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

Guojun D Bu, E. A. Maksymovitch, A. L. Schwartz

Research output: Contribution to journalArticle

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Abstract

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 ~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.

Original languageEnglish (US)
Pages (from-to)13002-13009
Number of pages8
JournalJournal of Biological Chemistry
Volume268
Issue number17
StatePublished - 1993
Externally publishedYes

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LDL-Receptor Related Proteins
LDL Receptors
Hep G2 Cells
Tissue Plasminogen Activator
Endocytosis
Plasminogen Activator Inhibitor 1
Lipoprotein Receptors
Plasminogen Activators
Hepatocellular Carcinoma
Proteins
Antibodies
Ligands

ASJC Scopus subject areas

  • Biochemistry

Cite this

Receptor-mediated endocytosis of tissue-type plasminogen activator by low density lipoprotein receptor-related protein on human hepatoma HepG2 cells. / Bu, Guojun D; Maksymovitch, E. A.; Schwartz, A. L.

In: Journal of Biological Chemistry, Vol. 268, No. 17, 1993, p. 13002-13009.

Research output: Contribution to journalArticle

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abstract = "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 ~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.",
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