RECEPTOR-MEDIATED ENDOCYTOSIS OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR BYLOW-DENSITY-LIPOPROTEIN RECEPTOR-RELATED PROTEIN ON HUMAN HEPATOMA HEPG2 CELLS

Hepatic parenchymal cells play an essential role in the clearance of c irculating tissue-type plasminogen activator (t-PA) in vivo as a major pathway in the regulation of plasma fibrinolytic activity. Previous s tudies 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 endocyt osis of the t-PA.PAI-1 complex are largely mediated by a recently iden tified low density lipoprotein receptor-related protein (LRP). A 39-kD a LRP receptor-associated protein that modulates ligand binding to LRP was found to bind specifically to HepG2 cells and to inhibit approxim ately 70-80% of specific I-125-t-PA.PAI-1 binding. This inhibition by the 39-kDa protein was not due to inhibition of complex formation betw een I-125-t-PA and PAI-1; instead, the 39-kDa protein inhibited I-125- t-PA.PAI-1 binding to LRP. Polyclonal anti-LRP antibody raised against purified human LRP also inhibited 70-80% of specific I-125-t-PA.PAI-1 binding. A similar extent of inhibition by the 39-kDa protein was als o observed for I-125-t-PA.PAI-1 endocytosis and degradation. Chemical cross-linking experiments demonstrated the direct interaction between I-125-t-PA.PAI-1 and LRP on HepG2 cells as anti-LRP antibody, in addit ion to anti-t-PA and anti-PAI-1 antibodies, was able to immunoprecipit ate the I-125-t-PA.PAI-1 complex following binding of I-125-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 [S-35]methionine-labeled HepG2 c ells. In addition, the direct binding of the 39-kDa protein to LRP on HepG2 cells was demonstrated by similar cross-linking experiments. Thu s, these data clearly show that LRP is the major cell-surface receptor responsible for t-PA.PAI-1 complex binding and endocytosis on human h epatoma HepG2 cells and extend the multifunctional nature of LRP as an endocytosis receptor for several structurally and functionally distin ct ligands.