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 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.
Highlights
Hepatic parenchymal cells play an essential role in Tissue-type plasminogen activator (t-PA)' is a serine prothe clearance of circulating tissue-type plasminogen tease that catalyzes the conversion of the zymogen plasminactivator (t-PA) in vivo as a major pathway in the ogen to the active serine protease plasmin
This inhibitionby the 39-kDa protein was not dueThe rapid removal of t-PA from the circulation has been to inhibition of complex formation between '261-t-PA demonstrated in uiuo in several animal species and man
Chemical cross-linking experiments demonstrated the direct interactionbetween '261-t-PA-PAI1 and lipoprotein receptor-related protein (LRP) on HepGZ cells as anti-LRP antibody, in addition to anti-t-PA and anti-plasminogen activatoirnhibitor type 1 (PAI-1) antibodies, was abletoimmunoprecipitate the '261-t-PA*PAI-1comaccepted that liver t-PA clearance is via receptor-mediated endocytosis
Summary
Following incubation for 5 h at 37 "C, cell monolayers were washed with binding buffer and used for ligand binding and chemical crosslinking experimentsas described below. After ligand binding at 4 "C, each cell monolayer was washed three times with PBSc buffer. Cell Culture-Maintenance of human hepatoma HepG2 cells has by incubating the cell monolayer with PBSc buffer containing 0.5 been described previously [18]. After 30 min at 4 "C, the reaction was quenched by minimum essential medium (with glutamine; GIBCO) supplemented washing the cell monolayer two times with Tris-buffered saline. Each aliquot of the cell lysate was brought to 0.5 ml with lysis buffer and mixed with 0.5 ml of PBSc buffer containing 1%(v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 1%(w/v) SDS, 0.5%(w/v) bovine serum albumin, and1mM phenylmethylsulfonyl fluoride (immunomixture). Scatchard analysis of the binding data frfoivme such experimentysielded 197,000
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