Protein interactions in cardiovascular signaling

这些研究的目标是了解与血浆相关的信号蛋白的相互作用。 血管内皮细胞中的丘疹小凹，最终目的是识别血管内皮细胞的异常 内皮信号是许多血管疾病状态的基础。凹陷有一种独特的脂肪含量， 并作为不同信号分子的隔离位置，包括受体和蛋白质 和脂蛋白激动酶。小窝蛋白在小窝中既起结构作用，又起信号作用，并调节 质膜和细胞骨架之间的相互作用。拟议的研究将使用细胞成像和 探索小窝蛋白在调节偶联蛋白相互作用中作用的生物化学方法 受体对信号反应的激活。在之前的项目期间，我们开发了基于siRNA的 小窝蛋白和其他关键信号蛋白“敲除”表达的途径，并探索了 对内皮细胞的细胞反应的影响。我们应用了荧光共振能量 转移(FRET)成像方法表征相互作用的空间和时间特征 钙调素和eNOS。我们发现了小窝蛋白在细胞周期调控中的潜在重要信号作用。 与细胞骨架相关的信号蛋白，如rac1，Vasp和Marcks，并确定了 血小板衍生脂鞘氨醇1-磷酸(S1P)和血管内皮生长因子信号之间的串扰。这个 拟议的研究将应用基于siRNA的方法来探索小窝蛋白在调节 细胞骨架相关的信号蛋白。我们将利用FRET方法来探索小窝蛋白调节的 钙调蛋白、MARCKS和eNOS之间的相互作用，并将决定钙调蛋白的作用 蛋白质相互作用上的磷酸化。我们将确定洞穴和马克在监管中的作用 以小凹为靶点的磷脂酰肌醇。我们将确定小窝蛋白对SIP1受体的影响。 靶向和周转，并将探索小窝蛋白对S1P介导的钙动员和 激活酶的激活。拟议的研究将确定小窝和小窝-- 相关蛋白调节激动剂介导的细胞过程，如血管生成和内皮 渗透性。我们对调节蛋白质相互作用的细胞和分子机制的研究 心血管信号转导可能为药物干预寻找新的靶点。

The goal of these studies is to understand the interactions of signaling proteins associated with plasma- lemmal caveolae in vascular endothelial cells, with an ultimate aim of identifying the abnormalities in endothelial signaling that underlie many vascular disease states. Caveolae have a distinctive lipid content, and serve as sites for the sequestration of diverse signaling molecules, including receptors as well as protein and lipid kinases. The protein caveolin serves both structural and signaling roles in caveolae, and modulates interactions between the plasmalemma and cytoskeleton. The proposed studies will use cellular imaging and biochemical approaches to explore the role of caveolin in modulating protein interactions that couple receptor activation to signaling responses. In the previous project period, we developed siRNA-based approaches to "knock-down" expression of caveolin and other key signaling proteins, and explored the consequences for cellular responses in endothelial cells. We applied fluorescence resonance energy transfer (FRET) imaging methods to characterize spatial and temporal features of the interactions between calmodulin and eNOS. We discovered potentially important signaling roles for caveolin in regulation of cytoskeleton-associated signaling proteins such as Rac1, VASP and MARCKS, and identified pathways for crosstalk between the platelet-derived lipid sphingosine 1-phosphate (S1P) and VEGF signaling. The proposed studies will apply siRNA-based methods to explore roles for caveolin in the regulation of cytoskeleton-associated signaling proteins. We will exploit FRET methods to explore caveolin-modulated interactions between calmodulin, MARCKS, and eNOS, and will determine the effects of calmodulin phosphorylation on its protein interactions. We will identify the roles of caveolin and MARCKS in regulation of phosphoinositide targeting to caveolae. We will determine the effects of caveolin on SIP-,receptor targeting and turnover, and will explore the effects of caveolin on S1P-mediated calcium mobilization and kinase activation. The proposed studies will identify mechanisms whereby caveolin and caveolae- associated proteins regulate agonist-mediated cellular processes such as angiogenesis and endothelial permeability. Our proposed studies of cellular and molecular mechanisms that regulate protein interactionsin cardiovascular signaling may lead to the identification of new points for pharmacological intervention.