Cell-associated proteolytic cleavage of urokinase between growth factor-like and kringle domains commits it for rapid internalisation by LRP/CD91-dependent mechanism

A. Poliakov, V. Stepanova, I. Beloglazova, S. Ragozin, S. Mukhina, Dmitry Traktuev, V. Tkachuk

Research output: Contribution to journalArticle

Abstract

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 languageEnglish (US)
Number of pages1
JournalFibrinolysis and Proteolysis
Volume14
Issue numberSUPPL. 1
StatePublished - Dec 1 2000
Externally publishedYes

Fingerprint

Kringles
Urokinase-Type Plasminogen Activator
Intercellular Signaling Peptides and Proteins
U937 Cells
Fibrinolysin
Binding Sites
Antifibrinolytic Agents
Aprotinin
Plasminogen
Protein Sequence Analysis
Endocytosis
Proteolysis
Digestion
Escherichia coli
Ligands
Amino Acids

ASJC Scopus subject areas

  • Hematology

Cite this

Cell-associated proteolytic cleavage of urokinase between growth factor-like and kringle domains commits it for rapid internalisation by LRP/CD91-dependent mechanism. / Poliakov, A.; Stepanova, V.; Beloglazova, I.; Ragozin, S.; Mukhina, S.; Traktuev, Dmitry; Tkachuk, V.

In: Fibrinolysis and Proteolysis, Vol. 14, No. SUPPL. 1, 01.12.2000.

Research output: Contribution to journalArticle

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abstract = "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.",
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TY - JOUR

T1 - Cell-associated proteolytic cleavage of urokinase between growth factor-like and kringle domains commits it for rapid internalisation by LRP/CD91-dependent mechanism

AU - Poliakov, A.

AU - Stepanova, V.

AU - Beloglazova, I.

AU - Ragozin, S.

AU - Mukhina, S.

AU - Traktuev, Dmitry

AU - Tkachuk, V.

PY - 2000/12/1

Y1 - 2000/12/1

N2 - 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.

AB - 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.

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