Intracellular and intercellular signaling mediated by Eph tyrosine kinase receptor in vascular endothelial cells

点击翻译按钮获取中文摘要

In this research project, we have identified a Rho guanine nucleotide exchange factor(GEF)espetially expressed in vascular smooth muscle cells. We named this exchange factor Vsm-RhoGEF(Vascular smooth muscle specific GEF) after its expression and characterized the exchange specificity and localization. Vsm-RhoGEF functions downstream of EphA4 receptor and is expressed exclusively in the arterial smooth muscle cells (VSMCs), where EphA4 receptor is localized/. Furtheremore, Vsm-RhoGEF exhibits GEF activity for Rac as well as Rho. Consistently, when VSMCs were stimulated with ephrin-A1,they showed remarkable membrane ruffling, which is a hallmark of Rac activation. Vsm-RhoGEF is localized on actin stress fiber in unstimulated VSMCs, whereas it is dissociated from stress fiber to peripheral ruffled membrane upon ephrin-A1 simulation. Thus, while the Vsm-RhoGEF on stress fiber may function as a RhoGEF for Rho-RhoKinase signaling to regulate acttin-myosin coupling, the dissocicated Vsm-RhoG … More EF activates Rac for membrane extension.We further investigated the Ras family GTPases, especially R-Ras family functioning downstream of Eph tyrosine kinase. R-Ras has been suggested to function for extracellular matrix-cell adhesion when simulated by ephrin. We hypothesized that actomyosin is regulated by R-Ras, because MysoinIX contains Ras binding domain in its ajnino-terminus of the myosin head. R-Ras family consists of R-Ras, M-Ras, and TC21. We found that among them R-Ras and TC21 preferentially bind to myosinIX. Other Ras family members, H-Ras, Rap1 and Ral do not bind to MysoinIX. Thus Eph-activated R-Ras and TC21 may promote myosinIX-based motor function such as vesicular frafficking or muscle contraction.Eph tyrosine kinase receptor is endocytosed similarly to other tyrosine kinase receptor family members. We first explored the endocytosis of carboxy-terminally EGFP-tagged EphB receptors upon ephrin-B1 stimulation. PECAM-1-EGFP was used as a negative control. The cells expressing either EphB1-EGFP or PECAM-1-EGFP stimulated with ephrin-B1 were time-lapse imaged. We found that EphB1-EGFP exhibited the oligomerization upon stimulation while PECAM-1 EGFP did not. In addition to oligomerization, we found that oligomerized EphB1-EGFP was endocytosed into the cell body f from the plasma membrane. These results suggest that Eph-ephrin-mediated intracellular signaling is triggerd upon cell-cell contact and modulated by endocytosis. We will prceed to examine the molecular mechanism how Vsm-RhoGEF functions as GEF switch for Rho and/or Rac. Less