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.
