Coreceptor Modification of TCR Tyrosine Kinase Signals

脂筏膜区域化和Maguk家族分子支架作为关键的组织者 神经元和上皮细胞的蛋白质和膜转运、细胞骨架重组和信号转导 交汇点。在这里，我们考虑了Dlghl Maguk家族成员在组织细胞骨架，脂质中的潜在作用 T细胞的筏子和信号转导：APC突触连接。在过去的融资周期中，我们展示了 Lck SHS结构域在介导TCR/共刺激诱导中的需求：突触RAFT聚集；进行性和 持续的信号传递；减少TCR参与所需的时间；ERK激活；以及IL-2的产生。我们成了 对Dlghl作为潜在的Lck SH3效应器感兴趣，因为我们确定它是Lck SH3配体并证明了 LockSH3：Dlghl相互作用是Dlghl RAFT微域膜定位所必需的。此外，我们有 最近发现黄蜂、Erk-1和p38是额外的Dlghl配体，并提示它们可能是Dlghl(RLCK) 效应器。在这项建议中，我们设计了旨在评估Dlghl在TCR/共刺激分子中的潜在作用的实验 诱导信号转导，阐明Dlghl活性的分子基础。此外，我们认为 不同的T细胞亚群不同地利用Dlghl活动来产生唯一适合的突触的可能性 用于实现特定的功能。为了解决这些问题，我们利用了一种涉及siRNA的三管齐下的方法 介导的Dlghl基因敲除、Dlghl过表达/再表达和dlghl基因敲除。我们包括以下分析 BI-141T杂交瘤、CD4+5CC7和CD8+OT-1TCR转基因T细胞及发育中T细胞的DlghL活性 子集。我们预测DLGL支架在不同发育阶段和T细胞效应人群中的直接比较 将阐明新的TCR信号转导机制和涉及突触专门化的分子细节。 具体地说，我们提出如下建议：1)研究Dlghl在抗原诱导的T细胞中的潜在作用 信号转导、免疫突触组装和效应器功能；目的2)确定 目标3)确定发育和效应T细胞(差异)是否依赖于 Dlghl脚手架活动。 我们的研究可能会使我们更好地理解TCR信号是如何调节调节功能的 结果，这对于旨在可预测地调节特定TCR反应的疗法的设计是必不可少的。 事实上，阐明突触组织和信号转导事件的分子介体将提供新的 旨在抑制不必要的T细胞激活反应的治疗靶点，包括自身免疫、移植物 排斥反应和T细胞转化。相反，可以在设计中使用单个激活事件的促进者 用于肿瘤或其他疫苗，旨在增强对次优呈现的抗原的反应。

Lipid raft membrane compartmentalization and MAGUK-family molecular scaffolds function as key organizers of protein and membrane trafficking, cytoskeletal reorganization and signal transduction in neuronal and epithelial cell junctions. Here we consider a potential role for the Dlghl MAGUK family member in organizing the cytoskeleton, lipid rafts and signal transducers at the T cell:APC synapticjunction. During the past funding cycle we demonstrated a requirement for the LckSHS domain in mediating TCR/costimulation induced: synaptic raft clustering; processive and sustained signaling; reduced required duration of TCR engagement; Erk activation; and IL-2 production. We became interested in Dlghl as a potential LckSH3 effector because we identified it as a LckSH3 ligand and demonstrated LckSH3:Dlghl interactions as essential for Dlghl raft microdomain membrane localization. Moreover, we have recently identified WASp, Erk-1 and p38 as additional Dlghl ligands and suggest they may function as Dlghl(rLck) effectors. In this proposal, we design experiments aimed at assessing a potential role for Dlghl in TCR/costimulator induced signal transduction and elucidating the molecular basis of Dlghl activity. Furthermore, we consider the possibility that distinct T cell subsets differentially capitalize on Dlghl activities to generate synapses uniquely suited for effecting particular functions. To address these issues we capitalize on a three pronged approach involving siRNA mediated Dlghl knockdown, Dlghl over-expression/re-expression and dlghl gene knockout. We include analysisof Dlghl activity in the BI-141 T hybridoma, CD4+ 5CC7 and CD8+ OT-1 TCR transgenic T cells and in developing T cell subsets. We predict that direct comparison of Dlghl scaffolds withindistinct developing and T effector populations will elucidate novel TCR signal transduction mechanisms and molecular details involved in specializing synapses. Specifically, we propose the following: Arm 1) To investigate potential roles for Dlghl in antigen-induced T cell signal transduction, immune synapse assembly, and effector function; Aim 2) To determine the molecular basis of Dlghl activity in T cells; and Aim 3) To determine whether developing and effector T cells (differentially)rely on Dlghl scaffolding activities. Our studies will likely lead to a better understanding of how TCR signals are regulated to mediate functional outcome, which is essential to the design of therapeutics aimed at predictably modulating particular TCR responses. Indeed, elucidation of molecular mediators of synaptic organization and signal transduction events will provide novel targets for therapeutics aimed at inhibiting unwanted T cell activation responses including autoimmunity, graft rejection, and T cell transformation. Conversely, facilitators of individual activation events could be used in the design of tumor or other vaccines aimed at potentiating responses against suboptimally presented antigens.