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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：
10251985
负责人：
Carsten Schultz
金额：
$ 30.8万
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/10251985
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 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 mutant new therapeutic target overexpression p38 Mitogen Activated Protein Kinase 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.

项目摘要肿瘤的进展部分受生长因子及其细胞内信号网络的调节。健康细胞通过内化和随后循环或破坏生长因子来控制生长因子信号转导受体以一种配基依赖的方式。最近，我们发现小分子脂质磷脂酰肌醇 3，4，5-三磷酸(PIP3)是生长因子受体内化的足够信号。莱兰德。PIP3是已知的最早的诱导生长受体内化的小分子之一。它对治疗癌症很有兴趣。在这里，我们将阐明PIP3如何导致表皮生长因子受体的内化。我们将使用各种独特的化学生物工具以获得对一些假说的机械性答案。第一个假设是PIP3直接与 EGFR并诱导内吞作用。因此，在目标1中，我们将合成一种薄膜--意指的、笼子里的、照片的-- PIP3的可交联和可点击的衍生品。我们的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构建块。PIP3衍生物将通过以下方式传递到细胞可生物激活的基团，通过光诱导结合，然后与任何结合蛋白进行光交联。质谱仪将显示细胞内生成具有生物活性的脂类。通过点击从化学到亲和探针，我们将提取脂质-蛋白质结合物，并用关注已知的生长因子受体。第二种假设是受体与效应器结合。一种蛋白质，通过笼蛋白包裹的凹坑将其作为货物进行内吞作用。我们将准备一些截断的荧光标记突变体鉴定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，我们将重点放在下列残基上：可能作为蛋白质结合的锚定点。为了避免荧光标签与在内吞机制中，我们将使用遗传密码扩展来引入快速反应的氨基酸，最低限度地侵入性标签。初步数据证实了该技术跟踪受体内化的可行性。通过共聚焦显微镜。因此，第三个假设是特定的丝氨酸和苏氨酸残基是被MAP激酶p38磷酸化。我们将在体外演示EGFR的磷酸化及其抑制作用以及在细胞中。为了演示功能相关性，我们将准备一个p38构造，该构造可以通过加入一个小分子(化学二聚体)，将酶转移到质膜上(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识别的蛋白质筛选对于PIP3诱导的内吞作用是必需的，例如PAR3和PAR6。作为读数，我们将使用荧光活细胞中来自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没有配体的情况下内化机制。任何对这个过程至关重要的蛋白质都必须被考虑降低细胞表面生长因子受体水平和癌症进展的新疗法的主要靶点。