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

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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.