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 p roteins (UTI-BPs) and initiates modulation of urokinase-type plasminogen ac tivator (uPA) expression, which results in UTI-mediated suppression of cell invasiveness, It has been established that suppression of uPA expression a nd invasiveness by UTI is mediated through inhibition of protein kinase C-d ependent 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 ident ical to link protein (LP), and a 45-kDa UTI-BP (UTI-BP40). Here we characte rize binding properties of UTI-BPs UTI complexes in the cells, In vitro lig and blot, cell binding and competition assays, and Scatchard analyses demon strate that both UTI-BP40 and UTP-BP45 bind I-125-UTL A deglycosylated form of UTI (NG-UTI), from which the chondroitin-sulfate side chain has been re moved, binds only to UTI-BP40 Additional experiments, using various reagent s to block binding of I-125-UTI and NG-UTI to the UTI-BP40 and UTI-BP45 con firm that the chondroitin sulfate Side chain of UTI is required for its bin ding to UTI-BP45 analysis of binding of I-125-UTI and NG-UTI to the cells s uggests 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-induc ed suppression of phorbol ester stimulated up-regulation of uPA is inhibite d by reagents that were shown to prevent binding of UTI to the 40- and 45-k Da proteins. We conclude that UTI must bind to both of the UTI-BPs to suppr ess uPA up-regulation.