A competitive chromogenic assay to study the functional interaction of urokinase-type plasminogen activator with its receptor.
Urokinase-type plasminogen activator (uPA) converts plasminogen to plasmin which degrades various extracellular matrix components. uPA is focused to the cell surface via binding to a specific receptor (uPAR, also termed CD87). uPAR-bound uPA mediates pericellular proteolysis in a variety of biological processes, e.g. cell migration, tissue remodeling and tumor invasion. We have developed a competitive microtiter plate-based chromogenic assay which allows the analysis of uPA/uPAR interaction. The plates are coated with recombinant uPAR expressed in Chinese hamster ovary (CHO) cells. Proteolytically active uPA (HMW-uPA) is added to the microtiter plate-attached uPAR. The amount of receptor-bound uPA is then determined indirectly via addition of plasminogen, which is activated to plasmin, followed by cleavage of a plasmin-specific chromogenic substrate. Substances interfering with binding of HMW-uPA to uPAR diminish the generation of plasmin, as indicated by a reduction of cleaved chromogenic substrate. This assay was used to analyze the inhibitory capacity of a variety of proteins and peptides, respectively, on the uPA/uPAR interaction: i) uPAR and uPAR-variants expressed in CHO cells, yeast or E. coli, ii) the aminoterminal fragment (ATF) of human uPA or yeast recombinant pro-uPA, iii) synthetic peptides derived from the sequence of the uPAR-binding region of uPA, and iv) antibodies directed against uPAR. This assay may be helpful in identifying uPA and uPAR analogues or antagonists which efficiently block uPA/uPAR interaction.
Rettenberger
P
P
Frauenklinik, Technischen Universität München, Germany.
Wilhelm
O
O
Oi
H
H
Weidle
U H
UH
Goretzki
L
L
Koppitz
M
M
Lottspeich
F
F
König
B
B
Pessara
U
U
Kramer
M D
MD
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D016466:CHO Cells; D002863:Chromogenic Compounds; D006224:Cricetinae; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D002863:Chromogenic Compounds; C524898:PLAUR protein, human; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins; D014568:Urokinase-Type Plasminogen Activator