Structural requirements for the growth factor activity of the amino-terminal domain of urokinase.
High molecular weight urokinase-type plasminogen activator (uPA) in which proteolytic activity was inactivated (diisopropyl fluorophosphate (DFP)-uPA), its amino-terminal fragment (ATF, amino acids (aa) 1-143), and fucosylated and defucosylated growth factor domains (GFD, aa 4-43) were tested for growth-promoting effects and binding in human SaOS-2 osteosarcoma cells and U-937 lymphoma cells. DFP-uPA, ATF, and both the fucosylated and defucosylated GFD were capable of competing with 125I-ATF for binding to both SaOS-2 and U-937 cells. DFP-uPA, ATF, and fucosylated GFD were also mitogenic in SaOS-2 cells and increased cell numbers. However, defucosylated GFD was nonmitogenic in SaOS-2 cells and did not stimulate cell proliferation, even though it bound to these cells in a manner equivalent to the fucosylated GFD. A nonglycosylated high molecular weight uPA expressed and purified from Escherichia coli inhibited 125I-ATF binding to SaOS-2 cells but was also nonmitogenic. No mitogenic activity was observed in U-937 cells treated with the uPA forms capable of eliciting a mitogenic response in SaOS-2 cells. Proteolytically prepared kringle domain (aa 47-135) and low molecular weight uPA (aa 144-411) did not compete for 125I-ATF binding and did not elicit any mitogenic response in either of the cell lines tested. In addition, tissue plasminogen activator (tPA), which has been shown to be homologous to uPA in its growth factor domain and is also fucosylated, did not inhibit 125I-ATF binding nor elicit any mitogenic response. These results demonstrate that the GFD, implicated in binding to the uPA receptor, is also responsible for growth factor like activity in SaOS-2 cells and that the fucosylation at Thr18 within this domain may serve as a molecular trigger in eliciting this response.
Rabbani
S A
SA
Department of Medicine, Royal Victoria Hospital, McGill University, Montreal, Canada.
Mazar
A P
AP
Bernier
S M
SM
Haq
M
M
Bolivar
I
I
Henkin
J
J
Goltzman
D
D
D001667:Binding, Competitive; D015152:Blotting, Northern; D002460:Cell Line; D004261:DNA Replication / Q000187:drug effects*; D006133:Growth Substances / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D007531:Isoflurophate / Q000494:pharmacology; D007700:Kinetics; D008223:Lymphoma; D008970:Molecular Weight; D012516:Osteosarcoma; D010446:Peptide Fragments / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D013936:Thymidine / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*
D006133:Growth Substances; C524898:PLAUR protein, human; D010446:Peptide Fragments; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D007531:Isoflurophate; D014568:Urokinase-Type Plasminogen Activator; D013936:Thymidine