Receptor binding and degradation of urokinase-type plasminogen activator by human mesangial cells.
The binding of [125I] labeled urokinase-type plasminogen activator (u-PA) was studied on human mesangial cells (MC) in culture. The binding of active [125I]u-PA at 37 degrees C reached a plateau after 30 minutes of incubation and remained stable for at least four hours. When the supernatant was analyzed with trichloracetic acid (TCA), TCA soluble radioactive material could be detected after a lag phase of 30 minutes, and then increased linearly for four hours. Analysis by electrophoresis on SDS PAGE and autoradiography of the cell associated radioactivity and of the intracellular content showed that active u-PA and u-PA complexed to plasminogen activator inhibitor type-1 (PAI-1) were bound to the cell surface, but only u-PA/PAI-1 complexes were internalized and degraded. Therefore, the Kd and the number of binding sites were determined by competitive inhibition curves at 4 degrees C using diisopropyl-fluorophosphate (DFP) u-PA. Scatchard plots showed a Kd = 400 +/- 30 pM, and Bmax = 240,000 +/- 25,000 sites/cell. Excess of the amino terminal fragment of u-PA (ATF) completely blocked the specific binding of [125I]u-PA, confirming that the binding of u-PA was independent of the presence of the active site and/or of the formation of complexes with PAI-1. 3H thymidine incorporation by mesangial cells after stimulation with 100 nM active u-PA showed that u-PA had a moderate but significant mitogenic effect, in contrast to inactive u-PA and ATF. However, this mitogenic effect was not accompanied by a proliferative effect.(ABSTRACT TRUNCATED AT 250 WORDS)
Nguyen
G
G
INSERM U 64, Hôpital Tenon, Paris, France.
Li
X M
XM
Peraldi
M N
MN
Zacharias
U
U
Hagège
J
J
Rondeau
E
E
Sraer
J D
JD
D001665:Binding Sites / Q000187:drug effects; D015152:Blotting, Northern; D002478:Cells, Cultured; D004705:Endocytosis; D005920:Glomerular Mesangium / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007531:Isoflurophate / Q000494:pharmacology; D010960:Plasminogen Activators / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D010738:Type C Phospholipases / Q000494:pharmacology; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
C524898:PLAUR protein, human; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D007531:Isoflurophate; D010738:Type C Phospholipases; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator