MOLECULAR BASIS OF THE ENDOTHELIAL SHEAR STRESS RECEPTOR

内皮剪切应力受体的分子基础

• 批准号: 2445348
• 负责人: John YJ Shyy
• 金额: $ 9.66万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2445348
• 关键词: 3T3 cells; biological signal transduction; chimeric proteins; colony stimulating factor; enzyme activity; epidermal growth factor; gene expression; growth factor receptors; guanine nucleotide binding protein; guanosinetriphosphatase activating protein; human tissue; immunoprecipitation; luciferin monooxygenase; northern blottings; phorbols; phosphorylation; plasmids; platelet derived growth factor; protein structure function; protein tyrosine kinase; receptor binding; receptor expression; tissue /cell culture; vascular endothelium; western blottings

DESCRIPTION: (Adapted from investigator's abstract) The objectives of the proposed research entitled "Molecular Basis of the Endothelial Shear Stress Receptor" are to elucidate the mechanisms of mechano-biochemical transduction through the study of receptor tyrosine kinases (TRKs) serving as shear stress receptors and the signal transduction through which the downstream genes are activated. We have been able to demonstrate in previous studies that the phorbol ester TPA responsive element (TRE) is a shear stress inducible cis-element. Our preliminary results illustrate further that p21ras is upstream to such TRE-mediated gene expression. In addition, the epidermal growth factor receptor (EGFR) is phosphorylated in response to shear stress. By using EGFR as a model RTK in conjunction with in vitro flow channel experiments, the proposed studies are designed to provide linkage between two membrane-associated events and to correlate the structural features of the extracellular domains of TRKs with the shear stress-induced cellular responses. In Specific Aim 1, we will investigate the recruitment of the src homology domain 2 (SH2)-containing molecules, including growth factor receptor binding protein-2 (Grb2), Shc, Son of sevenless (Sos), and RasGTPase activating protein (RasGAP) to the phosphotyrosines of the shear stress-activated RTKs. While the assembly of RTKs-Shc-Grb2-Sos quaternary complex and RTKs-Grb-2os ternary complex activates the Ras signaling pathway, the formation of RTKs-Ras-GAP binary complex antagonizes the activation of Ras. The resulting transient cellular responses will be examined by assessing the activities of p21ras, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases (ERKs), and the luciferase reporter activities driven by TRE. In Specific Aim 2, these cellular responses will be studies in cell lines expressing EGFR mutants and fusion receptors. The extracellular domains of EGFR in these molecules are either mutated or replaced with those of other RTKs to investigate their function as shear stress sensors. The effects of shear stress will be compared with those of ligand binding with respect to the tyrosine phosphorylation and receptor dimerization. In Specific Aim 3, we will examine whether the deduced molecular mechanisms are also functional in the vascular endothelial cells which are the cell type exposed to pathophysiological flow conditions in the body. The proposed research has considerable significance in elucidating both the fundamental mechano-biochemical transduction processes and the endothelial biology in atherogenesis.

描述：（改编自研究者的摘要） 提出的题为“内皮剪切应力的分子基础”的研究 受体”旨在阐明机械生化机制 通过研究受体酪氨酸激酶 (TRK) 进行转导 作为剪切应力受体和信号转导 下游基因被激活。 我们已经能够证明 先前的研究表明佛波酯 TPA 响应元件 (TRE) 是一种 剪切应力诱导顺式元件。 我们的初步结果表明 此外，p21ras 是 TRE 介导的基因表达的上游。 在 此外，表皮生长因子受体（EGFR）在 对剪切应力的响应。 通过使用 EGFR 作为模型 RTK 并结合 在体外流道实验中，所提出的研究旨在 提供两个膜相关事件之间的联系并将 TRKs胞外域的结构特征与剪切 压力诱导的细胞反应。 在具体目标 1 中，我们将调查 含有 src 同源结构域 2 (SH2) 的分子的募集， 包括生长因子受体结合蛋白-2 (Grb2)、Shc、Son of Sevenless (Sos) 和 RasGTPase 激活蛋白 (RasGAP) 剪切应力激活 RTK 的磷酸酪氨酸。 虽然组装 RTKs-Shc-Grb2-Sos 四元复合物和 RTKs-Grb-2os 三元复合物 激活Ras信号通路，形成RTKs-Ras-GAP二元 复合物拮抗Ras的激活。由此产生的瞬态细胞 将通过评估 p21ras 的活动来检查反应，c-Jun N 末端激酶 (JNK)、细胞外信号调节激酶 (ERK) 和 TRE 驱动的荧光素酶报告基因活动。 在具体目标 2 中，这些 细胞反应将在表达 EGFR 突变体的细胞系中进行研究 融合受体。 这些分子中 EGFR 的胞外结构域是 突变或替换为其他 RTK 以调查其 用作剪切应力传感器。 剪应力的影响将是 与酪氨酸配体结合相比 磷酸化和受体二聚化。 在具体目标 3 中，我们将 检查推论的分子机制是否也在 血管内皮细胞是暴露于的细胞类型 体内的病理生理流动状况。 拟议的研究有 对于阐明两者的基本原理具有重要意义 机械生化转导过程和内皮生物学 动脉粥样硬化。