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

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.