Coreceptor Modification of TCR Tyrosine Kinase Signals

描述(申请人提供)：脂筏膜分区和Maguk家族分子支架在神经元和上皮细胞连接中作为蛋白质和膜运输、细胞骨架重组和信号转导的关键组织者发挥作用。在这里，我们考虑了Dlgh1 Maguk家族成员在组织T细胞：APC突触连接的细胞骨架、脂筏和信号转导中的潜在作用。在过去的资金周期中，我们证明了在介导TCR/共刺激诱导的过程中需要Lck SHS结构域：突触RAFT聚集；过程和持续的信号；TCR参与的所需时间缩短；ERK激活；以及IL-2的产生。我们之所以对Dlgh1作为潜在的Lck SH3效应器感兴趣，是因为我们确定了它是一个Lck SH3配体，并证明了Lck SH3：Dlgh1相互作用对于Dlgh1 RAFT微域膜的定位是必不可少的。此外，我们最近发现黄蜂、Erk-1和p38是额外的Dlgh1配体，并提示它们可能作为Dlgh1(RLCK)效应分子发挥作用。在这项建议中，我们设计了实验，旨在评估Dlgh1在TCR/共刺激分子诱导的信号转导中的潜在作用，并阐明Dlgh1活性的分子基础。此外，我们考虑了这样一种可能性，即不同的T细胞亚群不同地利用Dlgh1的活动来产生唯一适合于实现特定功能的突触。为了解决这些问题，我们利用了一种三管齐下的方法，涉及siRNA介导的Dlgh1基因敲除、Dlgh1过度表达/再表达和Dlgh1基因敲除。我们对BI-141T杂交瘤、CD4+5CC7和CD8+OT-1TCR转基因T细胞以及发育中的T细胞亚群中的Dlgh1活性进行了分析。我们预测，在不同的发育和T效应人群中直接比较Dlgh1支架将阐明新的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Dlgh1在抗原诱导的T细胞信号转导、免疫突触组装和效应器功能中的潜在作用；2)确定T细胞中Dlgh1活性的分子基础；3)确定发育中的T细胞和效应T细胞(不同地)是否依赖Dlgh1支架活动。 我们的研究可能会更好地理解TCR信号是如何调节调节功能结果的，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质将为旨在抑制不必要的T细胞激活反应(包括自身免疫、移植物排斥和T细胞转化)的治疗提供新的靶点。相反，单个激活事件的促进剂可以用于肿瘤或其他疫苗的设计，目的是增强对次优呈现抗原的反应。

DESCRIPTION (provided by applicant): 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 Dlgh1 MAGUK family member in organizing the cytoskeleton, lipid rafts and signal transducers at the T cell:APC synaptic junction. 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 Dlgh1 as a potential LckSH3 effector because we identified it as a LckSH3 ligand and demonstrated LckSH3: Dlgh1 interactions as essential for Dlgh1 raft microdomain membrane localization. Moreover, we have recently identified WASp, Erk-1 and p38 as additional Dlgh1 ligands and suggest they may function as Dlgh1 (rLck) effectors. In this proposal, we design experiments aimed at assessing a potential role for Dlgh1 in TCR/costimulator induced signal transduction and elucidating the molecular basis of Dlgh1 activity. Furthermore, we consider the possibility that distinct T cell subsets differentially capitalize on Dlgh1 activities to generate synapses uniquely suited for effecting particular functions. To address these issues we capitalize on a three pronged approach involving siRNA mediated Dlgh1 knockdown, Dlgh1 over-expression/re-expression and Dlgh1 gene knockout. We include analysis of Dlgh1 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 Dlgh1 scaffolds within distinct 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 Dlgh1 in antigen-induced T cell signal transduction, immune synapse assembly, and effector function; Aim 2) To determine the molecular basis of Dlgh1 activity in T cells; and Aim 3) To determine whether developing and effector T cells (differentially) rely on Dlgh1 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 the 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.