Sphingolipid Signaling in Endothelial Function

说明(申请人提供)：鞘氨醇-1-磷酸(S1P)是一种生物活性的鞘氨醇脂类，调节多种细胞功能，如增殖、存活、趋化等。最近，研究表明S1P是G蛋白偶联受体EDG-1(内皮分化基因-1)的高亲和力配体。重要的是，S1P/EDG-1在血管发育和成熟中的新功能最近被提出。例如，S1P在体外促进内皮细胞的形态发生，并在体内增强血管内皮生长因子和成纤维细胞生长因子诱导的血管生成。此外，EDG-1的缺失导致胚胎死亡，因为形成了不成熟的血管系统。然而，S1P/EDG-1调控血管生成的分子机制仍不清楚。因此，我们的长期目标是了解这种鞘磷脂在内皮功能中的信号和作用。在这个提案中，我们将描述由EDG-1受体调控的信号通路。最初，通过亲和纯化程序，一组细胞多肽被发现与EDG-1受体的第三胞内环(I3结构域)特异性结合。其中，鉴定出两个表观分子量分别为120 kDa和56 kDa的蛋白激酶和一个21 kDa的磷酸蛋白。我们推测这些与EDG-1相关的蛋白激酶/多肽对S1P/EDG-1信号通路至关重要，特别是在调节内皮细胞的功能方面。初步数据表明，pp120激酶可能在EDG-1信号转导中起重要作用。因此，我们建议在血管生物学的背景下优先并重点确定pp120激酶在EDG-1信号转导中的身份和功能。本研究的目的是：1)纯化和克隆pp120EDG-1相关蛋白，2)鉴定pp120蛋白在S1P/EDG-1信号转导中的作用，3)研究pp120蛋白在内皮细胞激活中的作用，4)确定EDG-I-I3的结构与功能关系。这些研究的完成有望更好地了解血管生成的分子机制，最终可能导致未来的治疗开发。

DESCRIPTION (provided by applicant): 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.