Koopman J L, Slomp J, de Bart A C, Quax P H, Verheijen J H
Gaubius Laboratory, TNO Prevention and Health, 2301 CE Leiden, The Netherlands.
J Biol Chem. 1998 Dec 11;273(50):33267-72. doi: 10.1074/jbc.273.50.33267.
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.
分析了尿激酶型纤溶酶原激活剂（u-PA）的结构和功能特性，这些特性与该蛋白水解酶对人黑色素瘤细胞M14和IF6的促有丝分裂作用有关，还分析了u-PA受体（u-PAR）在转导该信号中的作用。从尿液中纯化的天然u-PA在静止的IF6和M14细胞中诱导了促有丝分裂反应，其范围为胎牛血清所获得促有丝分裂反应的25%至40%。M14和IF6细胞中达到最大反应一半时的u-PA浓度分别约为35和60 nM。阻断u-PA的蛋白水解活性导致促有丝分裂作用降低30%，而抑制纤溶酶活性并未改变促有丝分裂作用。缺乏生长因子结构域和kringle结构域的低分子量u-PA未引发促有丝分裂反应。u-PA的ATF结构域诱导的促有丝分裂反应与完整的u-PA相似。去岩藻糖基化的ATF和从大肠杆菌中纯化的缺乏所有翻译后修饰的重组u-PA未诱导促有丝分裂反应。使用特异性单克隆抗体阻断u-PA与u-PAR的相互作用，并未改变u-PA诱导的促有丝分裂作用。放射性标记的u-PA与M14和IF6细胞的结合特征为通过u-PAR介导的高亲和力结合以及与未知结合位点的低亲和力结合。这些结果表明，蛋白水解无活性的u-PA能够通过一种涉及ATF结构域但独立于与u-PAR的高亲和力结合的机制，在体外静止黑色素瘤细胞中诱导促有丝分裂反应。此外，这表明u-PA能够以低亲和力与一种迄今未鉴定的膜相关蛋白结合，该蛋白可能参与u-PA诱导的信号转导。
