Regulation of EGFR Signaling by the Endocytic Pathway

项目摘要 我们实验室的长期目标是了解细胞表面受体的信号转导是如何 监管.最终，我们希望开发出选择性激活或抑制这些细胞的策略， 活性和绕过受体的限制，如受体数量少或受体脱敏。 每个细胞外配体（生长因子、激素、神经递质等）都与独特的细胞表面结合 一种诱导细胞内生化变化的受体，这些变化被整合以引起细胞内的特定变化 physiology.虽然该系统的精确特异性早已得到赞赏，但其分子机制 它是如何发生的，我们知之甚少。了解一组重叠的生化反应 产生一种特定的生理机能是这个问题的关键。为了更好地理解这一重要过程，我们 使用原型受体酪氨酸激酶，表皮生长因子受体（EGFR）作为模型。的 EGFR对许多发育和稳态过程至关重要; EGFR的刺激导致 多种细胞变化，包括细胞增殖、分化、迁移和活力。此外，本发明还 EGFR的过表达与许多癌症有关。 我们和其他人认为，这些生化中间体信号的强度和持续时间 决定了细胞生理学的改变调节受体信号传导的幅度和持续时间的一种方式是 是通过配体结合后受体的内化和降解。除了激活 在细胞内信号传导途径中，配体结合也导致大多数细胞表面受体通过 网格蛋白包被的凹坑一旦进入细胞，配体：受体复合物通过一系列明确的 内吞阶段，直到其最终到达溶酶体，在那里其经历降解。已经表明 先前许多研究小组认为，破坏这一过程可以改变激活的EGFR对 牢房然而，这些研究仅限于区分细胞表面和细胞内 受体。我们研究的首要假设是，内吞途径是一个关键的积极和积极的影响。 细胞表面受体信号传导的负调节剂。 在目标I中，我们将使用培养的细胞并通过内吞途径选择性地破坏EGFR的运输。 我们将评估EGFR信号在这些内吞阶段的每个阶段。此外，我们将确定是否 信号传导的差异是由于受体本身、受体：效应物相互作用或 信号的持续时间/幅度。目的二，我们将建立在我们最近的研究结果，空间限制EGFR 在癌细胞（MDA-MB-468乳腺癌细胞）中， 特性.我们将建立在这个模型，以确定哪些信号从细胞表面发出，哪些是 在细胞内产生。最后，在目标III中，我们将探讨内吞途径如何负调节 EGFR信号传导。在这些研究中，我们将确定信号失活的机制，并确定它是否 对于受体或效应物独特的蛋白质以相同的方式失活。 这些研究的完成将揭示EGFR信号是如何在受体下游调节的。这 将指导更多靶向治疗的产生，以抑制EGFR信号传导（即癌症）或激活 EGFR信号传导（即角膜伤口愈合）。

Project Summary The long-term goal of our laboratory is understand how signaling transduction by cell surface receptors is regulated. Ultimately, we would like to develop strategies for selectively activating or inhibiting these cellular activities and bypass limitations of the receptor, such as low receptor number or receptor desensitization. Every extracellular ligand (growth factor, hormone, neurotransmitter, etc) binds to a unique cell surface receptor that induces intracellular, biochemical changes that are integrated to invoke a specific change in cell physiology. While the exquisite specificity of this system has been long appreciated, the molecular mechanism by which it occurs is poorly understood. Understanding how an overlapping set of biochemical responses produces a specific physiology is the key to this problem. To better understand this important process, we are using the prototypical receptor tyrosine kinase, the epidermal growth factor receptor (EGFR), as a model. The EGFR is critical for many developmental and homeostatic processes; stimulation of the EGFR leads to a variety of cellular changes including cell proliferation, differentiation, migration, and viability. Further, overexpression of the EGFR is associated with many cancers. We, and others, believe that the magnitude and duration of signaling to these biochemical intermediates dictates how cell physiology is altered. One way the magnitude and duration of receptor signaling is modulated is through the internalization and degradation of the receptor following ligand binding. In addition to activating intracellular signaling pathways, ligand binding also causes most cells surface receptors to internalize via clathrin-coated pits. Once inside the cell, the ligand:receptor complex moves through a series of well-defined endocytic stages until it ultimately reaches the lysosome where it undergoes degradation. It has been shown previously by a number of groups that disrupting this process can alter the effect that activated EGFRs have on the cell. However, these studies have been limited to distinguishing between cell surface and intracellular receptors. The overarching hypothesis of our research is that the endocytic pathway is a key positive and negative regulator of cell surface receptor signaling. In Aim I, we will use cultured cells and selectively disrupt EGFR trafficking through the endocytic pathway. We will assess EGFR signaling at each of these endocytic stages. Further, we will determine whether differences in signaling occur due to changes in the receptor itself, receptor:effector interactions, or the duration/magnitude of signaling. Aim II, we will build on our recent findings that spatially restricting the EGFR in cancer cells (MDA-MB-468 mammary adenocarcinoma cells) dramatically changes cell growth and viability properties. We will build on this model to determine which signals emanate from the cell surface and which are produced within the cell. Finally, in Aim III, we will explore how the endocytic pathway negatively regulates EGFR signaling. In these studies, we will determine the mechanism of signal inactivation and determine if it is unique for the receptor or effectors are inactivated in this same manner. Completion of these studies will reveal how EGFR signaling is regulated downstream of the receptor. This will guide the production of more targeted therapies to inhibit EGFR signaling (i.e. cancer) or activation of EGFR signaling (i.e. corneal wound healing).