CD45 ISOFORMS AND T CELL ACTIVATION

CD45 同工型和 T 细胞激活

• 批准号: 2672359
• 负责人: DAVID M ROTHSTEIN
• 金额: $ 20.75万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-30 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2672359
• 关键词: CD antigens; T cell receptor; T lymphocyte; biological signal transduction; clone cells; human tissue; laboratory mouse; leukocyte activation /transformation; phosphoprotein phosphatase; protein isoforms; transfection

DESCRIPTION (Adapted from the Applicant's Abstract): The CD45 family of transmembrane protein tyrosine phosphatases (PTPases) plays a key role in T cell activation by regulating the activity of critical protein tyrosine kinases (PTKs) and their phosphorylated substrates. However, the exact nature of this regulation, and the identity of additional in vivo substrates remains uncertain. CD45 is composed of multiple isoforms which differ only in their extracellular domains. These are differentially distributed on subsets of T cells having distinct functions. It has been proposed that the CD45 extracellular domains differentially regulate signaling through the cytoplasmic domain. However, the actual role of the individual isoforms remains unclear. Such studies have been hampered by the previous inability to individually express different CD45 isoforms in the same cell line. The principal investigator has developed a unique model whereby endogenous CD45 expression in the Jurkat human T cell leukemia line has been specifically blocked by transfection of a CD45-antisense gene. Antisense transfected cells lacking CD45 were then reconstituted to uniquely express either the largest or smallest human CD45 isoform. The applicant has found isoform-specific differences in IL-2 secretion and in the tyrosine phosphorylation of several key signaling molecules. These findings indicate preferential regulation of TCR-generated signaling pathways by these two isoforms. Using this model system, the investigator seeks to extend these findings and determine the role of CD45 and its isoforms in regulating T cell activation. In Aim 1, additional Jurkat derivatives, uniquely expressing the CD45(0) and CD45(ABC) isoforms in the context of a cloned Ag-specific TCR will be created, to see if the current observations can be reproduced in an Ag-specific system. Single isoform transfectants will be compared in experiments designed to determine the role of individual CD45 isoforms in regulating the activity and phosphorylation of PTKs, particular critical substrates, and IL-2 secretion. In Aim 2, the role of each of the two cytoplasmic CD45 PTPase domains in activation signaling and substrate recruitment in vivo will be determined by examining anti-sense transfectants reconstituted with defined PTPase mutants. In addition, the potential role of tyrosine phosphorylation of CD45 in signaling will be determined using specific Tyr to Phe point mutants. To evaluate possible differential utilization or regulation of these PTPase domains, these mutants will be expressed in the context of two different CD45 isoforms. In Aim 3, the function of the remaining three human CD45 isoforms in regulating IL-2 production, PTK activity, and early tyrosine phosphorylation events will be determined.

描述（改编自申请人的摘要）： 跨膜蛋白酪氨酸磷酸酶（PTPases）在T细胞中起关键作用， 通过调节关键蛋白酪氨酸的活性激活细胞 激酶（PTK）及其磷酸化底物。 但具体 这种调节的性质，以及其他体内底物的特性 仍然不确定。 CD 45由多种同种型组成，仅不同 在它们的细胞外结构域中。 这些差异分布在 具有不同功能的T细胞亚群。 已经提出 CD 45细胞外结构域通过细胞外信号通路差异调节信号传导。 胞质结构域 然而，单个同种型的实际作用 仍不清楚 这些研究由于以前无法 在同一细胞系中单独表达不同的CD 45亚型。 的 一位主要研究者开发了一种独特的模型， 在Jurkat人T细胞白血病细胞系中的表达已经被特异性地 通过转染CD 45反义基因阻断。 反义转染 然后重建缺乏CD 45的细胞，以独特地表达 最大或最小的人CD 45同种型。 申请人已经发现 IL-2分泌和酪氨酸蛋白酶的亚型特异性差异 几种关键信号分子的磷酸化。 这些发现表明 这两种细胞对TCR产生的信号通路的优先调节 同种型。 使用这个模型系统，研究人员试图扩展这些 研究结果并确定CD 45及其亚型在调节T细胞中的作用， 细胞激活 在目标1中，额外的Jurkat衍生物，唯一地 表达CD 45（0）和CD 45（ABC）同种型， 将创建Ag特异性TCR，以查看当前观察结果是否可以 在特定的Ag系统中复制。 单一同种型转染子将是 在旨在确定单个CD 45的作用的实验中进行比较， 在调节PTKs的活性和磷酸化方面， 关键底物和IL-2分泌。 在目标2中， 两个细胞质CD 45 PTB结构域在活化信号传导和底物 通过检查反义转染子来确定体内募集 用确定的PTSD突变体重构。 此外，潜在作用 CD 45在信号传导中的酪氨酸磷酸化将使用 特异性Tyr至Phe点突变体。 评估可能的差异 利用或调节这些PTK结构域，这些突变体将被 在两种不同的CD 45同种型的情况下表达。 在目标3中， 其余三种人CD 45同种型在调节IL-2中的功能 生产，PTK活性，和早期酪氨酸磷酸化事件将是 测定