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Mitogenic effects of urokinase on melanoma cells are independent of high affinity binding to the urokinase receptor

Author: Koopman, J.L. · Slomp, J. · Bart, A.C.W. de · Quax, P.H.A. · Verheijent, J.H.
Institution: Gaubius Instituut TNO
Source:Journal of Biological Chemistry, 50, 273, 33267-33272
Identifier: 234852
doi: doi:10.1074/jbc.273.50.33267
Keywords: Health · Cell Division · Humans · Melanoma · Mitogens · Plasminogen Activator Inhibitor 1 · Protein Binding · Receptors, Cell Surface · Signal Transduction · Tissue Plasminogen Activator · Tumor Cells, Cultured · Urinary Plasminogen Activator · Escherichia coli · Rickettsia sp. PAR


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.