ROLE OF CELL SURFACE IN REGULATION CELL PROLIFERATION

该项目的主要目标仍然是确定 分子事件的因果关系参与调节 启动细胞增殖。 这些研究的重点是 凝血酶与细胞表面特异性受体相互作用 成纤维细胞及其引起的跨膜信号 互动 我们已经确定了高亲和力结合域 凝血酶，并发现它是同源的其他调节 分子包括组织纤溶酶原激活剂，这表明， 调节分子家族可以产生它们的生物学活性， 通过与这个共同受体的相互作用传递信号。 在 此外，凝血酶结合或与其他分子的相互作用 细胞涉及止血的各个方面， 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促有丝分裂信号 以及这些信号如何与其他 凝血酶相互作用以及生长因子和激素，例如 表皮生长因子（EGF）和胰岛素。 拟议 研究我们将：1）纯化和表征高亲和力的 使用HPLC和亲和色谱法， 检查凝血酶受体中的碳水化合物参与 相互作用，测序受体的一部分，构建DNA 基于该序列的探针筛选基因组和表达 文库，并试图克隆和测序的基因， 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 磷酸肌醇周转，Ca++动员，甘油二酯 释放、蛋白激酶C激活和特异性蛋白 磷酸化，3）重建凝血酶结合至纯化的 受体来决定蛋白水解和构象 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）确定细胞骨架元件的作用 在受体锚定和跨膜信号传导中，以及6） 鉴定并纯化与之相互作用的其它调节分子， 凝血酶受体，以帮助评估这种受体的效用 系统在各种组织和不同的细胞功能。

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.