的 腹膜，并通过 muPECAM-1的体外和体内表达状态的测定 同种型。 这些研究将使人们更好地了解 细胞-细胞粘附和结构/功能的机制 PECAM-I的关系，因此最终可能在设计中具有价值 治疗炎症和恶性疾病。