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

在本研究项目中，我们发现了一种在血管平滑肌细胞中特异表达的Rho鸟嘌呤核苷酸交换因子。根据该交换因子的表达，我们将其命名为VSM-Rhogef(血管平滑肌特异性交换因子)，并对其交换的特异性和定位进行了研究。VSM-Rhogef位于EphA4受体下游，仅在EphA4受体所在的动脉平滑肌细胞(VSMCs)中表达。此外，VSM-Rhogef还展示了RAC和Rho的全环基金活性。一致地，当VSMCs被ePhin-A1刺激时，它们表现出显著的膜褶皱，这是RAC激活的一个标志。在未受刺激的VSMCs中，VSM-Rhogef定位于肌动蛋白应激纤维上，而在ePhin-A1模拟中，它从应激纤维上解离到外周褶皱的膜上。因此，尽管应力纤维上的vsm-Rhogef可能作为Rho-RhoKinase信号调节肌动蛋白-肌球蛋白偶联的Rhogef，但解离的vsm-RhoG…我们进一步研究了Ras家族的GTP酶，特别是Eph酪氨酸激酶下游的R-RAS家族的功能。R-RAS已被认为是在细胞外基质-细胞间的黏附中起作用。我们推测肌球蛋白受R-RAS调控，因为MysoinIX在其肌球蛋白头部的末端含有RAS结合域。R-RAS家族由R-RAS、M-RAS和TC21组成。我们发现其中R-RAS和TC21优先与肌球蛋白IX结合。其他RAS家族成员H-RAS、RAP1和RAL不与MysoinIX结合。因此，Eph激活的R-RAS和TC21可能促进基于肌球蛋白IX的运动功能，如囊泡收缩或肌肉收缩。Eph酪氨酸激酶受体类似于其他酪氨酸激酶受体家族成员的内吞。我们首先研究了羧基末端的EGFP标记的EphB受体在ephin-B1刺激下的内吞作用。以PECAM-1-EGFP为阴性对照。对EphB1-EGFP或PECAM-1-EGFP表达的细胞进行时间推移成像。我们发现EphB1-EGFP在刺激下表现出寡聚化，而PECAM-1 EGFP则没有。除了寡聚作用外，我们还发现寡聚化的EphB1-EGFP从细胞膜内吞入细胞体f。这些结果表明，Eph-eaffin介导的细胞内信号是在细胞-细胞接触时触发的，并受内吞作用的调节。我们将继续研究VSM-Rhogef作为Rho和/或Rac的环境基金开关的分子机制。较少

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