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European Journal of Biochemistry, 1992
The binding of urokinase-type plasminogen activator (uPA) to its specific cell-surface receptor (... more The binding of urokinase-type plasminogen activator (uPA) to its specific cell-surface receptor (uPAR) localises the proteolytic cascade initiated by uPA to the pericellular environment. Inhibition of uPA activity or prevention ofuPA binding to uPAR might have a beneficial effect on disease states wherein this activity is deregulated, e.g. cancer and some inflammatory diseases. To this end, a bifunctional hybrid molecule consisting of the uPAR-binding growth-factor domain of uPA (amino acids 1-47; GFuPA) at the N-terminus of plasminogen-activator inhibitor type 2 (PAI-2) was produced in Succharomyces cerevisiae. The purified protein inhibited uPA with kinetics similar to placental or recombinant PAI-2 and was also found to bind to U937 cells and to FL amnion cells. GFuPA-PAI-2 competed with uPA, the N-terminal fragment of uPA and a proteolytic fragment of uPA (amino acids 4-43) in cell binding experiments, indicating that the molecule bound to the cells via uPAR. Hence, both the uPA-inhibitory and uPAR-binding domains of the hybrid molecule were functional, demonstrating the feasibility of the novel concept of introducing an unrelated, functional domain onto a member of the serine-protease-inhibitor superfamily. Cell-surface-bound urokinase (uPA) activity is believed to play a central role in the processes of tissue remodelling important for wound healing, mammary gland involution, ovulation and development and repair of the nervous system. Such activity is also important in a number of disease states such as cancer and the inflammatory diseases pemphigus and rheumatoid arthritis [1-31. uPA is concentrated locally by binding to a specific cell-surface receptor [4] where it initiates pericellular proteolysis by the activation of cell-surface-associated plasminogen. The binding of uPA to its receptor (uPAR) is via the N-terminal growth-factor (GF) domain, and residues 12-32 have been found to be important for this interaction [S]. Although this domain shows similarity to epidermal growth factor, the latter does not compete with uPA for binding to uPAR and, indeed, the specificity of the interaction is such that mouse uPA does not bind to human uPAR [5]. Urokinase can bind to uPAR in the proenzyme form [single-chain (sc) uPA] and the activation process and subsequent conversion of plasminogen to plasmin appear to be potentiated by these proteins being bound to the cell surface [6, 71. uPAR is a glycoprotein of approximately 55 kDa; the
European Journal of Biochemistry, 1992
The binding of urokinase-type plasminogen activator (uPA) to its specific cell-surface receptor (... more The binding of urokinase-type plasminogen activator (uPA) to its specific cell-surface receptor (uPAR) localises the proteolytic cascade initiated by uPA to the pericellular environment. Inhibition of uPA activity or prevention ofuPA binding to uPAR might have a beneficial effect on disease states wherein this activity is deregulated, e.g. cancer and some inflammatory diseases. To this end, a bifunctional hybrid molecule consisting of the uPAR-binding growth-factor domain of uPA (amino acids 1-47; GFuPA) at the N-terminus of plasminogen-activator inhibitor type 2 (PAI-2) was produced in Succharomyces cerevisiae. The purified protein inhibited uPA with kinetics similar to placental or recombinant PAI-2 and was also found to bind to U937 cells and to FL amnion cells. GFuPA-PAI-2 competed with uPA, the N-terminal fragment of uPA and a proteolytic fragment of uPA (amino acids 4-43) in cell binding experiments, indicating that the molecule bound to the cells via uPAR. Hence, both the uPA-inhibitory and uPAR-binding domains of the hybrid molecule were functional, demonstrating the feasibility of the novel concept of introducing an unrelated, functional domain onto a member of the serine-protease-inhibitor superfamily. Cell-surface-bound urokinase (uPA) activity is believed to play a central role in the processes of tissue remodelling important for wound healing, mammary gland involution, ovulation and development and repair of the nervous system. Such activity is also important in a number of disease states such as cancer and the inflammatory diseases pemphigus and rheumatoid arthritis [1-31. uPA is concentrated locally by binding to a specific cell-surface receptor [4] where it initiates pericellular proteolysis by the activation of cell-surface-associated plasminogen. The binding of uPA to its receptor (uPAR) is via the N-terminal growth-factor (GF) domain, and residues 12-32 have been found to be important for this interaction [S]. Although this domain shows similarity to epidermal growth factor, the latter does not compete with uPA for binding to uPAR and, indeed, the specificity of the interaction is such that mouse uPA does not bind to human uPAR [5]. Urokinase can bind to uPAR in the proenzyme form [single-chain (sc) uPA] and the activation process and subsequent conversion of plasminogen to plasmin appear to be potentiated by these proteins being bound to the cell surface [6, 71. uPAR is a glycoprotein of approximately 55 kDa; the