CONTROL OF EXTRACELLULAR REGULATORY PROTEASES

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This proposal is based on our recent studies that have described a novel class of cell-secreted proteins, the "protease nexins.' The nexins are secreted by a variety of cells including normal human foreskin fibroblast, vascular endothelial cells of bovine and human origin, and fibroblast-like cells derived from rodent embryos. The nexine function to monitor and regulate specific extracellular serine proteases through a mechanism that involves linkage to the specific protease, followed by cellular binding, internalization, and intracellular degradation of the protease nexin: protease complex. This process is mediated by cellular receptors specific for the nexins. We have identified three different protease nexins, PN-I (Mr 38,000), PN-II (Mr 95,000), and PN-III (Mr. 31,000), each of which show specificity for linkage to different serine proteases. My research plan is to purify two of the nexins (PN-I and PN-II) to homogeneity from the serum-free conditioned medium of human foreskin fibroblasts, raise antibodies to them, and develop radioimmunoassays for them. This will enable me to perform quantitative studies on their cellular release and biosynthesis. I also plan to generate peptide maps to determine if the different nexins are structurally related, as well as to study the site specific cleavage in the nexins upon interaction with their target proteases, and determine the role of this cleavage in the regulation of their cellular binding. In the final phases of my studies, I will conduct experiments to answer questions regarding the control of nexin biosynthesis and release. Since it is known that nexin release is stimulated by certain agents, including the proteases for which they show linkage specificity, I will try to identify the cellular receptor system that mediates this stimulation. Finally, I will address the question of specificity of the induction of nexin release, i.e., coordinate or individual, the determine if this regulation occurs at the level of transcription, translation, or some post-translational modification.