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

• 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
• 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

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.

项目摘要 涉及细胞信号的许多蛋白质包含蛋白质 - 蛋白质相互作用模块的串联重复序列 属于PDZ家族（PSD95-DLG1-ZO1）域，该域识别短的氨基酸基序 通常，但不仅仅是位于蛋白质的C末端。 PDZ域的亲和力和特异性 对于规范（C末端）的肽配体，已经广泛研究了。虽然有证据表明 分子PDZ肽相互作用可以通过竞争分子内相互作用（结构）调节 这种自身抑制作用和激活机制的基础尚不清楚。解决这个问题 问题，我们专注于Na+/H+交换器调节因子1（NHERF；也称为NHERF1或EBP50），一个 调节重要细胞信号事件的衔接蛋白在膜细胞骨架界面处 上皮细胞。 NHERF包含两个PDZ结构域和一个C末端Ezrin结合基序（EBM），以及长时间 无序地区。该项目的第一个目的是建立结构/动态之间的联系 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，顺磁性放松研究 和H-D交流将提供详细的见解（i）NHERF识别肽的机制 源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，（iii）激活机制 由细胞骨架相互作用伙伴触发。第二个目的是阐明NHERF在调节中的作用 表皮生长因子受体（EGFR）的信号传导，内吞和降解，这是一个重要的 许多癌症的致癌驱动力。细胞生物学研究确定了C末端的潜在相互作用位点 NHERF的第一个PDZ域的EGFR的调节区域。但是，没有生物物理 体外研究以及这些相互作用的结构细节和生物学意义很差 理解。我们试图通过使用NMR和荧光技术来填补这些知识的空白（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）膜环境和效应子的影响的生物物理研究 NHERF-EGFR相互作用上的蛋白质，以及（III），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的内在化，回收和/或退化的影响 人类癌细胞系。这些细胞生物学方法，结合结构和机械性 深入了解NHERF的激活及其与所提出的生物物理研究中获得的EGFR相互作用， 将对NHERF在调节稳定性，细胞定位和细胞中的作用进行详细的新见解 EGFR的生长刺激活动。