ROLE OF CELL SURFACE IN REGULATING CELL PROLIFERATION

细胞表面在调节细胞增殖中的作用

• 批准号: 3227607
• 负责人: Darrell H. Carney
• 金额: $ 15.39万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-07-01 至 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3227607
• 关键词: affinity chromatography; autoradiography; cell growth regulation; cell membrane; dexamethasone; electron microscopy; enzyme mechanism; epidermal growth factor; flow cytometry; fluorescence microscopy; growth factor; high performance liquid chromatography; insulin; laboratory mouse; laboratory rabbit; membrane activity; microtubules; mitogens; nucleic acid probes; phospholipase C; phosphorylation; protein kinase C; radiotracer; receptor; thrombin; tissue /cell culture

The primary objective of this project remains to determine the molecular events that are causally involved in regulating initiation of cell proliferation. These studies are focused on the interaction of thrombin with specific surface receptors on fibroblastic cells and the transmembrane signals elicited by this interaction. We have identified the high-affinity binding domain of thrombin and found it to be homologous to other regulatory molecules including tissue plasminogen activator, suggesting that a family of regulatory molecules may generate their biological signals through interactions with this common receptor. In addition, thrombin binding or interaction with molecules on other cells have been implicated in various aspects of hemostasis and wound healing. Thus, it is more important than ever to determine how thrombin-receptor interaction generates mitogenic signals and how these signals compare with those generated by other thrombin interactions and by growth factors and hormones such as epidermal growth factor (EGF) and insulin. In the proposed studies we will: 1) purify and characterize the high-affinity receptor for thrombin using HPLC and affinity chromatography, examine carbohydrate involvement in thrombin-receptor interaction, sequence a portion of the receptor, construct DNA probes based on this sequence screen genomic and expression libraries and attempt to clone and sequence the gene for the thrombin receptor, 2) characterize signals generated by thrombin enzymic activity an receptor occupancy including phosphoinositide turnover, Ca++ mobilization, diacylglycerol release, protein kinase C activation and specific protein phosphorylation, 3) reconstitute thrombin binding to purified receptors to determine proteolytic and conformational alterations, 4) utilize binding domain synthetic peptides to determine correlations between affinity, size, conformation and signal generation, 5) determine the role of cytoskeletal elements in receptor anchorage and transmembrane signalling and 6) identify and purify other regulatory molecules which interact with thrombin receptors to help evaluate the utility of this receptor system in various tissues and different cellular functions.

该项目的主要目标仍然是确定 与调控有关的分子事件 启动细胞增殖。这些研究主要集中在 凝血酶与血管内皮细胞表面受体的相互作用 成纤维细胞及其引发的跨膜信号 互动。我们已经鉴定了高亲和力结合结构域 发现它与其他调节因子同源 包括组织纤溶酶原激活剂在内的分子，表明 一系列调节分子可能会产生它们的生物学特性 通过与这种共同受体的相互作用来传递信号。在……里面 此外，凝血酶与其他分子的结合或相互作用 细胞与止血的各个方面有关，而且 伤口愈合。因此，比以往任何时候都更重要的是确定 凝血酶-受体相互作用如何产生有丝分裂信号 以及这些信号与其他设备产生的信号相比如何 凝血酶相互作用以及生长因子和激素，如 表皮生长因子和胰岛素。在建议的 研究内容：1)纯化和鉴定高亲和力 凝血酶受体的高效液相色谱和亲和层析， 检测碳水化合物与凝血酶受体的关系 相互作用，对一部分受体进行测序，构建DNA 基于该序列的探针筛选基因组和表达 文库，并尝试克隆和测序该基因 凝血酶受体，2)表征凝血酶产生的信号 酶活性和受体占有率包括 肌醇磷脂周转、钙离子动员、甘油二酯 释放、蛋白激酶C激活和特定蛋白 磷酸化，3)重建凝血酶与纯化的结合 决定蛋白质降解和构象的受体 改变，4)利用结合结构域合成肽 确定亲和力、大小、构象和 信号产生，5)决定细胞骨架元素的作用 受体锚定和跨膜信号和6) 鉴定和纯化与之相互作用的其他调节分子 凝血酶受体有助于评估该受体的效用 系统在不同的组织和不同的细胞功能。