Molecular biological research of biological functions of the sphingoshine-1-phosphate receptors

我们研究了三种G蛋白偶联的鞘氨醇-1-磷酸(S1P)受体，即EDG1、EDG3和EDG5的细胞活性及其跨膜信号机制，并利用基因打靶技术进行了功能分析。我们建立了稳定表达EDG1、EDG3和EDG5受体的中国仓鼠卵巢(CHO)、K562和HEL细胞系。通过使用这些细胞，我们发现这些受体是S1P的特异性受体，并激活受体类型的特异性信号机制。EDG1和EDG3介导趋化作用，而EDG5独特地介导抑制细胞迁移。后者的观察有力地表明，正是EDG5受体介导了先前报道的S1P对各种细胞类型的抑制活性。S1P诱导三维基质培养的血管内皮细胞形成毛细血管样管。S1P可抑制血管平滑肌细胞的迁移。S1P还可刺激血小板衍生生长因子-B链基因的表达。EDG1和EDG3以依赖于Gi和PI-3的方式介导小G蛋白Rac的激活。值得注意的是，EDG5很可能通过刺激RAC的GTP酶激活蛋白来抑制RAC的激活。EDG5是第一个被证明能抑制RAC活性的受体。我们正试图培育出EDG5基因敲除小鼠。我们成功地产生了嵌合小鼠。我们已经构建了一个靶向载体，用于产生鞘氨醇激酶基因敲除小鼠。

We performed investigations of cellular activities of three G protein-coupled receptors for sphingosine-1-phosphate (S1P), i.e.EDG1, EDG3 and EDG5, and their transmembrane signaling mechanisms, and functional analysis using the gene targeting technique.1. We established Chinese hamster ovary (CHO), K562 and HEL cell lines that stably express each of EDG1, EDG3 and EDG5 receptors. By using these cells, we found that these receptors are specific for S1P and activate receptor-type specific signaling mechanisms.2. EDG1 and EDG3 mediated chemotaxis, whereas EDG5 uniquely mediated inhibition of cell migration. The latter observation strongly suggests that it is the EDG5 receptor that mediates previously reported inhibitory activities of S1P on various cell types.3. S1P induced capillary-like tube formation of vascular endothelial cells in the 3-dimensional matrigel cultures. S1P induced inhibition of migration of vascular smooth muscle cells. S1P also induced stimulation of platelet-derived growth factor-B chain gene expression.4. EDG1 and EDG3 mediated activation of the small G protein Rac in a manner dependent on Gi and PI-3 kinase. Strikingly, EDG5 mediated inhibition of Rac activation most likely through stimulation of GTPase-activating protein for Rac. EDG5 is the first example of the receptor that is demonstrated to inhibit Rac activity.5. We are trying to generate EDG5-knock-out mice. We successfully generated chimeric mouse.6. We have constructed a targeting vector for generating sphingosine kinase-knock out mice.