Receptor-mediated endocytosis of tissue-type plasminogen activator by low density lipoprotein receptor-related protein on human hepatoma HepG2 cells

• Published in: The Journal of biological chemistry Vol. 268; no. 17; pp. 13002 - 13009
• Main Authors: GUOJUN BU, MAKSYMOVITCH, E. A, SCHWARTZ, A. L
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 15.06.1993
• Subjects: Animals; Binding, Competitive; Biological and medical sciences; Carcinoma, Hepatocellular; Cell receptors; Cell structures and functions; Endocytosis; Fundamental and applied biological sciences. Psychology; Glutathione Transferase - biosynthesis; Humans; Iodine Radioisotopes; Kinetics; Liver Neoplasms; Low Density Lipoprotein Receptor-Related Protein-1; Miscellaneous; Molecular and cellular biology; Plasminogen Activator Inhibitor 1 - metabolism; Rats; Receptors, Immunologic - biosynthesis; Receptors, Immunologic - metabolism; Receptors, LDL - metabolism; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Recombinant Proteins - pharmacology; Tissue Plasminogen Activator - metabolism; Tumor Cells, Cultured; Human; Lipoprotein LDL; Ligand binding; Membrane receptor; Endocytosis; Immunoprecipitation reaction; Enzyme; Plasminogen activator
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)31486-8

Hepatic parenchymal cells play an essential role in the clearance of circulating tissue-type plasminogen activator (t-PA) in vivo as a major pathway in the regulation of plasma fibrinolytic activity. Previous studies have identified plasminogen activator inhibitor type 1 (PAI-1)-dependent t-PA-binding sites in the human hepatoma cell line HepG2. In this study, we demonstrate that receptor-mediated binding and endocytosis of the t-PA-PAI-1 complex are largely mediated by a recently identified low density lipoprotein receptor-related protein (LRP). A 39-kDa LRP receptor-associated protein that modulates ligand binding to LRP was found to bind specifically to HepG2 cells and to inhibit approximately 70-80% of specific 125I-t-PA.PAI-1 binding. This inhibition by the 39-kDa protein was not due to inhibition of complex formation between 125I-t-PA and PAI-1; instead, the 39-kDa protein inhibited 125I-t-PA.PAI-1 binding to LRP. Polyclonal anti-LRP antibody raised against purified human LRP also inhibited 70-80% of specific 125I-t-PA.PAI-1 binding. A similar extent of inhibition by the 39-kDa protein was also observed for 125I-t-PA.PAI-1 endocytosis and degradation. Chemical cross-linking experiments demonstrated the direct interaction between 125I-t-PA.PAI-1 and LRP on HepG2 cells as anti-LRP antibody, in addition to anti-t-PA and anti-PAI-1 antibodies, was able to immunoprecipitate the 125I-t-PA.PAI-1 complex following binding of 125I-t-PA.PAI-1 to HepG2 cells and cross-linking. This interaction of the t-PA.PAI-1 complex with LRP on HepG2 cells was also observed when the unlabeled t-PA.PAI-1 complex was cross-linked to [35S]methionine-labeled HepG2 cells. In addition, the direct binding of the 39-kDa protein to LRP on HepG2 cells was demonstrated by similar cross-linking experiments. Thus, these data clearly show that LRP is the major cell-surface receptor responsible for t-PA.PAI-1 complex binding and endocytosis on human hepatoma HepG2 cells and extend the multifunctional nature of LRP as an endocytosis receptor for several structurally and functionally distinct ligands.