Characterization of Binding Properties of Urinary Trypsin Inhibitor to Cell-associated Binding Sites on Human Chondrosarcoma Cell Line HCS-2/8

Urinary trypsin inhibitor (UTI) forms membrane complexes with UTI-binding proteins (UTI-BPs) and initiates modulation of urokinase-type plasminogen activator (uPA) expression, which results in UTI-mediated suppression of cell invasiveness. It has been established that suppression of uPA expression and invasiveness by UTI is mediated through inhibition of protein kinase C-dependent signaling pathways and that human chondrosarcoma cell line HCS-2/8 expresses two types of UTI-BPs; a 40-kDa UTI-BP (UTI-BP₄₀), which is identical to link protein (LP), and a 45-kDa UTI-BP (UTI-BP₄₅). Here we characterize binding properties of UTI-BPs·UTI complexes in the cells. In vitro ligand blot, cell binding and competition assays, and Scatchard analyses demonstrate that both UTI-BP₄₀ and UTI-BP₄₅ bind ¹²⁵I-UTI. A deglycosylated form of UTI (NG-UTI), from which the chondroitin-sulfate side chain has been removed, binds only to UTI-BP ₄₀. Additional experiments, using various reagents to block binding of ¹²⁵I-UTI and NG-UTI to the UTI-BP₄₀ and UTI-BP ₄₅ confirm that the chondroitin sulfate side chain of UTI is required for its binding to UTI-BP₄₅. Analysis of binding of ¹²⁵I-UTI and NG-UTI to the cells suggests that low affinity binding sites are the UTI-BP₄₀ (which can bind NG-UTI), and the high affinity sites are the UTI-BP₄₅. In addition, UTI-induced suppression of phorbol ester stimulated up-regulation of uPA is inhibited by reagents that were shown to prevent binding of UTI to the 40- and 45-kDa proteins. We conclude that UTI must bind to both of the UTI-BPs to suppress uPA up-regulation.