A non-catalytic, human urokinase plasminogen activator derivative produced by mouse cells has full receptor binding activity

N. Pedersen, M. T. Masucci, L. B. Møller, F. Blasi

Research output: Contribution to journalArticlepeer-review

Abstract

Urokinase plasminogen activator (u-PA) binds a specific surface receptor present on many cell types as well as specific plasminogen activator inhibitors. However, different regions of the molecule are involved in these interactions: the amino terminal region binds the receptor, while the carboxy terminus binds the inhibitors. We designed an expression vector to code for a truncated human u-PA containing the receptor binding regions, but lacking the catalytic domain of u-PA. The mutated u-PA is expressed and secreted when the vector is stably transfected into mouse LB6 cells. The resulting protein (ΔF-u-PA) contains the amino terminal 164 amino acids of the human u-PA and an additional 30 amino acid extension at the C-terminus derived from the cloning procedure. The ΔF-u-PA efficiently binds to the human receptor and competes for binding of recombinant pro-u-PA, of active u-PA and of the proteolytically derived amino terminal fragment of u-PA (ATF) in both in vivo binding and in vitro crosslinking assays with the human u-PA receptor. It binds and competes for binding on receptors produced by a human monocyte like cell line (U937) and on a recombinant human u-PA receptor produced by mouse LB6 cells. The transfected LB6 cells produce 200-400 ng ΔF-u-PA per ml medium per day and the receptor-binding activity is retained after antibody affinity column purification and precipitation. ΔF-u-PA binding to receptors result in the inhibition of cell surface u-PA activity, as shown by a caseinolytic plaque assay. Co-cultivation of the ΔF-u-PA expressing cells with cells expressing the human u-PA receptor results in constitutive saturation of these receptors. In addition to providing a source of recombinant receptor binding u-PA derivative, these cells will be of use in understanding the role of the receptor in cell migration and invasion.

Original languageEnglish
Pages (from-to)155-164
Number of pages10
JournalFibrinolysis and Proteolysis
Volume5
Issue number3
DOIs
Publication statusPublished - 1991

ASJC Scopus subject areas

  • Hematology

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