Sphingolipid metabolites have recently been shown to regulate a wide variety of cellular processes, including cell growth, differentiation and apoptosis. Recently we discovered that the orphan receptor endothelial differentiation gene-1 (Edg-1) is the receptor for sphingosine-1-phosphate (SPP). Edg-1 is coupled through a Gi protein to cause decreased cAMP and activation of the MAP kinase ERK2. SPP signaling through Edg-1 causes morphological changes resembling differentiation of endothelial cells in vitro, through a mechanism which requires the small GTPase Rho. The long-term objectives of this project are to determine the binding site for SPP on Edg-1, and how Edg-1 signaling regulates gene expression. These goals will be addressed by using site-directed mutagenesis to determine which of several candidate residues of Edg-1 may be involved in binding SPP. The effects of these mutations on SPP binding and on Edg-1 signaling will be determined. In addition, gene expression regulated by the serum response element (SRE) promoter will be examined. The SRE regulates transcription of several early response genes including c-fos, expression of which is regulated by SPP in some cell types. SPP / Edg-1-induced gene transcription will be measured using reporter plasmids regulated by the wild type or a mutant SRE, which can discriminate between Rho-mediated and MAP kinase- mediated signaling pathways, in order to elucidate the mechanisms used by Edg-1 to regulate this important promoter. Dominant negative mutants of the small GTPase Rho and the kinase Raf-1 will also be used to specifically block Rho- and MAP kinase- mediated signaling respectively, in order to define the role of these pathways in Edg-1 regulated gene expression.
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