There are at least two receptor systems recognising urokinase (u-PA) on the cell surface: the high affinity binding site U-PAR/CD87 and the low affinity binding site LRP/CD91. This elaborate system tightly regulates u-PA binding, proteolytic activation and interaction with its inhibitors resulting in u-PA inactivation and prompt internalisation. In the present study using recombinant u-PA forms expressed in E. coli, i.e. full-length single-chain u-PA (r-uPA), and singlechain u-PA form without growth factor-like domain (r-uPA(GFD-)), we have defined a new mechanism of u-PA internalisation dependent on plasmin associated proteolysis within linker region between u-PA's growth factor-like and kringle domains. Using ligand precipitation approach we have shown that in contrast to the full length r-uPA, the r-uPA(GFD-) form efficiently precipitated LRP/CD91 from U937 cell lysates and did not interact with U-PAR/CD87. Utilising biotinylated and 125I-labeled uPA forms we have found that both r-uPA and u-PA(GFD-) specifically bound to the U937 cell surface but exhibited different rates of their internalisation and degradation. When l2'l-r-uPA and '25J-ruPA(GFD') were preincubated with U937 cells at 4°C and subsequently transferred to 37°C, more than 80% of the bound 125I-r-uPA(GFD') was internalised and degraded during 30 min of incubation at 37°C However, at that time significant part of the 125I-r-uPA was retained on the cell surface and only 10% of the bound protein was degraded after 60 min of incubation at 37°C. Taking into account that binding of the l25I-r-uPA(GFD-) to U937 cells was completely inhibited by excess of RAP, we consider that 125I-r-uPA(GFD-) binding, internalisation and degradation are mediated mostly by LRP/CD91. We have demonstrated that a conversion of r-uPA into r-uPA(GFD') took place upon binding to U937 cells at 4°C Besides, the generated r-uPA(GFD-) degraded quickly when cells were transferred to 37°C. The r-uPA degradation was strongly dependent on cell-associated plasmin formation because the generation of r-uPA(GFD') was accelerated upon preincubation of cells with plasminogen and diminished in the presence of plasmin inhibitor aprotinin. Partial amino acid sequencing of proteolytic products of r-uPA exposed to in vitro digestion with plasmin has revealed that r-uPA(GFD') (amino acids 47-411) was one of the main forms generated. To sum up, cell associated proteolytic cleavage of r-uPA within linker region between growth factor-like and kringle domains may result in dissociation of uPA from uPAR/CD87 with concomitant binding to LRP/CD91 and rapid endocytosis. Furthermore, it might be supposed that initial interaction with U-PAR/CD87 not only regulates u-PA's proteolytic and signalling functions but could protect u-PA from further internalisation keeping it for a long time on the cell surface.
|Original language||English (US)|
|Number of pages||1|
|Journal||Fibrinolysis and Proteolysis|
|Issue number||SUPPL. 1|
|State||Published - Dec 1 2000|
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