MOLECULAR BASIS OF THE ENDOTHELIAL SHEAR STRESS RECEPTOR

描述：（改编自研究者摘要） 一项名为“内皮剪切应力的分子基础”的研究 受体”的研究是为了阐明机械-生物化学作用的机制 通过研究受体酪氨酸激酶（TRKs）的转导， 作为切应力受体以及通过切应力受体的信号传导 下游基因被激活。 我们已经能够证明， 先前的研究表明佛波醇酯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，p21 ras是这种TRE-mediated基因表达的上游。 在 此外，表皮生长因子受体（EGFR）在细胞内被磷酸化， 对剪切应力的反应。 通过使用EGFR作为模型RTK， 在体外流动通道实验中，所提出的研究旨在 提供两个膜相关事件之间的联系， TRKs胞外结构域的结构特征与剪切 应激诱导的细胞反应。 在具体目标1中，我们将研究 包含src同源结构域2（SH 2）的分子的募集， 包括生长因子受体结合蛋白-2（Grb 2）、Shc、 Sevenless（Sos）和RasGT 3激活蛋白（RasGAP）。 剪切应力激活的RTK的磷酸酪氨酸。 虽然大会 RTKs-Shc-Grb 2-Sos四元配合物和RTKs-Grb-2 os三元配合物 激活Ras信号通路，形成RTKs-Ras-GAP二元 复合物拮抗Ras的激活。由此产生的短暂细胞 将通过评估p21 ras、c-Jun的活性来检查反应 N-末端激酶（JNK），细胞外信号调节激酶（ERK），和 由TRE驱动的荧光素酶报告活性。 在第二个目标中，这些 将在表达EGFR突变体的细胞系中研究细胞应答， 融合受体 这些分子中EGFR的细胞外结构域是 要么突变或与其他RTK取代，以调查他们的 用作剪切应力传感器。 剪切应力的影响将是 与配体结合的酪氨酸相比， 磷酸化和受体二聚化。 在具体目标3中，我们 检查推断的分子机制是否也在 血管内皮细胞是暴露于 体内的病理生理流动条件。 拟议的研究已 在阐明基本的 机械-生化转导过程和内皮生物学 动脉粥样硬化

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.