Interaction of a 39 kDa protein with the low-density-lipoprotein-receptor-related protein (LRP) on rat hepatoma cells

S. P. Iadonato, G. Bu, E. A. Maksymovitch, A. L. Schwartz

Research output: Contribution to journalArticle

59 Scopus citations

Abstract

We have recently described a PAI-1-independent pathway of tissue-type plasminogen activator (t-PA) uptake and degradation on the rat MH1C1 hepatoma cell line. Further studies have implicated the low-density-lipoprotein-receptor-related protein (LRP) as the mediator of plasminogen-activator inhibitor type-1-independent t-PA endocytosis. The LRP is a multi-functional receptor which is shared by a variety of ligands, including α2-macroglobulin, apoprotein E-enriched β-very-low-density lipoprotein, t-PA and Pseudomonas exotoxin A. In each case, binding of ligand to this receptor can be inhibited by addition of the 39 kDa LRP-receptor-associated protein. This protein, which co-purifies with the LRP receptor, is the focus of our present study. 125I-labelled 39 kDa protein binds specifically and with high affinity to a single kinetic binding species on the rat MH1C1 cell surface. Scatchard analysis reveals an equilibrium dissociation constant (K(d)) of 3.3 ± 0.9 (S.D.) nM, with 380000 ± 190000 (S.D.) binding sites per cell. Cross-linking studies indicate that the specific interaction between MH1C1 cells and the 39 kDa protein is mediated by an association with the LRP receptor. The 39 kDa protein strongly inhibits binding of 125I-t-PA, with an apparent K(i) value of 0.5 nM. In addition, both unlabelled t-PA and 125I-labelled 39 kDa protein can be co-bound and cross-linked to the same cell-associated LRP receptor. Endocytosis of cell-surface-associated 39 kDa protein was shown to be rapid, with internalized ligand subsequently degraded and released to the extracellular milieu. The rate of uptake and degradation of 125I-labelled 39 kDa protein at 37°C was determined to be 52 fmol/min per 106 cells, and supports a model for active recycling of the LRP receptor.

Original languageEnglish (US)
Pages (from-to)867-875
Number of pages9
JournalBiochemical Journal
Volume296
Issue number3
DOIs
StatePublished - Jan 1 1993

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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