The apoE isoform binding properties of the VLDL receptor reveal marked differences from LRP and the LDL receptor

Jose Ruiz,Diana Kouiavskaia,Molly Migliorini,Susan Robinson,Evgueni L Saenko,Natalia Gorlatova,Donghua Li,Daniel Lawrence,Bradley T Hyman,Karl H Weisgraber,Dudley K Strickland

doi:10.1194/jlr.m500114-jlr200

Abstract

Apolipoprotein E (apoE) associates with lipoproteins and mediates their interaction with members of the LDL receptor family. ApoE exists as three common isoforms that have important distinct functional and biological properties. Two apoE isoforms, apoE3 and apoE4, are recognized by the LDL receptor, whereas apoE2 binds poorly to this receptor and is associated with type III hyperlipidemia. In addition, the apoE4 isoform is associated with the common late-onset familial and sporadic forms of Alzheimer's disease. Although the interaction of apoE with the LDL receptor is well characterized, the specificity of other members of this receptor family for apoE is poorly understood. In the current investigation, we have characterized the binding of apoE to the VLDL receptor and the LDL receptor-related protein (LRP). Our results indicate that like the LDL receptor, LRP prefers lipid-bound forms of apoE, but in contrast to the LDL receptor, both LRP and the VLDL receptor recognize all apoE isoforms. Interestingly, the VLDL receptor does not require the association of apoE with lipid for optimal recognition and avidly binds lipid-free apoE. It is likely that this receptor-dependent specificity for various apoE isoforms and for lipid-free versus lipid-bound forms of apoE is physiologically significant and is connected to distinct functions for these receptors.

Highlights

Apolipoprotein E associates with lipoproteins and mediates their interaction with members of the LDL receptor family
Binding of Apolipoprotein E (apoE) isoforms to LDL receptorrelated protein (LRP) and the VLDL receptor Normally, apoE only binds to the LDL receptor when incorporated into lipoprotein particles
The results reveal that all three apoE isoforms are readily internalized in VLDL receptor-transfected cells in a process that is inhibited by receptor-associated protein (RAP) (Fig. 4)

Summary

EXPERIMENTAL PROCEDURES

The soluble VLDL receptor fragment containing ligand binding repeats 1–8 (sVLDLr1–8) was prepared and characterized as described [26]. Anti-VLDL receptor IgG 5F3 and anti-apoE IgG 3H1 were radiolabeled with 125I (Amersham Pharmacia Biotech, Piscataway, NJ) to a specific activity ranging from 2 to 10 ␮Ci/␮g protein using Iodogen (Pierce Chemical Co., Rockford, IL) For these assays, wild-type 293 cells, 293/VLDLR transfected cells, LRP-expressing mouse embryonic fibroblasts, or PEA-13 cells (LRP-deficient) were seeded onto 6-well plates (precoated with 0.1% gelatin) as indicated and grown overnight at 37ЊC in 5% CO2. Cells were incubated with assay medium containing radiolabeled proteins in the absence or presence of excess unlabeled competitors as indicated at 37ЊC In those experiments measuring the internalization of apoE using radiolabeled antibody 3H1, we confirmed that the uptake of this monoclonal antibody was totally dependent upon the addition of exogenous apoE. This medium was harvested after 48 h of incubation, subjected to immunoblot analysis using anti-myc antibody to detect recombinant proteins, and used in the binding assays

RESULTS

25 Ϯ 10 nb nb

DISCUSSION