Chemical biology tools for studying growth factor receptor internalization

用于研究生长因子受体内化的化学生物学工具

基本信息

批准号：
9894257
负责人：
Carsten Schultz
金额：
$ 5万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-20 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9894257
关键词：
Affinity Alkynes Amber Amino Acids Automobile Driving Back Binding Binding Proteins Biological Biology Cell Proliferation Cell Surface Receptors Cell membrane Cell surface Cells Cellular biology Chemicals Chemistry Chimera organism Chimeric Proteins Clathrin Communities Complex Confocal Microscopy Cytosol Data Diazomethane Diglycerides Dimerization Endocytosis Enzymes Epidermal Growth Factor Receptor Epitopes Esters Event Extracellular Domain Generations Genetic Code Growth Growth Factor Growth Factor Receptors Head Hela Cells In Vitro Label Learning Ligands Light Lighting Lipid Binding Lipids Location MAPK14 gene Malignant Neoplasms Mass Spectrum Analysis Measures Medicine Membrane Membrane Proteins Metabolism Monitor Nuclear Envelope PARD6A gene Pathway interactions Phosphatidylinositols Phosphorylation Physiology Process Proliferating Protein translocation Proteins Proteomics RNA interference screen Receptor Activation Recycling Role Signal Transduction Stimulus Techniques Testing Time Tyrosine Phosphorylation Ubiquitination Variant Work axon growth coated pit crosslink dimer interest minimally invasive mitochondrial membrane mitogen-activated protein kinase p38 mutant new therapeutic target overexpression phosphatidylinositol 3,4,5-triphosphate phosphatidylinositol 4-phosphate pointed protein prevent protein complex receptor receptor internalization receptor recycling small molecule tool tumor progression upstream kinase

项目摘要

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.

项目概要癌症进展部分受到生长因子及其细胞内信号网络的调节。健康细胞通过生长因子的内化和随后的回收或破坏来控制生长因子信号传导受体以配体依赖性方式。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足够的信号来内化生长因子受体配体。 PIP3 是最早已知能特异性诱导生长受体内化的小分子之一这对于治疗癌症很有意义。在这里，我们将阐明PIP3如何引起的机制表皮生长因子受体（EGFR）的内化。我们将使用各种独特的化学生物学工具获得许多假设的机械答案。第一个假设是 PIP3 直接结合 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性、笼状、光- PIP3 的可交联且可点击的衍生物。我们实验室过去已经制备了类似的PIP3衍生物并综合了几个关键的构建模块。 PIP3衍生物将通过以下途径递送至细胞：可生物激活的基团，通过光释放以诱导结合，然后与任何结合蛋白光交联。质谱分析将证明细胞内生物活性脂质的产生。通过点击从化学到亲和探针，我们将提取脂蛋白缀合物并使用关注已知的生长因子受体。第二个假设是受体与效应器结合蛋白质通过网格蛋白包被的凹坑将其作为内吞作用的货物进行启动。我们会准备一些截断的荧光标记突变体以识别内吞作用所需受体的最小细胞内表位（目标 2）。在没有配体和酪氨酸磷酸化的情况下，我们将重点关注 Ser 和 Thr 残基可能作为蛋白质结合的锚定点。为了避免荧光标记的干扰内吞机制，我们将使用遗传密码扩展来引入快速反应的氨基酸，以最小的速度侵入性标记。初步数据证明了该技术追踪受体内化的可行性通过共焦显微镜。因此，第三个假设是特定的 Ser 和 Thr 残基是被 MAP 激酶 p38 磷酸化。我们将在体外演示 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 构建体，可以通过以下方式打开它：添加一种小分子（化学二聚剂），将酶转移到质膜上（目标 3）。一旦成功，我们将使用类似的构建体来易位我们已经通过 RNAi 识别的蛋白质筛选对于 PIP3 诱导的内吞作用至关重要，例如PAR3 和 PAR6。作为读数，我们将使用荧光活细胞中 Aim 2 标记的受体变体。综合结果将有助于更好地了解受体在没有配体的情况下的内化机制。必须考虑此过程所必需的任何蛋白质降低细胞表面生长因子受体水平和癌症进展的新型疗法的主要目标。