Regulation of EGFR Signaling by the Endocytic Pathway

描述（由申请人提供）：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调节的。我们希望开发选择性激活或抑制这些细胞活性的策略，并绕过受体的限制（即受体数量少或受体脱敏）。细胞外配体（生长因子、激素、神经递质等）与独特的细胞表面受体结合，诱导细胞内的生物化学变化，这些变化被整合以引起细胞生理学的特定变化。虽然该系统的精确特异性早已被人们所认识，但其发生的分子机制却知之甚少。理解一组重叠的生化反应如何产生特定的生理学是解决这个问题的关键。为了理解这一过程，我们正在使用原型受体酪氨酸激酶，表皮生长因子受体（EGFR），作为一个模型。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。EGFR的过度表达与许多癌症相关。 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是如何改变的。发生这种情况的一种方式是通过配体结合后受体的内化和降解。除了激活细胞内信号传导途径外，配体结合还导致大多数细胞表面受体通过网格蛋白包被的小凹内化。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，直到它最终到达溶酶体，在那里它经历降解。先前已经表明，破坏EGFR内吞作用可以改变细胞的反应。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，内吞途径是细胞表面受体信号传导的关键正和负调节因子。 在目标1中，我们将选择性地破坏EGFR通过内吞途径的运输。我们将在几个内吞阶段评估EGFR信号传导，并确定信号传导的差异是否由于受体本身的变化、受体：效应物相互作用或信号传导的持续时间/幅度而发生。在目标2中，我们将基于我们最近的发现，即在空间上限制癌细胞（MDA-MB-468细胞）中的EGFR显著改变细胞生长和活力特性。我们将确定细胞内EGFR如何产生凋亡信号，以及改变其他细胞系中EGFR的运输和信号传导是否会导致它们发生EGFR介导的凋亡。最后，在目标3中，我们将探讨内吞途径如何负调控EGFR信号传导。在这些研究中，我们将确定信号失活的机制，并确定改变失活速率是否足以改变EGFR信号传导。 公共卫生相关性：表皮生长因子受体（EGFR）是一种基本的细胞表面蛋白，其通过检测细胞外生长因子的存在并将信息转化为细胞内的生化变化来发挥功能。EGFR的正常功能对于整个生物体的发育和体内平衡是必需的; EGFR的过表达/超活化与许多癌症相关。这项研究的直接目标是更好地了解调节EGFR生物学和病理学功能的分子机制，并将这些信息用于治疗与EGFR相关的疾病。

DESCRIPTION (provided by applicant): The long-term goal of our laboratory is to understand how signal transduction by cell surface receptors is regulated. We would like to develop strategies for selectively activating or inhibiting these cellular activities and bypass limitations of the receptor (i.e. low receptor number or receptor desensitization). Extracellular ligands (growth factor, hormone, neurotransmitter, etc.) bind to unique cell surface receptors that induce intracellular, biochemical changes that are integrated to invoke specific changes 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 understand this 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. Overexpression of the EGFR is associated with many cancers. The magnitude and duration of signaling to these biochemical intermediates dictates how cell physiology is altered. One way this occurs 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 that disrupting EGFR endocytosis can alter the response of 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 1, we will selectively disrupt EGFR trafficking through the endocytic pathway. We will assess EGFR signaling at several endocytic stages and determine whether differences in signaling occur due to changes in the receptor itself, receptor:effector interactions, or the duration/magnitude of signaling. In Aim 2 we will build on our recent findings that spatially restricting the EGFR in cancer cells (MDA-MB-468 cells) dramatically changes cell growth and viability properties. We will determine how the intracellular EGFRs produce apoptotic signals and whether changing the trafficking and signaling of EGFRs in other cell lines can cause them to undergo EGFR-mediated apoptosis. Finally, in Aim 3, 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 altering the rate of inactivation is sufficient to change EGFR signaling. PUBLIC HEALTH RELEVANCE: The epidermal growth factor receptor (EGFR) is a fundamental cell surface protein that functions by detecting the presence of growth factors outside the cell and converting the information into biochemical changes within the cell. Proper function of the EGFR is necessary for development and homeostasis of the whole organism; overexpression/hyperactivation of the EGFR is associated with many cancers. The immediate goal of this research is to better understand the molecular mechanisms that regulate the biological and pathological functions of the EGFR and use that information for treatment of diseases associated with the EGFR.