Protein interactions in cardiovascular signaling

这些研究的目的是了解与血浆相关的信号蛋白的相互作用， 血管内皮细胞膜小窝，最终目的是确定异常， 血管内皮细胞信号传导是许多血管疾病状态的基础。小窝有独特的脂质含量， 并作为不同信号分子的隔离位点，包括受体和蛋白质 和脂质激酶。蛋白质小窝蛋白在小窝中起结构和信号传导作用，并且调节 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， 生物化学方法来探索小窝蛋白在调节蛋白质相互作用中的作用， 受体激活信号反应。在上一个项目期间，我们开发了基于siRNA的 方法“敲低”表达的小窝蛋白和其他关键信号蛋白，并探讨了 内皮细胞中细胞反应的后果。我们将荧光共振能量 转移（FRET）成像方法，以表征空间和时间特征的相互作用之间 钙调素和eNOS。我们发现了小窝蛋白在调节细胞凋亡中潜在的重要信号作用。 细胞凋亡相关的信号蛋白，如Rac 1，VASP和MARCKS，并确定了通路， 血小板衍生的脂质鞘氨醇1-磷酸（S1 P）和VEGF信号之间的串扰。的 拟议的研究将应用基于siRNA的方法来探索小窝蛋白在调节 细胞凋亡相关信号蛋白。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 磷酸化对其蛋白质相互作用的影响。我们将确定小窝蛋白和MARCKS在调控中的作用 磷酸肌醇靶向小窝。我们将研究小窝蛋白对SIP-1受体的影响， 靶向和营业额，并将探讨小窝蛋白对S1 P介导的钙动员的影响， 激酶活化拟议的研究将确定小窝蛋白和小窝的机制- 相关蛋白调节激动剂介导的细胞过程，如血管生成和内皮细胞增殖。 磁导率我们建议研究调节蛋白质相互作用的细胞和分子机制， 心血管信号传导可能导致识别药理学干预的新点。

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.