Characterization of the receptor for protease nexin-I: Protease complexes on human fibroblasts

✍ Scribed by Eric W. Howard; Daniel J. Knauer

• Publisher: John Wiley and Sons
• Year: 1987
• Tongue: English
• Weight: 787 KB
• Volume: 131
• Category: Article
• ISSN: 0021-9541
• DOI: 10.1002/jcp.1041310219

✦ Synopsis

Fibroblasts as well as several other cell types, secrete a number of protease inhibitors into their culture media. Among these inhibitors are the protease nexins, a class of proteins which covalently bind serine proteases, thereby inactivating their specific targets. Protease nexin-I, first discovered in human foreskin fibroblasts, binds thrombin, plasmin, and urokinase with high affinity, forming covalently linked complexes. Human fibroblasts bind complexes of protease nexin-l and its target protease via a cell-surface, high-affinity receptor. We have analyzed a number of characteristics of this receptor, and found them to be typical of class I I receptors in general. At 4OC binding of PN4:protease complexes was competable by heparin. In addition, binding was independent of the particular protease bound to the PN-I; purified complexes of PN-I with thrombin or urokinase competed equipotently for [12511-thrombin:PN-l binding. As the pH of the binding buffer was lowered, binding to cells increased. A twofold increase in binding was attained by lowering the pH from 7.5to4.5.This phenomenon was not due to irreversible, pH-induced changes to either the cell surface or the labeled complexes. At 37OC, the removal of labeled complexes from culture medium was rapid; approximately 80% was removed by 4 hours under given conditions. The internalization of Complexes was also very rapid, with an estimated k, (endocytic rate constant) of 1.0 min-'. At neutral pH, fibroblasts bind complexes in a saturable manner. Scatchard analysis yields a receptor number of 250,000 per cell and a Kd of 1 nM.