Coreceptor Modification of TCR Tyrosine Kinase Signals

描述（由申请人提供）：脂筏膜区室化和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，我们认为Dlgh 1 MAGUK家族成员在组织细胞骨架，脂筏和信号转导在T细胞：APC突触连接的潜在作用。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：突触筏聚集;进行性和持续性信号传导;减少TCR参与所需的持续时间; Erk激活;和IL-2产生。我们对Dlgh 1作为潜在的LckSH 3效应子产生了兴趣，因为我们将其鉴定为LckSH 3配体，并证明LckSH 3：Dlgh 1相互作用对Dlgh 1筏微结构域膜定位至关重要。此外，我们最近发现WASp，Erk-1和p38作为额外的Dlgh 1配体，并建议他们可能作为Dlgh 1（rLck）效应。在这个建议中，我们设计的实验旨在评估Dlgh 1在TCR/共刺激分子诱导的信号转导中的潜在作用，并阐明Dlgh 1活性的分子基础。此外，我们认为不同的T细胞亚群差异利用Dlgh 1活动产生突触的可能性，独特的适合影响特定的功能。为了解决这些问题，我们利用三管齐下的方法，包括siRNA介导的Dlgh 1敲低，Dlgh 1过表达/再表达和Dlgh 1基因敲除。我们包括BI-141 T杂交瘤、CD 4 + 5CC 7和CD 8 + OT-1 TCR转基因T细胞和发育中T细胞亚群中Dlgh 1活性的分析。我们预测，直接比较Dlgh 1支架内不同的发展和T效应人群将阐明新的TCR信号转导机制和分子细节参与专门的突触。 具体而言，我们提出了以下建议：1）研究Dlgh 1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Dlgh 1在T细胞中活性的分子基础; 3）确定发育中的T细胞和效应器T细胞是否（有差异地）依赖于Dlgh 1支架活性。 我们的研究可能会导致更好地理解TCR信号是如何调节以介导功能结果的，这对于设计旨在可预测地调节特定TCR反应的治疗方法至关重要。事实上，阐明突触组织和信号转导事件的分子介体将为旨在抑制不需要的T细胞活化应答（包括自身免疫、移植物排斥和T细胞转化）的治疗提供新的靶标。相反，个体活化事件的促进剂可用于设计肿瘤或其他疫苗，旨在增强针对次优呈递抗原的应答。

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.