Structural Plasticity and Functional Interactions of the Signaling Adapter NHERF

信号适配器 NHERF 的结构可塑性和功能相互作用

基本信息

批准号：
9176248
负责人：
HEINRICH RODER
金额：
$ 35.25万
依托单位：
RESEARCH INST OF FOX CHASE CAN CTR
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-08-01 至 2020-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9176248
关键词：
Abbreviations Address Affect Affinity Amino Acid Motifs Binding Biochemical Biological Biological Assay Boxing C-terminal Cancer cell line Cell Culture Techniques Cell surface Cells Chemicals Complex Confocal Microscopy Cystic Fibrosis Transmembrane Conductance Regulator Cytoskeleton DLG4 gene Data Defect Detection Disease Dissociation Drug resistance EGF gene Endocytosis Environment Epidermal Growth Factor Receptor Epithelial Cells Equilibrium Event Family Flow Cytometry Fluorescence Fluorescence Polarization Fluorescence Spectroscopy Foundations Growth Human Image Cytometry In Vitro Ion Channel Kinetics Knowledge Label Length Ligand Binding Ligands Link Liposomes Malignant Neoplasms Measures Membrane Molecular Molecular Conformation Mutation Neurofibromin 2 Nucleic Acid Regulatory Sequences Oncogenic PDGFRB gene PTEN gene Peptides Phosphatidylinositol 4,5-Diphosphate Physiological Property Proteins Publishing Receptor Protein-Tyrosine Kinases Receptor Signaling Recombinants Recycling Regulation Relaxation Reporting Role Signal Pathway Signal Transduction Signaling Protein Site Specificity Stream Structure Surface Tail Tandem Repeat Sequences Techniques Testing Thermodynamics Time Tumor Suppressor Proteins Variant adapter protein base biophysical analysis biophysical properties biophysical techniques carcinogenesis cell growth cellular imaging ezrin functional plasticity improved insight intermolecular interaction mutant predictive marker protein protein interaction receptor receptor internalization research study sodium-hydrogen exchanger regulatory factor targeted treatment tumor tumor progression

项目摘要

PROJECT SUMMARY Numerous proteins involved in cell signaling contain tandem repeats of protein-protein interaction modules belonging to the family of PDZ (PSD95-Dlg1-Zo1) domains, which recognize short amino acid motifs that are typically, but not exclusively, located at the C-terminus of a protein. The affinity and specificity of PDZ domains for canonical (C-terminal) peptide ligands has been studied extensively. While there is evidence that inter- molecular PDZ-peptide interactions can be modulated by competing intramolecular interactions, the structural basis of such autoinhibitory effects and the mechanism of activation are not well understood. To address this question, we focus on Na+/H+ exchanger regulatory factor 1 (NHERF; also known as NHERF1 or EBP50), an adapter protein involved in regulating important cell signaling events at the membrane-cytoskeleton interface of epithelial cells. NHERF contains two PDZ domains and a C-terminal ezrin-binding motif (EBM), as well as long disordered regions. The first aim of this project is to establish a linkage between the structural/dynamic properties of NHERF and its affinity for physiological interaction partners. Fluorescence-based binding assays, rapid mixing and various NMR techniques, including chemical shift analysis, paramagnetic relaxation studies and H-D exchange, will provide detailed insight into (i) the mechanism by which NHERF recognizes peptide ligands derived from the C-termini of biological interaction partners, (ii) the structural and thermodynamic basis of regulation of the binding affinity and specificity of the PDZ domains, and (iii) mechanisms of activation triggered by cytoskeletal interaction partners. The second aim is to elucidate the roles of NHERF in regulating signaling, endocytosis and degradation of the epidermal growth factor receptor (EGFR), an important oncogenic driver of many cancers. Cell-biological studies identified potential interaction sites in the C-terminal regulatory region of EGFR for the first PDZ domain of NHERF. However, there have been no biophysical studies in vitro, and the structural details and biological implications of these interactions are poorly understood. We seek to fill these gaps in knowledge (i) by using NMR and fluorescence techniques to elucidate the structural and energetic basis of interactions between NHERF and the cytoplasmic regulatory region EGFR interactions, (ii) biophysical studies of the influence of the membrane environment and effector proteins on NHERF-EGFR interactions, and (iii) by using flow cytometry and cellular imaging approaches to explore the impact of interactions with NHERF on internalization, recycling and/or degradation of EGFR in human cancer cell lines. These cell-biological approaches, combined with the structural and mechanistic insight into activation of NHERF and its interactions with EGFR gained from the proposed biophysical studies, will yield detailed new insight into the role NHERF in regulating the stability, cellular localization and cell growth-stimulating activities of EGFR.

项目概要许多参与细胞信号转导的蛋白质含有蛋白质-蛋白质相互作用模块的串联重复序列属于 PDZ (PSD95-Dlg1-Zo1) 结构域家族，该结构域识别短氨基酸基序通常（但不限于）位于蛋白质的 C 末端。 PDZ结构域的亲和力和特异性经典（C 端）肽配体已被广泛研究。虽然有证据表明，分子 PDZ-肽相互作用可以通过竞争性分子内相互作用来调节，结构这种自抑制作用的基础和激活机制尚不清楚。为了解决这个问题问题中，我们重点关注 Na+/H+ 交换调节因子 1（NHERF；也称为 NHERF1 或 EBP50），接头蛋白参与调节膜-细胞骨架界面的重要细胞信号转导事件上皮细胞。 NHERF 包含两个 PDZ 结构域和一个 C 端埃兹蛋白结合基序 (EBM)，以及长链无序区域。该项目的首要目标是在结构/动态之间建立联系 NHERF 的特性及其对生理相互作用伙伴的亲和力。基于荧光的结合测定，快速混合和各种 NMR 技术，包括化学位移分析、顺磁弛豫研究和 H-D 交换，将提供对 (i) NHERF 识别肽的机制的详细见解衍生自生物相互作用伙伴的 C 末端的配体，(ii) 结构和热力学基础 PDZ 结构域的结合亲和力和特异性的调节，以及 (iii) 激活机制由细胞骨架相互作用伙伴触发。第二个目标是阐明 NHERF 在调节中的作用表皮生长因子受体 (EGFR) 的信号传导、内吞作用和降解，表皮生长因子受体是一种重要的许多癌症的致癌驱动因素。细胞生物学研究确定了 C 端潜在的相互作用位点 NHERF 的第一个 PDZ 结构域的 EGFR 调节区。但目前还没有生物物理体外研究，这些相互作用的结构细节和生物学意义还很差明白了。我们寻求通过使用 NMR 和荧光技术来填补这些知识空白 (i) 阐明 NHERF 与细胞质调节之间相互作用的结构和能量基础区域 EGFR 相互作用，(ii) 膜环境和效应器影响的生物物理学研究 NHERF-EGFR 相互作用的蛋白质，以及 (iii) 通过使用流式细胞术和细胞成像方法探索与 NHERF 的相互作用对 EGFR 内化、再循环和/或降解的影响人类癌细胞系。这些细胞生物学方法与结构和机制相结合从拟议的生物物理学研究中获得对 NHERF 激活及其与 EGFR 相互作用的深入了解，将对 NHERF 在调节稳定性、细胞定位和细胞 EGFR 的生长刺激活性。