Receptor-mediated endocytosis of tissue-type plasminogen activator by the human hepatoma cell line Hep G2.
Receptor-mediated endocytosis of tissue-type plasminogen activator (t-PA) was characterized with the human hepatoma cell line Hep G2. At 4 degrees C binding of 125I-t-PA to Hep G2 cells is rapid, specific, saturable, and reflective of a homogeneous population of 76,000 high-affinity surface sites per cell (Kd = 3.7 nM). The kinetics of 125I-t-PA binding to its receptor are characterized by rate constants for association (k1 = 1.2 x 10(6) min-1 M-1) and dissociation (k-1 = 0.001 min-1). A specific glycosylation pattern does not appear to be required for binding. Binding does not appear to be mediated by other recognized hepatic receptor systems. At 37 degrees C a single cohort of bound 125I-t-PA molecules disappears rapidly from the cell surface. Ligand then accumulates intracellularly. Thereafter, the intracellular concentration of ligand declines simultaneously with the release of ligand degradation products into the media. In the continued presence of 125I-t-PA at 37 degrees C the concentration of cell-associated ligand plateaus after 30 min with the concomitant appearance of low molecular weight 125I-labeled fragments in the media. Cumulative degradation then increases linearly with time. Under steady state conditions half-maximal ligand uptake and degradation is 26.6 nM and maximal rate of catabolism is 1.2 pmol/10(6) cells/h. At saturating ligand concentrations uptake and degradation by Hep G2 cells continue linearly for at least 6 h even in the absence of protein synthesis. During this period the cumulative ligand uptake exceeds the total cellular capacity of binding sites, consistent with receptor recycling. We conclude that t-PA clearance in human Hep G2 cells involves ligand binding, uptake, and degradation mediated by a novel high-capacity, high-affinity specific receptor system.
Owensby
D A
DA
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
Sobel
B E
BE
Schwartz
A L
AL
D001667:Binding, Competitive; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D004705:Endocytosis; D006801:Humans; D007700:Kinetics; D008113:Liver Neoplasms / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000378:metabolism*
D003513:Cycloheximide; D010959:Tissue Plasminogen Activator