The purpose of the proposed project is to continue to identify and characterize the melanoma cell surface receptor for tissue-type plasminogen activator. The study is important because (i) plasminogen activation is by far the most important mechanism of extracellular proteolysis in human tumor cells, (ii) the essential role of the urokinase-type (uPA) and tissue-type (tPA) plasminogen activators in the process of plasminogen activation has now become well established, (iii) plasminogen activation is a cascade-like process which takes place on the cell surface wit surface-bound reactants, (iv) uPA binds to the cell surface through a high affinity receptor but a specific receptor protein for tPA has not been identified, (v) human melanoma cells synthesize and secrete predominantly tPA and utilize cell-bound tPA to generate cell- bound plasmin. Thus, we hypothesize that human melanoma cells represents an excellent cell system to study and isolate the cell surface receptor for tPA. The protein representing the melanoma tPA cell-surface receptor will be isolated from the human Bowes melanoma cell line by affinity chromatography, using a protein fragment corresponding to the Kringle-2 domain of tPA for binding. The N-terminus of the eluted and purified receptor will be microsequenced followed by cloning of the corresponding gene. The gene for the tPA receptor will be isolated by screening an expression library of Bowes melanoma cells with either the Kringle-2 peptide domain, monoclonal and polyclonal antibodies produced during these studies, or with oligonucleotides synthesized based on the available amino acid sequences. The gene will be sequenced, compared with Genbank sequences, and full-length cDNA will be expressed in mammalian cells. A functional link between the tPA receptor and the plasminogen activation process will be investigated. These results should shed light on the role of tPA receptor in progression of human melanoma cells.
| Bizik, J; Trancikova, D; Felnerova, D et al. (1997) Spatial orientation of tissue-type plasminogen activator bound at the melanoma cell surface. Biochem Biophys Res Commun 239:322-8 |
