CD45 ISOFORMS AND T CELL ACTIVATION

描述(改编自申请人摘要)：CD45家族 跨膜蛋白酪氨酸磷酸酶(PTPase)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。然而，准确的 这一规定的性质，以及体内其他底物的特性 目前仍不确定。CD45由多种不同的亚型组成 在它们的胞外区域。这些数据以不同方式分布在 具有不同功能的T细胞亚群。有议员建议， CD45胞外区通过不同途径调节信号转导 细胞质结构域。然而，个体亚型的实际作用 目前仍不清楚。这样的研究由于之前的无能为力而受到阻碍 在同一细胞系中分别表达不同的CD45亚型。这个 首席研究员开发了一种独特的模型，内源性CD45 在Jurkat人T细胞白血病细胞系中的表达具有特异性 通过CD45反义基因的导入而被阻断。反义基因转染 然后，缺乏CD45的细胞被重组以唯一地表达 人类最大或最小的CD45亚型。申请人已找到 IL-2分泌和酪氨酸的异构体特异性差异 几个关键信号分子的磷酸化。这些发现表明 二者对TCR产生的信号通路的优先调控 异构体。使用这个模型系统，调查员试图扩展这些 CD45及其异构体在调节T细胞中的作用 细胞激活。在目标1中，额外的Jurkat衍生品，唯一 CD45(0)和CD45(ABC)亚型在克隆细胞中的表达 将创建特定于AG的TCR，以查看当前观测是否可以 在特定于银的系统中复制。单一的异构体转染体将是 在旨在确定单个CD45的作用的实验中进行比较 调节PTKs活性和磷酸化的异构体，特别是 关键底物和IL-2分泌。在目标2中，每个人的角色 激活信号和底物中的两个胞质CD45 PTPase结构域 体内招募将通过检测反义转染体来确定 用已定义的PTPase突变体重组。此外，潜在的作用是 CD45在信号传递中的酪氨酸磷酸化程度将用 特定的Tyr到Phe点突变。评估可能的差异 利用或调节这些PTPase结构域，这些突变体将被 在两种不同的CD45亚型背景下表达。在目标3中， 其余三种人CD45亚型在调节IL-2中的作用 产量、PTK活性和早期酪氨酸磷酸化事件将是 下定决心。

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.