Sphingolipid Signaling in Endothelial Function

说明（申请人提供）：鞘氨醇-1-磷酸（S1 P）是一种生物活性鞘脂，可调节多种细胞功能，如增殖、存活、趋化性等。最近，研究表明S1 P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，最近已经提出了S1 P/EDG-1在血管发育和成熟中的新功能。例如，S1 P在体外促进内皮细胞的形态发生，并在体内增强VEGF和FGF诱导的血管生成。此外，EDG-1的缺失由于未成熟脉管系统的形成而导致胚胎死亡。然而，S1 P/EDG-1调节血管生成的分子机制仍然是难以捉摸的。因此，我们的长期目标是了解这种鞘脂在内皮功能中的信号传导和作用。在这项提案中，我们将表征由EDG-1受体调节的信号通路。最初，通过利用亲和纯化程序，发现一组细胞多肽与EDG-1受体的第三胞内环（i3结构域）特异性缔合。其中，两个蛋白激酶的表观分子量为120-和56-kDa和21-kDa磷蛋白被确定。我们假设这些EDG-1相关激酶/多肽对于S1 P/EDG-1信号传导至关重要，特别是在调节内皮细胞功能方面。初步数据表明pp 120激酶可能在EDG-1信号传导中具有重要功能。因此，我们建议优先考虑并专注于确定pp 120激酶在血管生物学背景下EDG-1信号传导中的身份和功能。本研究的主要目的是：纯化和克隆pp 120 EDG-1相关激酶，研究pp 120激酶在S1 P/EDG-1信号转导中的作用，研究pp 120激酶在内皮细胞活化中的作用，确定EDG-I-i3的结构与功能关系。预计这些研究的完成将使我们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发展。

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.