Cleavage of cell surface proteins by thrombin.
This study was based on our previous findings that the mitogenic action of thrombin on cultured fibroblasts can result from interaction of thrombin with the cell surface in the absence of internalization, and that the proteolytic activity of thrombin is required for stimulation of cell division. This prompted us to look for thrombin-mediated cleavages using 2-dimensional gel electrophoresis of labeled cell surface proteins. Surface membrane components were labeled by 3 procedures: 1) proteins were labeled by lactoperoxidase-catalyzed iodination using 125I-; 2) galactose and galactosamine residues of glycoproteins were oxidized with galactose oxidase and reduced with 3H-NaBH4; and 3) glycoproteins were metabolically labeled by incubating cells with 3H-fucose. labeling with the first 2 procedures was carried out after thrombin treatment; in contrast, cells metabolically labeled with 3H-fucose were subsequently treated with thrombin to look for proteolytic cleavages. Collectively, these studies indicated that only about 5 cell surface proteins were thrombin-sensitive, consistent with the high specificity of this protease. Each of the labeling procedures revealed a thrombin-sensitive cell surface glycoprotein which was identified as fibronectin by immunoprecipitation experiments. In addition, cell surface proteins of about 140K and 55K daltons were thrombin-sensitive. However, cell surface proteins of about 45K daltons and 130K to 150K daltons were increased after thrombin treatment. These experiments were conducted on an established line of Chinese hamster lung cells with the eventual goal of studying thrombin-mediated cleavages of cell surface proteins in a large number or in cloned populations derived from this line that are either responsive or unresponsive to the mitogenic action of thrombin. This approach should permit identification of proteolytic cleavages tha are necessary for thrombin-stimulated cell division.
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D005692:Galactose Oxidase; D006023:Glycoproteins / Q000032:analysis*; D008168:Lung; D008565:Membrane Proteins / Q000032:analysis*; D008934:Mitogens; D008970:Molecular Weight; D013917:Thrombin / Q000378:metabolism*
D006023:Glycoproteins; D008565:Membrane Proteins; D008934:Mitogens; D005692:Galactose Oxidase; D013917:Thrombin