Endocytosis and lysosomal delivery of tissue plasminogen activator-inhibitor 1 complexes in Hep G2 cells.
Receptor-mediated endocytosis of tissue-type plasminogen activator (t-PA)-plasminogen activator inhibitor type 1 (PAI-1) complexes results in their clearance by Hep G2 cells. After complexes are internalized, the t-PA component is degraded. However, neither the locus of intracellular catabolism nor the fate of PAI-1 has been elucidated. To characterize these aspects of t-PA-PAI-1 catabolism, the subcellular distribution of a prebound cohort of ligand molecules was delineated after internalization at 37 degrees C. 125I-t-PA.PAI-1 and t-PA.125I-PAI-1 were compared in separate experiments. After ligand uptake, intracellular vesicles were separated on density gradients. Internalized 125I-t-PA.PAI-1 concentrated initially in endosomes. After 20 minutes of uptake, the complex began to appear in lysosomes. Subsequently, low molecular weight labeled ligand fragments were detected in culture media. A panel of lysosomotropic agents, including primaquine, chloroquine, ammonium chloride, and a combination of leupeptin and pepstatin A, inhibited degradation. When t-PA.125I-PAI-1 rather than 125I-t-PA.PAI-1 was internalized, strikingly different results were observed. Although the kinetics of internalization and the intracellular itinerary were indistinguishable for the differently labeled complexes, the 125I-PAI-1 component of t-PA.125I-PAI-1 resisted rapid degradation. After a rapid loss of t-PA, the 125I-PAI-1 moiety persisted in lysosomes for up to 180 minutes. Thus, internalized t-PA.PAI-1 is targeted to lysosomes in which PAI-1 is relatively more stable than t-PA.
Underhill
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DM
Edward Mallinckrodt Departments of Cell Biology, St Louis Children's Hospital, Washington University School of Medicine, MO.
Owensby
D A
DA
Morton
P A
PA
Schwartz
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AL
D000643:Ammonium Chloride / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular; D002738:Chloroquine / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D008113:Liver Neoplasms; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D010436:Pepstatins / Q000494:pharmacology; D017395:Plasminogen Activator Inhibitor 1 / Q000302:isolation & purification / Q000378:metabolism*; D011319:Primaquine / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000302:isolation & purification / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
D007976:Leupeptins; D010436:Pepstatins; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D000643:Ammonium Chloride; C014195:Streptomyces pepsin inhibitor; D002738:Chloroquine; D001619:beta-N-Acetylhexosaminidases; D010959:Tissue Plasminogen Activator; C032854:leupeptin; D011319:Primaquine; C031375:pepstatin