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

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 alpha 2-macroglobulin, apoprotein E-enriched beta-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 (Kd) of 3.3 +/- 0.9 (S.D.) nM, with 380,000 +/- 190,000 (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 Ki 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 degrees C was determined to be 52 fmol/min per 10(6) cells, and supports a model for active recycling of the LRP receptor.

SUBMITTER: Iadonato SP

PROVIDER: S-EPMC1137774 | biostudies-other | 1993 Dec

REPOSITORIES: biostudies-other

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Project description:BackgroundTriacylglyerol-rich very low density lipoprotein (VLDL) particles are the primary carriers of fatty acids in the circulation and as such serve as a rich energy source for peripheral tissues. Receptor-mediated uptake of these particles is dependent upon prior association with apolipoprotein E (apoE-VLDL) and is brought about by cell surface heparan sulfate proteoglycans (HSPG) in some cell types and by the low density lipoprotein receptor-related protein (LRP) in others. Although LRP's role in apoE-VLDL uptake has been well studied, the identity of the HSPG family member that mediates apoE-VLDL uptake has not been established. We investigated if syndecan-1 (Syn-1), a transmembrane cell surface HSPG, is able to mediate the internalization of apoE-VLDL and examined the relationship between Syn-1 and LRP toward apoE-VLDL uptake. For this study, we used a human fibroblast cell line (GM00701) that expresses large amounts of LRP, but possesses no LDL receptor activity to eliminate its contributions toward apoE-VLDL uptake.ResultsAlthough LRP in these cells is fully active as established by substantial alpha2macroglobulin binding and internalization, uptake of apoE-VLDL is absent. Expression of human Syn-1 cDNA restored apoE-VLDL binding and uptake by these cells. Competition for this uptake with an LRP ligand-binding antagonist had little or no effect, whereas co-incubation with heparin abolished apoE-VLDL internalization. Depleting Syn-1 expressing cells of K+, to block clathrin-mediated endocytosis, showed no inhibition of Syn-1 internalization of apoE-VLDL. By contrast, treatment of cells with nystatin to inhibit lipid raft function, prevented the uptake of apoE-VLDL by Syn-1.ConclusionThese data demonstrate that Syn-1 is able to mediate apoE-VLDL uptake in human fibroblasts with little or no contribution from LRP and that the endocytic path taken by Syn-1 is clathrin-independent and relies upon lipid raft function. These data are consistent with previous studies demonstrating Syn-1 association with lipid raft domains.

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

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