The adaptor protein PID1 regulates receptor-dependent endocytosis of postprandial triglyceride-rich lipoproteins.

ObjectiveInsulin resistance is associated with impaired receptor dependent hepatic uptake of triglyceride-rich lipoproteins (TRL), promoting hypertriglyceridemia and atherosclerosis. Next to low-density lipoprotein (LDL) receptor (LDLR) and syndecan-1, the LDLR-related protein 1 (LRP1) stimulated by insulin action contributes to the rapid clearance of TRL in the postprandial state. Here, we investigated the hypothesis that the adaptor protein phosphotyrosine interacting domain-containing protein 1 (PID1) regulates LRP1 function, thereby controlling hepatic endocytosis of postprandial lipoproteins.MethodsLocalization and interaction of PID1 and LRP1 in cultured hepatocytes was studied by confocal microscopy of fluorescent tagged proteins, by indirect immunohistochemistry of endogenous proteins, by GST-based pull down and by immunoprecipitation experiments. The in vivo relevance of PID1 was assessed using whole body as well as liver-specific Pid1-deficient mice on a wild type or Ldlr-deficient (Ldlr-/-) background. Intravital microscopy was used to study LRP1 translocation in the liver. Lipoprotein metabolism was investigated by lipoprotein profiling, gene and protein expression as well as organ-specific uptake of radiolabelled TRL.ResultsPID1 co-localized in perinuclear endosomes and was found associated with LRP1 under fasting conditions. We identified the distal NPxY motif of the intracellular C-terminal domain (ICD) of LRP1 as the site critical for the interaction with PID1. Insulin-mediated NPxY-phosphorylation caused the dissociation of PID1 from the ICD, causing LRP1 translocation to the plasma membrane. PID1 deletion resulted in higher LRP1 abundance at the cell surface, higher LDLR protein levels and, paradoxically, reduced total LRP1. The latter can be explained by higher receptor shedding, which we observed in cultured Pid1-deficient hepatocytes. Consistently, PID1 deficiency alone led to increased LDLR-dependent endocytosis of postprandial lipoproteins and lower plasma triglycerides. In contrast, hepatic PID1 deletion on an Ldlr-/- background reduced lipoprotein uptake into liver and caused plasma TRL accumulation.ConclusionsBy acting as an insulin-dependent retention adaptor, PID1 serves as a regulator of LRP1 function controlling the disposal of postprandial lipoproteins. PID1 inhibition provides a novel approach to lower plasma levels of pro-atherogenic TRL remnants by stimulating endocytic function of both LRP1 and LDLR in the liver

Low Density Lipoprotein Receptor-Related Protein 1 Dependent Endosomal Trapping and Recycling of Apolipoprotein E

BACKGROUND: Lipoprotein receptors from the low density lipoprotein (LDL) receptor family are multifunctional membrane proteins which can efficiently mediate endocytosis and thereby facilitate lipoprotein clearance from the plasma. The biggest member of this family, the LDL receptor-related protein 1 (LRP1), facilitates the hepatic uptake of triglyceride-rich lipoproteins (TRL) via interaction with apolipoprotein E (apoE). In contrast to the classical LDL degradation pathway, TRL disintegrate in peripheral endosomes, and core lipids and apoB are targeted along the endocytic pathway for lysosomal degradation. Notably, TRL-derived apoE remains within recycling endosomes and is then mobilized by high density lipoproteins (HDL) for re-secretion. The aim of this study is to investigate the involvement of LRP1 in the regulation of apoE recycling. PRINCIPAL FINDINGS: Immunofluorescence studies indicate the LRP1-dependent trapping of apoE in EEA1-positive endosomes in human hepatoma cells. This processing is distinct from other LRP1 ligands such as RAP which is efficiently targeted to lysosomal compartments. Upon stimulation of HDL-induced recycling, apoE is released from LRP1-positive endosomes but is targeted to another, distinct population of early endosomes that contain HDL, but not LRP1. For subsequent analysis of the recycling capacity, we expressed the full-length human LRP1 and used an RNA interference approach to manipulate the expression levels of LRP1. In support of LRP1 determining the intracellular fate of apoE, overexpression of LRP1 significantly stimulated HDL-induced apoE recycling. Vice versa LRP1 knockdown in HEK293 cells and primary hepatocytes strongly reduced the efficiency of HDL to stimulate apoE secretion. CONCLUSION: We conclude that LRP1 enables apoE to accumulate in an early endosomal recycling compartment that serves as a pool for the intracellular formation and subsequent re-secretion of apoE-enriched HDL particles

TRL-derived apoE is internalized into peripheral endosomal compartments.

<p>Cy5-apoE-TRL and Cy3-RAP were prepared and protein labelling was analyzed by SDS-PAGE and subsequent in-gel fluorescence detection (A). Incubation of HuH7 cells without (B) and with Cy5-apoE-TRL (C) for 30 min at 37°C resulted in a punctuated endosomal pattern indicating receptor-mediated endocytosis. Nuclei were visualized by DAPI and appear in blue. Bar is 20 µm.</p

LRP1-dependent apoE recycling.

<p>HEK293 cells were transfected with pFB-LRP-EGFP, an shRNA vector against LRP1 or EGFP as control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029385#pone.0029385-Laatsch2" target="_blank">[31]</a>. Pulse-chase experiments were performed by incubating HEK293 cells with Cy3-apoE-TRL for 60 min at 37°C. Cells were washed with heparin and incubated for additional 60 min at 37°C with media in the presence or absence of 20 µg/ml HDL₃. Then cell culture media were harvested and the amount of re-secreted Cy3-apoE was determined by SDS-PAGE and subsequent in-gel fluorescence detection. The position of apoE is indicated. Representative gels with the corresponding apoE bands are shown for LRP1-EGFP overexpression (A) and reduced LRP1 expression (B). The highest and lowest intensity in each gel is represented as maximum black and white, respectively; therefore only intra-gel comparisons were performed. The quantification of these experiments is shown as percent recycling versus the corresponding mock transfected cells, which was set to 100% in order to ensure a valid comparison of the changes in apoE recycling between the different experiments (C). *: p<0.05, **: p<0.01 for Student's t-test (n≥4) ± S.E.M.</p

Internalized TRL-derived apoE co-localizes with LRP1 in EEA1-positive endosomes.

<p>HuH7 cells were incubated with Cy5-apoE-TRL for 10 min at 37°C. As indicated by the arrows, subsequent immunofluorescence analysis revealed co-localization of apoE (A) with LRP1 (B) in EEA1-positive endosomes (C). The merged image is shown in D. Nuclei were visualized by DAPI and appear in blue. Bar is 20 µm.</p

HDL stimulates the exit of apoE from LRP1- containing endosomes.

<p>HuH7 cells were incubated with Cy5-apoE-TRL for 20 minutes. ApoE recycling was then induced by Cy3-HDL. After 5 min (A–C) and 15 min (D–F), LRP1 (arrows) did no longer co-localize with Cy5-apoE which was instead found associated with Cy3-HDL (arrowheads; nuclei in blue). This was confirmed by high-magnification video-microscopy of LRP1-EGFP expressing HEK293 cells (G–I; due to short time-lapse between acquisition of red and blue channels, a minor signal offset due to endosomal movements was observed). Bar is 5 (G–I) and 20 µm (A–F), respectively.</p

Internalized RAP but not TRL-derived apoE is sorted towards lysosomes.

<p>Incubation of HuH7 cells with Cy5-apoE-TRL or Cy3-RAP was performed for 30 min at 37°C. Subsequent confocal immunofluorescence analysis revealed colocalization of LRP1 with apoE (A–B, see arrows in merged image C). Internalized Cy3-apoE did not appear within lysosomes as indicated by a counterstain with LAMP-1 (D–F). In contrast, after 30 min Cy3-RAP co-localized with LAMP-1 (G–H, see arrows in the merged image I), indicating lysosomal targeting of the LRP1 ligand RAP. Nuclei were visualized by DAPI and appear in blue. Bar is 20 µm.</p

ApoE recycling is reduced in LRP1^−/− primary hepatocytes.

<p>LRP1<i>flox</i> mice were infected with AdEGFP or AdCre and three days after infection primary hepatocytes were isolated. Sixteen hours after seeding, the infection with AdCre resulted in a dramatic loss of LRP1 protein expression as determined by Western blotting (A) and indirect immunofluorescence (B–C). Pulse-chase experiments were performed by incubating LRP1-positive (LRP^+/+) and LRP1–negative (LRP^−/−) hepatocytes with Cy3-apoE-TRL for 60 min at 37°C. Cells were washed with heparin and incubated for additional 60 min at 37°C with media in the presence or absence (w/o) of 20 µg/ml HDL₃. Then supernatants were harvested and the amount of re-secreted Cy3-apoE was determined by SDS-PAGE and subsequent quantification as described above. HDL-induced apoE recycling was reduced in LRP1^−/− cells as seen by in-gel fluorescence of chase media (D). Quantification of 4 independent experiments ± S.E.M. revealed a strong reduction of HDL-induced apoE recycling in LRP1^−/− hepatocytes ± HDL (E).</p

LRP1-dependent sorting of apoE and RAP.

<p>Cell lysates from EGFP (lane 1 in A, B and C) or LRP1-EGFP (lane 2 in A, B and C) transfected cells were subjected to SDS-PAGE, and Western blotting was performed with antibodies against the 85 kDa subunit of LRP1 (A), the 515 kDa subunit of LRP1 (B) and the LDLR (C). The endogenous LRP1 precursor protein is cleaved by furin into 515 kDa and 85 kDa fragments (shown in lane 1 of A and B). The recombinant LRP-EGFP can be detected at approximately 600 kDa (lane 2 in A and B) and for the cleaved 85 kDa fragment fused to EGFP at 115 kDa (LRP1₈₅-EGFP; lane 2 in A). The overexpression of LRP1-EGFP expression slightly reduced the expression of LDLR (C). Western blotting using an antibody against beta actin verified equal protein loading. LRP1-EGFP transfected HEK293 cells were incubated with Cy3-apoE-TRL for 30 min. Confocal microscopy revealed strong colocalization of LRP1-EGFP and Cy3-apoE in endosomal compartments (D–E; see arrows in panel F; nuclei are stained with DAPI). High-magnification confocal live-cell microscopy of LRP1-EGFP expressing cells incubated with Cy3-RAP (G) or Cy3-apoE-TRL (H) revealed that RAP is still attached to the inner leaflet of the vesicular membrane and does not diffuse freely within the lumen (G, arrows and inlet). Additionally, RAP was detected in small, dense vesicles not containing LRP1 (G, arrowheads). In contrast, apoE was only present within LRP1-EGFP endosomes and evenly distributed inside the lumen (H, arrows). Bar is 5 (G–H) and 20 µm (D–F), respectively.</p