The E3 ubiquitin ligase IDOL induces the degradation of the low density lipoprotein receptor family members VLDLR and ApoER2 - PubMed (original) (raw)

. 2010 Jun 25;285(26):19720-6.

doi: 10.1074/jbc.M110.123729. Epub 2010 Apr 28.

Sarah Duit, Pilvi Jalonen, Ruud Out, Lilith Scheer, Vincenzo Sorrentino, Rima Boyadjian, Kees W Rodenburg, Edan Foley, Laura Korhonen, Dan Lindholm, Johannes Nimpf, Theo J C van Berkel, Peter Tontonoz, Noam Zelcer

Affiliations

The E3 ubiquitin ligase IDOL induces the degradation of the low density lipoprotein receptor family members VLDLR and ApoER2

Cynthia Hong et al. J Biol Chem. 2010.

Abstract

We have previously identified the E3 ubiquitin ligase-inducible degrader of the low density lipoprotein receptor (LDLR) (Idol) as a post-translational modulator of LDLR levels. Idol is a direct target for regulation by liver X receptors (LXRs), and its expression is responsive to cellular sterol status independent of the sterol-response element-binding proteins. Here we demonstrate that Idol also targets two closely related LDLR family members, VLDLR and ApoE receptor 2 (ApoER2), proteins implicated in both neuronal development and lipid metabolism. Idol triggers ubiquitination of the VLDLR and ApoER2 on their cytoplasmic tails, leading to their degradation. We further show that the level of endogenous VLDLR is sensitive to cellular sterol content, Idol expression, and activation of the LXR pathway. Pharmacological activation of the LXR pathway in mice leads to increased Idol expression and to decreased Vldlr levels in vivo. Finally, we establish an unexpected functional link between LXR and Reelin signaling. We demonstrate that LXR activation results in decreased Reelin binding to VLDLR and reduced Dab1 phosphorylation. The identification of VLDLR and ApoER2 as Idol targets suggests potential roles for this LXR-inducible E3 ligase in the central nervous system in addition to lipid metabolism.

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Figures

FIGURE 1.

FIGURE 1.

The LXR pathway modulates the level of the VLDLR and ApoER2. A, immunoblot analysis of total SNB19 cell lysates cells following treatment with 1 μ

m

GW3965 (GW) or 1 μ

m

T0901317 (T0) for 24 h. B, expression of ABCA1, IDOL, and VLDLR was analyzed in SNB19 cells treated for 24 h with 1 μ

m

of the indicated ligand (n = 4). C, NIH-3T3 cells that stably produce either Vldlr or ApoER2 were treated for 24 h with 1 μ

m

ligand. Subsequently, total cell lysates were analyzed by immunoblotting. D, gene expression was determined in SNB19 cells that were infected with the indicated adenoviruses for 24 h and subsequently treated for an additional 24 h as shown. E, immunoblot analysis of these cells (n = 3). The blots are representative of at least two independent experiments. The bars and error bars represent the means ± S.D. ***, p < 0.001. DMSO, dimethyl sulfoxide.

FIGURE 2.

FIGURE 2.

The LXR-IDOL pathway modulates the level of the VLDLR in vivo. A, C57Bl/6 mice (n = 4–6 mice/group) were orally gavaged for 3 days with the indicated ligand (20 mg/kg/day). Expression of Abca1, Idol, and Vldlr in several metabolic tissues was determined. S. Muscle, skeletal muscle; WAT, white adipose tissue. B and C, immunoblot analysis of Vldlr in skeletal muscle of C57Bl/6 mice pharmacologically dosed with an LXR ligand. The intensity of Vldlr was normalized to that of tubulin and is plotted. Skeletal muscle from a Vldlr−/− mouse was used as a negative control. The bars and error bars represent the means ± S.D. *, p < 0.05. DMSO, dimethyl sulfoxide; GW, GW3965; T0, T0901317.

FIGURE 3.

FIGURE 3.

IDOL targets the LDLR, VLDLR, and ApoER2 for degradation and is evolutionarily conserved. A, alignment of the intracellular domains of the human (h) LDLR, human VLDLR, mouse (m) ApoER2, L. migratoria LpR (lmLpr), and mouse Lrp1b. The triangles represent the conserved lysine following the NPVY endocytic motif and the cysteine that is ubiquitinated in the LDLR (13). The ApoER2 sequence is cut because of space considerations. B–E, HEK 293T cells were co-transfected with the indicated plasmids, and the total cell lysates were analyzed by immunoblotting as indicated. WT, wild type; MT, mutant. The blots are representative of at least two independent experiments.

FIGURE 4.

FIGURE 4.

Post-translations regulation of the VLDLR by IDOL is dependent on ubiquitination of a single conserved lysine residue. A, adenovirally mediated expression of Idol in SNB19 cells leads to a decreased level of endogenous Vldlr. B, HEK 293T cells were co-transfected with a VLDLR and a control or Idol expression plasmid. 48 h after transfection cells were pulsed with [35S]methionine and [35S]cysteine for 15 min and chased as indicated. The samples were immunoprecipitated (IP) at the indicated time points after labeling. p and m represent the precursor and mature VLDLR protein, respectively. C, HEK 293T cells were co-transfected with VLDLR-GFP, Idol, and HA-ubiquitin expression plasmids as indicated. Subsequently, the cells were treated with vehicle or 25 μ

m

MG132 for 6 h. D, HEK 293T cells were co-transfected with Idol and wild type or mutant VLDLR-HA expression plasmids. Total cell lysates were analyzed by immunoblotting (IB). The blots are representative of at least two independent experiments.

FIGURE 5.

FIGURE 5.

Functional cross-talk between the LXR pathway and Reelin signaling. A, gene expression analysis of primary rat hippocampal neurons treated with 1 μ

m

GW3965 (GW) ligand for 24 h. The fold change in mRNA expression following GW treatment is plotted. Expression of the indicated genes in dimethyl sulfoxide-treated cells (DMSO) was set to 1 (n = 4–6). B, immunoblot analysis of primary rat hippocampal neurons treated with 1 μ

m

GW ligand for 24 h. The arrow indicates the Vldlr isoform that is modulated by LXR treatment (n = 4–6). C, SNB19 cells were treated with 1 μ

m

of the indicated LXR ligands or dimethyl sulfoxide for 24 h. Subsequently, binding of Reelin to the cells and the level of the indicated proteins were determined by immunoblotting (n = 3). D, NIH 3T3 cells that stably produce Dab1 and VLDLR (3T3V/D) or ApoER2 (3T3A/D) were treated with or without 1 μ

m

GW and Reelin or mock conditioned media as indicated. The level of total Dab1 and phospho-Dab1 were determined by immunoblotting. The blots are representative of at least two independent experiments. The bars and error bars represent the means ± S.D. **, p < 0.01; ***, p < 0.001. T0, T0901317.

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