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.
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