Chemical biology tools for studying growth factor receptor internalization

项目摘要 癌症进展部分受生长因子及其细胞内信号网络调节。健康细胞 通过内化和随后的生长因子再循环或破坏来控制生长因子信号传导 受体以配体依赖的方式。最近，我们发现小分子脂质磷脂酰肌醇 3，4，5-三磷酸（PIP3）在缺乏生长因子受体的情况下提供了足够的信号来内化生长因子受体。 配体。PIP3是已知的第一个特异性诱导生长受体内化的小分子之一 其对治疗癌症有意义。在这里，我们将阐明PIP3如何引起 表皮生长因子受体（EGFR）的内化。我们将使用各种独特的化学生物学工具 来获得一些假设的机械答案。第一个假设是PIP3直接结合于 EGFR并诱导内吞作用。因此，在目标1中，我们将合成一种膜渗透的、笼状的、光- PIP3的可交联和可点击的衍生物。我们实验室过去已经制备了类似的PIP3衍生物 并合成了几个关键的组成部分。PIP3衍生物将通过以下途径递送至细胞： 可生物活化的基团，通过光释放以诱导结合，然后光交联至任何结合蛋白。 质谱法将证明生物活性脂质物质的细胞内产生。经由点击 化学亲和探针，我们将提取脂质-蛋白质结合物，并进行蛋白质组学分析与 关注已知的生长因子受体。第二个假设是受体与效应物结合 通过网格蛋白包被的小凹使其作为内吞作用的货物的蛋白质。我们将准备一些截短的 荧光标记的突变体，以鉴定内吞作用所需的受体的最小细胞内表位 (Aim 2）。在缺乏配体和酪氨酸磷酸化的情况下，我们将重点关注丝氨酸和苏氨酸残基， 可能作为蛋白质结合的锚定点。为了避免荧光标记物干扰 内吞机制，我们将使用遗传密码扩展，以最小限度地引入快速反应氨基酸， 侵入性标记。初步的数据证明了这项技术的可行性，以遵循受体内化 通过共聚焦显微镜因此，第三种假设是，特定的Ser和Thr残基是 由MAP激酶p38磷酸化。我们将在体外证明EGFR磷酸化及其抑制 和细胞中。为了证明功能相关性，我们将准备一个p38结构，它可以通过以下方式被打开： 加入一种小分子（化学二聚剂），使酶转移到质膜上（目标3）。 一旦成功，我们将使用类似的结构来转移我们已经通过RNAi鉴定的蛋白质， 筛选为PIP3诱导的内吞作用所必需的，例如PAR3和PAR6。作为读数，我们将使用荧光 来自Aim 2的标记受体变体。综合结果将有助于更好地了解受体 在缺乏配体的情况下的内化机制。必须考虑到这一过程所必需的任何蛋白质 一个新的治疗的主要目标，以减少细胞表面生长因子受体水平和癌症的进展。

Project Summary Cancer progression is partly regulated by growth factors and their intracellular signaling networks. Healthy cells control growth factor signaling by internalization and subsequent recycling or destruction of the growth factor receptor in a ligand-dependent fashion. Recently, we found that the small molecule lipid phosphatidylinositol 3,4,5-trisphosphate (PIP3) presents a sufficient signal to internalize growth factor receptors in the absence of a ligand. PIP3 is one of the very first small molecules known to induce growth receptor internalization specifically which is of interest for treating cancer. Here, we will elucidate the mechanism of how PIP3 causes the internalization of epidermal growth factor receptor (EGFR). We will use a variety of unique chemical biology tools to acquire mechanistic answers to a number of hypotheses. The first hypothesis is that PIP3 binds directly to EGFR and induces endocytosis. In Aim 1, we will therefore synthesize a membrane-permeant, caged, photo- crosslinkable and clickable derivative of PIP3. Our lab has already prepared similar PIP3 derivatives in the past and has synthesized several of the key building blocks. The PIP3 derivative will be delivered to cells via bioactivatable groups, uncaged by light to induce binding and then photo-crosslinked to any binding protein. Mass spectrometry will demonstrate the intracellular generation of biologically active lipid species. Via click chemistry to affinity probes, we will extract the lipid-protein conjugates and perform proteomic analysis with a focus on known growth factor receptors. The second hypothesis is that the receptor is binding to an effector protein that primes it as cargo for endocytosis via clathrin-coated pits. We will prepare a number of truncated fluorescently labeled mutants to identify the minimal intracellular epitope of the receptor required for endocytosis (Aim 2). In the absence of a ligand and tyrosine phosphorylation, we will focus on Ser and Thr residues that might serve as anchoring points for protein binding. In order to avoid interference of the fluorescent label with the endocytic machinery, we will use genetic code expansion to introduce fast reacting amino acids for minimally invasive labeling. Preliminary data demonstrated the feasibility of this technique to follow receptor internalization by confocal microscopy. Therefore, the third hypothesis is that specific Ser and Thr residues are phosphorylated by the MAP kinase p38. We will demonstrate EGFR phosphorylation and its inhibition in vitro and in cells. To demonstrate functional relevance, we will prepare a p38 construct that can be switched on by adding a small molecule (a chemical dimerizer) that translocates the enzyme to the plasma membrane (Aim 3). Once successful, we will use similar constructs to translocate proteins that we already identified by an RNAi screen as essential for PIP3-induced endocytosis, e.g. PAR3 & PAR6. As a readout, we will use the fluorescently labeled receptor variants from Aim 2 in live cells. The combined results will help to better understand receptor internalization mechanisms in the absence of a ligand. Any protein essential for this process must be considered a prime target for novel therapeutics to reduce cell surface growth factor receptor levels and cancer progression.