MOLECULAR BASIS OF THE ENDOTHELIAL SHEAR STRESS RECEPTOR

描述：(改编自调查员摘要) 建议进行题为“内皮细胞切应力的分子基础”的研究 受体“是为了阐明机械生化的机制。 受体酪氨酸激酶(TRKs)作用的转导研究 作为切应力受体和信号转导 下游基因被激活。我们已经能够在 以往的研究表明佛波酯TPA反应元件(TrE)是一种 剪应力诱导顺式单元。我们的初步结果说明 此外，p21ras位于TrE介导的基因表达的上游。在……里面 此外，表皮生长因子受体(EGFR)在 对剪应力的响应。通过使用EGFR作为RTK模型，并结合 在体外流道实验中，拟议的研究旨在 提供两个膜相关事件之间的联系，并将 剪切作用下TRKs胞外区的结构特征 应激诱导的细胞反应。在具体目标1中，我们将调查 包含src同源结构域2(SH2)的分子的招募， 包括生长因子受体结合蛋白-2(Grb2)，Shc，Son Seven less(SOS)和RasGTP酶激活蛋白(RasGAP) 剪切应力激活的RTK的磷酸酪氨酸。当大会上的 RTKs-Shc-Grb2-SOS四元络合物和RTKs-Grb-2OS三元络合物 激活RAS信号通路，形成RTKs-RAS-GAP双元 复合体拮抗RAS的激活。由此产生的瞬时细胞 将通过评估p21ras的活动来审查答复，c-jun N末端激酶(JNK)、细胞外信号调节激酶(ERKs)和 由tre推动的荧光素酶记者活动。在具体目标2中，这些 细胞反应将在表达EGFR突变体和 融合受体。这些分子中EGFR的胞外结构域是 与其他RTK的突变或替换，以研究其 具有剪切应力传感器的功能。剪切应力的影响将是 与与酪氨酸结合的配体相比 磷酸化和受体二聚化。在具体目标3中，我们将 检查所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状态。拟议中的研究已经 对阐明这两个基本问题具有相当重要的意义 机械生化转导过程与血管内皮细胞生物学 动脉粥样硬化的形成。

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.