Processing of type 1 plasminogen activator inhibitor (PAI-1) into the regulated secretory pathway.
To understand the processing of type 1 plasminogen activator inhibitor (PAI-1) into the storage granules of platelets, we utilized a eukaryotic expression vector (pRC/CMV) to transfer the human cDNA for PAI-1 into AtT-20 cells, a mouse pituitary cell line known to sort proteins in a regulated fashion. Immunofluorescence staining of PAI-1-transfected AtT-20 clones revealed co-localization of PAI-1 with an endogenously produced and stored hormone (i.e. adrenocorticotropic hormone, ACTH). Stimulation of PAI-1-transfected AtT-20 cells with a secretagogue resulted in the release of both active PAI-1 and the latent form. In comparison, PAI-1-transfected Chinese hamster ovary cells (i.e. a nonpackaging cell line) did not release PAI-1 in response to a secretagogue and exhibited immunoreactivity for PAI-1 primarily confined to the Golgi region. Percoll density gradient fractionation of AtT-20 cells revealed a codistribution of PAI-1 and ACTH in cellular compartments of the same density. The half-life of PAI-1 activity at 37 degrees C was prolonged in intact granules (t1/2, 5 h) in comparison with its half-life in lysed granules (t1/2, 2 h). These studies demonstrate the presence of a new functional property associated with the PAI-1 molecule that directs this inhibitor into the storage secretory pathway.
Gombau
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Scripps Research Institute, Department of Vascular Biology, La Jolla, California 92037.
Schleef
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RR
D000324:Adrenocorticotropic Hormone / Q000032:analysis / Q000096:biosynthesis; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism*; D002460:Cell Line; D003594:Cytoplasmic Granules / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D007700:Kinetics; D007797:Laminin / Q000032:analysis; D051379:Mice; D010911:Pituitary Neoplasms; D017395:Plasminogen Activator Inhibitor 1 / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D007797:Laminin; D017395:Plasminogen Activator Inhibitor 1; D000324:Adrenocorticotropic Hormone