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 h epatoma cell line. Further studies have implicated the low-density-lip oprotein-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, in cluding alpha2-macroglobulin, apoprotein E-enriched beta-very-low-dens ity lipoprotein, t-PA and Pseudomonas exotoxin A. In each case, bindin g of ligand to this receptor can be inhibited by addition of the 39 kD a LRP-receptor-associated protein. This protein, which co-purifies wit h the LR.P receptor, is the focus of our present study. I-125-labelled 39 kDa protein binds specifically and with high affinity to a single kinetic binding species on the rat MH1C1 cell surface. Scatchard analy sis reveals an equilibrium dissociation constant (K(a)) of 3.3 +/- 0.9 (S.D.) nM, with 380000 +/- 190000 (S.D.) binding sites per cell. Cros s-linking studies indicate that the specific interaction between MHC, cells and the 39 kDa protein is mediated by an association with the LR P receptor. The 39 kDa protein strongly inhibits binding of I-125-t-PA , with an apparent K(i) value of 0.5 nM. In addition, both unlabelled t-PA and I-125-labelled 39 kDa protein can be co-bound and cross-linke d to the same cell-associated LRP receptor. Endocytosis of cell-surfac e-associated 39 kDa protein was shown to be rapid, with internalized l igand subsequently degraded and released to the extracellular milieu. The rate of uptake and degradation of I-125-labelled 39 kDa protein at 37-degrees-C was determined to be 52 fmol/min per 10(6) cells, and su pports a model for active recycling of the LRP receptor.