Molecular Cloning and Signaling Mechanisms of G Protein-Coupled Receptors for Sphingosine 1-Phoshate

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By this research project for two years, we found some new things as follows.1. Molecular cloning of sphingosine 1-phospahte (S1P) receptor- It has been reported that the endothelial differentiation gene-1 (Edg-1), Edg-3, Edg-5 were the S1P receptors. On the other hand, we obtained some evidences that some cellular responses elicited by S1P might not be mediated by these receptors. So we tried to identify another S1P receptor and found that Edg-6, which was recently identified as an orphan G-protein-coupled receptor, was the another S1P receptor.2. Signaling Mechanisms of the S1P receptors- To evaluate and compare the intrinsic activity of the each S1P receptor subtypes in the regulation of multiple signaling pathways, we prepared the Chinese hamster ovary (CHO) cell lines that permanently express the respective S1P receptor subtype to a comparative level. Using these cell lines, we found these things as follows. (1) There was no significant difference in the expressing numbers of the S1P receptors and their affinities to S1P. (2) S1P selectively regulated multiple signaling pathways according to the receptor subtype. For example, the rank order of their intrinsic activity of S1P receptor subtype was Edg-3>Edg-5>Edg-1>Edg-6 to induce activation of PLC/Ca ィイD12+ィエD1 system. On the other hand, as for cell migration activity, Edg-3 and Edg-1 were equally potent, but Edg-5 and Edg-6 were ineffective.