Mitogenic effects of urokinase on melanoma cells are independent of high affinity binding to the urokinase receptor.
The structural and functional properties of the urokinase-type plasminogen activator (u-PA) that are involved in the mitogenic effect of this proteolytic enzyme on human melanoma cells M14 and IF6 and the role of the u-PA receptor (u-PAR) in transducing this signal were analyzed. Native u-PA purified from urine induced a mitogenic response in quiescent IF6 and M14 cells that ranged from 25 to 40% of the mitogenic response obtained by fetal calf serum. The half-maximum response in M14 and IF6 cells was reached at u-PA concentrations of approximately 35 and 60 nM, respectively. Blocking the proteolytic activity of u-PA resulted in a 30% decrease of the mitogenic effect, whereas inhibition of plasmin activity did not alter the mitogenic effect. No mitogenic response was elicited by low molecular weight u-PA, lacking the growth factor domain and the kringle domain. The ATF domain of u-PA induced a mitogenic response that was similar to complete u-PA. Defucosylated ATF and recombinant u-PA purified from Escherichia coli lacking all post-translational modifications did not induce a mitogenic response. Blocking the interaction of u-PA with u-PAR, using a specific monoclonal antibody, did not alter the mitogenic effect induced by u-PA. The binding of radiolabeled u-PA to M14 and IF6 cells was characterized by high affinity binding mediated by u-PAR and low affinity binding to an unknown binding site. These results demonstrate that proteolytically inactive u-PA is able to induce a mitogenic response in quiescent melanoma cells in vitro by a mechanism that involves the ATF domain but is independent of high affinity binding to u-PAR. Furthermore, it suggests that u-PA is able to bind with low affinity to a hitherto unidentified membrane associated protein that could be involved in u-PA-induced signal transduction.
Koopman
J L
JL
Gaubius Laboratory, TNO Prevention and Health, 2301 CE Leiden, The Netherlands.
Slomp
J
J
de Bart
A C
AC
Quax
P H
PH
Verheijen
J H
JH
D002455:Cell Division / Q000187:drug effects; D006801:Humans; D008545:Melanoma / Q000201:enzymology / Q000378:metabolism / Q000473:pathology*; D008934:Mitogens / Q000378:metabolism / Q000494:pharmacology*; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D015398:Signal Transduction; D010959:Tissue Plasminogen Activator / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism / Q000494:pharmacology*
D008934:Mitogens; C524898:PLAUR protein, human; D017395:Plasminogen Activator Inhibitor 1; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator