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

OBJECTIVE: Insulin 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. METHODS: Localization 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. RESULTS: PID1 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. CONCLUSIONS: By 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.