Enhancement of the enzymatic activity of single-chain urokinase plasminogen activator by soluble urokinase receptor

A. A R Higazi*, R. L. Cohen, J. Henkin, D. Kniss, B. S. Schwartz, D. B. Cines

*Corresponding author for this work

    Research output: Contribution to journalArticlepeer-review

    85 Scopus citations

    Abstract

    Single-chain urokinase (scuPA), the unique form of urokinase secreted by cells, is converted to an active two-chain molecule through the cleavage of a single peptide bond by plasmin and other specific proteinases. Although scuPA may express limited enzymatic activity, its contribution to plasminogen activation on cell surfaces remains uncertain. Further, although it is well known that scuPA binds to a specific extracellular urokinase-type plasminogen activator receptor, the effect of this interaction on the enzymatic activity of scuPA has not been described. In the present paper we report that the binding of scuPA to cellular and to recombinant soluble urokinase-type plasminogen activator receptors (suPAR) increases its catalytic activity as measured by the cleavage of a urokinase-specific chromogenic substrate. suPAR increased the V(max) of scuPA 5-fold with little change in its K(m). suPAR also stimulated the plasminogen activator activity of scuPA by decreasing its K(m) for Glu-plasminogen from 1.15 μM to 0.022 mM and by increasing the k(cat) of this reaction from 0.0015 to 0.022 s-1. Preincubation of scuPA with suPAR also enhances its susceptibility to inhibition by plasminogen activator inhibitor type 1, consistent with exposure of its catalytic site. The activity of scuPA bound to suPAR is not accompanied by cleavage of scuPA, which continues to migrate as a single band in SDS-polyacrylamide gel electrophoresis under reducing conditions. Moreover, suPAR increases the plasminogen activator activity of a plasmin-insensitive variant, scuPA (scuPA-Glu158), as well. Enhancement of scuPA activity by suPAR is both prevented and reversed by its amino-terminal fragment (amino acids 1-135), which competes for receptor binding, suggesting that continued binding to the receptor is required for expression of scuPA's enzymatic activity. Thus, our data suggest that scuPA may undergo a reversible transformation between a latent and an active state. The urokinase receptor may induce or stabilize scuPA in its active conformation, thereby contributing to the initiation of plasminogen activation on cell surfaces.

    Original languageEnglish (US)
    Pages (from-to)17375-17380
    Number of pages6
    JournalJournal of Biological Chemistry
    Volume270
    Issue number29
    DOIs
    StatePublished - 1995

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology

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