Sphingosine-1-phosphate (S1P), a bioactive sphingolipid, regulates a variety of cellular functions such as proliferation, survival, chemotaxis etc. Recently, it has been shown that S1P is the high affinity ligand for the G-protein coupled receptor, EDG-1 (Endothelial Differentiation Gene-1). Importantly, a novel function of S1P/EDG-1 in blood vessel development and maturation has been proposed recently. For example, S1P promotes morphogenesis of endothelial cells in vitro and potentiates VEGF and FGF-induced angiogenesis in vivo. Furthermore, deletion of EDG-1 resulted in embryonic lethality due to the formation of immature vasculature. However, the molecular mechanisms underlying S1P/EDG-1 regulated angiogenesis remain elusive. Thus, our long-term goal is to understand the signaling and roles of this sphingolipid in endothelial functions. In this proposal, we will characterize signaling pathways regulated by the EDG-1 receptor. Initially, by utilizing the affinity purification procedure, a panel of cellular polypeptides was found to specifically associate with the third intracellular loop (i3 domain) of EDG-1 receptor. Among them, two protein kinases with apparent molecular masses of 120- and 56-kDa and a 21-kDa phosphoprotein were identified. We hypothesize that these EDG-1 associated kinases/polypeptides are critical for the S1 P/EDG-1 signaling, in particular in regulating the function of endothelial cells. Preliminary data suggest that pp120 kinase may be functionally important in EDG-1 signaling. Thus, we propose to prioritize and focus on determining the identity and functions of pp120 kinase in EDG-1 signaling in the context of vascular biology.
The research aims are I) purify and clone the pp120EDG-1 associated kinase, II) characterize the relevance of pp120 kinase in S1 P/EDG-1 signaling, III) study the functions of pp120 kinase in endothelial cells activation, and IV) determine the structure-function relationship of EDG-I-i3. The completion of these studies is anticipated to better our knowledge of molecular mechanisms underlying angiogenesis, which ultimately may lead to future therapeutic development.
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