Coreceptor Modification of TCR Tyrosine Kinase Signals

描述（由申请人提供）：脂筏膜区隔化和maguk家族分子支架是神经元和上皮细胞连接中蛋白质和膜运输、细胞骨架重组和信号转导的关键组织者。在这里，我们考虑Dlgh1 MAGUK家族成员在T细胞APC突触连接组织细胞骨架、脂筏和信号转导中的潜在作用。在过去的资助周期中，我们证明了LckSHS结构域在介导TCR/共刺激诱导中的需求：突触筏聚类；持续的信号传导；缩短了TCR业务所需的持续时间；Erk激活;和IL-2的产生。我们之所以对Dlgh1作为潜在的LckSH3效应物产生兴趣，是因为我们将其鉴定为LckSH3配体，并证明LckSH3: Dlgh1相互作用对于Dlgh1筏微域膜定位至关重要。此外，我们最近发现了WASp， Erk-1和p38作为额外的Dlgh1配体，并表明它们可能作为Dlgh1 （rLck）效应体起作用。在本文中，我们设计了旨在评估Dlgh1在TCR/共刺激剂诱导的信号转导中的潜在作用并阐明Dlgh1活性的分子基础的实验。此外，我们考虑了不同的T细胞亚群不同地利用Dlgh1活性来产生独特适合于影响特定功能的突触的可能性。为了解决这些问题，我们采用了三管齐下的方法，包括siRNA介导的Dlgh1敲除、Dlgh1过表达/再表达和Dlgh1基因敲除。我们分析了Dlgh1在BI-141 T杂交瘤、CD4+ 5CC7和CD8+ OT-1 TCR转基因T细胞和发展中的T细胞亚群中的活性。我们预测，直接比较不同发育群体和T效应群体中的Dlgh1支架将阐明新的TCR信号转导机制和特化突触中涉及的分子细节。

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.