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-BP(40)), which is identical to link protein (LP), and a 45-kDa UTI-BP (UTI-BP(45)). 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-BP(40) and UTI-BP(45) bind (125)I-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 (125)I-UTI and NG-UTI to the UTI-BP(40) and UTI-BP(45) confirm that the chondroitin sulfate side chain of UTI is required for its binding to UTI-BP(45). Analysis of binding of (125)I-UTI and NG-UTI to the cells suggests that low affinity binding sites are the UTI-BP(40) (which can bind NG-UTI), and the high affinity sites are the UTI-BP(45). 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.
Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Handacho 3600, Hamamatsu, Shizuoka, 431-3192, Japan.
Y
Y
Hirashima
H
H
Kobayashi
M
M
Suzuki
Y
Y
Tanaka
N
N
Kanayama
M
M
Fujie
T
T
Nishida
M
M
Takigawa
T
T
Terao
D001665:Binding Sites; D001667:Binding, Competitive; D015153:Blotting, Western; D019765:Chondroitin ABC Lyase / Q000494:pharmacology; D002809:Chondroitin Sulfates / Q000737:chemistry; D002813:Chondrosarcoma / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D006821:Hyaluronoglucosaminidase / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D008954:Models, Biological; D010703:Phorbol Esters / Q000494:pharmacology; D010960:Plasminogen Activators / Q000378:metabolism; D011485:Protein Binding; D015398:Signal Transduction; D013997:Time Factors; D014407:Tumor Cells, Cultured; D015854:Up-Regulation / Q000187:drug effects
D006023:Glycoproteins; D008024:Ligands; D010703:Phorbol Esters; D002809:Chondroitin Sulfates; D006821:Hyaluronoglucosaminidase; D010960:Plasminogen Activators; D019765:Chondroitin ABC Lyase; C028665:urinastatin