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

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.