title: Characterization of 125I-tissue plasminogen activator binding to cerebellar granule neurons.
abstract: Mouse cerebellar cells in culture secrete tissue plasminogen activator (tPA) into the culture medium. Fibrin overlays have shown tPA to be associated with granule neurons in these cultures. This cell associated tPA can be displaced by extensive washing of the cells or by a brief lowering of the pH (less than 4), which leads to a loss of fibrinolytic activity by the cells. Incubation of these fibrinolytically inactive cells with exogenously added murine tPA leads to the restoration of the fibrinolytic activity, indicating the presence of tPA binding sites on these granule neurons. Using 125I-tPA, the binding to cerebellar granule neurons is rapid, saturable, specific, high affinity (Kd = 50 pM) and reversible. Both murine and human tPA compete with 125I-tPA for binding, with both murine and human urokinase (uPA) as well as human thrombin and plasminogen fail to compete. Neither the catalytic site nor the carbohydrate moiety of tPA appear to be involved in the binding, since both diisopropyl-fluorophosphate-treated tPA and endoglycosidase-H-treated tPA compete with 12I-tPA for binding. Furthermore, epidermal growth factor does not compete well with tPA for binding even at a 10:1 molar excess, suggesting that the epidermal growth factor-like (EGF) domain of tPA may not be involved in the binding mechanism. Autoradiography and antibody immunofluorescence show the specific tPA binding is to granule neurons in these cultures. Thus, granule neurons possess tPA receptors on their surface, where this protease binds retaining is functional activity and may play a role in cell and axon migration.
authors.affiliation: Department of Biochemistry, Biophysics, and Genetics, University of Colorado Health Science Center, Denver 80262.
authors.forename: S
authors.initials: S
authors.lastname: Verrall
authors.forename: N W
authors.initials: NW
authors.lastname: Seeds
mesh_terms: D000818:Animals; D001345:Autoradiography; D002462:Cell Membrane / Q000378:metabolism; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D007700:Kinetics; D051379:Mice; D011956:Receptors, Cell Surface / Q000378:metabolism*; D010959:Tissue Plasminogen Activator / Q000378:metabolism*
chemical_list: D011956:Receptors, Cell Surface; D010959:Tissue Plasminogen Activator