Core--Cytoskeletal Facility

核心--细胞骨架设施

• 批准号: 6585990
• 负责人: ANIRBAN BANERJEE
• 金额: $ 15.83万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6585990
• 关键词: 3T3 cells; G protein coupled receptor kinase; biological signal transduction; biomedical facility; clathrin; confocal scanning microscopy; cytoskeleton; digital imaging; enzyme induction /repression; fluorescence resonance energy transfer; human tissue; immunocytochemistry; macrophage; mitogen activated protein kinase; multiple organ failure; phosphorylation; protein kinase C; protein structure function; receptor mediated endocytosis; tissue /cell culture; trauma

Trauma provokes the release of a large number of bioactive mediators, such as lipids, hormones and cytokines (Project I-A). These ligands elicit immediate changes in function and metabolism, but also modify the cellular response(s) to subsequent stimuli (both destructively or constructively). The mechanism(s) of these adapted responses (termed priming or preconditioning, respectively) remain the focus of our investigations (with Projects II, III, IV, VII, and IX). Deservedly, ligand activated signaling by second messengers, ion-flux and protein phosphorylation continue to receive extensive study. But ligand binding also triggers receptor internalization. Using transformed models we (and others) now accept that activated receptors for several classes of bioactive mediators (such as catechols, lipids and TNFa) are internalized by clathrin coated vesicles. Clathrin coated endosomes are transported about the cell on microtubular tracks. Surprisingly, recent studies indicate that receptor internalization is necessary to initiate a novel phase of signaling. Signals emanating from internalized G-protein coupled receptors (GPCR) affect transcriptionally directed protein kinase cascades (including PKC and the MAPK modules) causing sustained changes in cell responsiveness (with Project V). However it is unclear how previous signaling events influence the interactions between internalized receptors, the endocytotic machinery and the microtubular cytoskeleton, in mammalian, especially human cells. Moreover it is unclear whether pro-inflammatory cells such as macrophages respond similarly to vulnerable targets such as endothelial or smooth muscle cells (with Project VI). We hypothesize that circulating mediators (adrenergic, lysolipid and cytokines ) (with Projects V, VII and IX) released after trauma augment internalization for membranes, receptors and subsequent signal traffic. We initially focus on ligands selected for i. pertinence to trauma, ii. mediation by GPCR, and iii, activation of PKC and MAPK modules. We will examine how these test ligands influence a. constitutive membrane/receptor trafficking and b. internalization dependent signaling in a set of myeloid and non myeloid cells from humans and rodents. These studies will form the basis for evaluating whether currently accessible therapeutic strategies can be directed against clathrin coated endocytosis and microtubule based trafficking.

创伤会引发大量生物活性介质的释放，例如脂质、激素和细胞因子（项目 I-A）。这些配体引起功能和代谢的立即变化，但也改变细胞对后续刺激的反应（破坏性或建设性的）。这些适应性反应的机制（分别称为启动或预处理）仍然是我们研究的重点（项目 II、III、IV、VII 和 IX）。配体激活的第二信使信号传导、离子通量和蛋白质磷酸化理所当然地继续受到广泛的研究。但配体结合也会触发受体内化。使用转化模型，我们（和其他人）现在承认几类生物活性介质（例如儿茶酚、脂质和 TNFa）的激活受体被网格蛋白包被的囊泡内化。网格蛋白包被的内涵体通过微管轨道在细胞周围运输。令人惊讶的是，最近的研究表明受体内化对于启动新的信号传导阶段是必要的。内化 G 蛋白偶联受体 (GPCR) 发出的信号影响转录定向的蛋白激酶级联（包括 PKC 和 MAPK 模块），导致细胞反应性持续变化（使用 Project V）。然而，目前尚不清楚先前的信号传导事件如何影响哺乳动物细胞，尤其是人类细胞中内化受体、内吞机制和微管细胞骨架之间的相互作用。此外，尚不清楚巨噬细胞等促炎细胞是否对内皮细胞或平滑肌细胞（项目 VI）等脆弱靶标有类似的反应。我们假设创伤后释放的循环介质（肾上腺素、溶血脂和细胞因子）（项目 V、VII 和 IX）增强了膜、受体和随后信号传输的内化。我们最初关注为 i 选择的配体。与创伤的相关性，ii. GPCR 的介导，以及 iii，PKC 和 MAPK 模块的激活。我们将研究这些测试配体如何影响 a。组成型膜/受体运输和b．来自人类和啮齿动物的一组骨髓和非骨髓细胞中的内化依赖性信号传导。这些研究将为评估当前可用的治疗策略是否可以针对网格蛋白包被的内吞作用和基于微管的运输奠定基础。