ROLE OF CELL SURFACE IN REGULATION CELL PROLIFERATION

该项目的主要目标仍然是确定 因果关系参与调节的分子事件 细胞增殖的开始。 这些研究主要集中在 凝血酶与特定表面受体的相互作用 成纤维细胞和由此引起的跨膜信号 互动。 我们已经确定了高亲和力结合域 凝血酶并发现它与其他调节剂同源 分子包括组织纤溶酶原激活剂，表明 调节分子家族可能会产生其生物 通过与这种共同受体的相互作用发出信号。 在 此外，凝血酶与其他分子结合或相互作用 细胞参与止血和止血的各个方面 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信号产生的信号进行比较 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。 在提议的 研究我们将：1）纯化和表征高亲和力 使用 HPLC 和亲和色谱法检测凝血酶受体， 检查碳水化合物参与凝血酶受体 相互作用、对受体的一部分进行测序、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 酶活性和受体占据，包括 磷酸肌醇周转、钙动员、二酰甘油 释放、蛋白激酶 C 激活和特定蛋白 磷酸化，3) 重建与纯化的凝血酶结合 受体以确定蛋白水解和构象 改变，4)利用结合域合成肽 确定亲和力、大小、构象和之间的相关性 信号产生，5）确定细胞骨架元件的作用 受体锚定和跨膜信号传导以及 6) 识别和纯化与相互作用的其他调节分子 凝血酶受体以帮助评估该受体的效用 不同组织和不同细胞功能的系统。

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.