Urokinase activates calcium-dependent potassium channels in U937 cells via calcium release from intracellular stores.
The urokinase receptor (uPAR) is highly expressed in the human promyelocytic cell line U937 and contributes to transmembrane signalling. However, the signalling mechanisms are poorly understood. We used the patch-clamp technique to demonstrate that urokinase (uPA) binds to uPAR and thereby stimulates Ca(2+)-activated K+ channels in U937 cells. uPA transiently increased K+ currents within 30 s. The K+ currents were pertussis toxin-sensitive and were also observed in Ca(2+)-free solution. However, when cells were dialysed with EGTA, uPA did not affect K+ currents. The intracellular Ca2+ response to uPA was independent of extracellular Ca2+, was pertussis toxin-sensitive, and was blocked by both thapsigargin and the phospholipase C inhibitor U-73122. The uPA-induced increase in intracellular Ca2+ was independent of uPA proteolytic activity. Furthermore, uPA initiated a rapid formation of inositol 1,4, 5-trisphosphate [Ins(1,4,5)P3]. The amino-terminal uPA fragment and uPA inactivated with diisopropyl fluorophosphate or with inhibitory antibody, elicited the same Ca2+ signal. On the other hand, Ca2+ signalling required the intact uPAR because the effects were abrogated by PtdIns-specific phospholipase C, which removes the uPAR from the cell surface. The prevention of glycosyl phosphatidylinositol moiety synthesis and interference with uPAR anchoring to the cell surface using mannosamine also abolished Ca2+ signals. Taken together, our findings indicate that uPA binds to uPAR and stimulates the production of Ins(1,4,5)P3 via a G-protein- and phospholipase C-dependent mechanism. Ins(1,4,5)P3 in turn liberates Ca2+ from intracellular stores, which leads to the stimulation of Ca(2+)-activated K+ channels.
Christow
S P
SP
Charité Humboldt University Berlin, Franz Volhard Clinic and Max-Delbrück Center for Molecular Medicine, Germany.
Bychkov
R
R
Schroeder
C
C
Dietz
R
R
Haller
H
H
Dumler
I
I
Gulba
D C
DC
D002118:Calcium / Q000378:metabolism*; D004553:Electric Conductivity; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D015640:Ion Channel Gating; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism*; D015221:Potassium Channels / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D015398:Signal Transduction; D020298:U937 Cells; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
C524898:PLAUR protein, human; D015221:Potassium Channels; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D015544:Inositol 1,4,5-Trisphosphate; D014568:Urokinase-Type Plasminogen Activator; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D011188:Potassium; D002118:Calcium