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.     

Lipoprotein lipase-mediated selective uptake from low density lipoprotein requires cell surface proteoglycans and is independent of scavenger receptor class B type 1

Lipoprotein lipase (LpL) hydrolyzes chylomicron and very low density lipoprotein triglycerides to provide fatty acids to tissues. Aside from its lipolytic activity, LpL promotes lipoprotein uptake by increasing the association of these particles with cell surfaces allowing for the internalization by receptors and proteoglycans. Recent studies also indicate that LpL stimulates selective uptake of lipids from high density lipoprotein (HDL) and very low density lipoprotein. To study whether LpL can mediate selective uptake of lipids from low density lipoprotein (LDL), LpL was incubated with LDL receptor negative fibroblasts, and the uptake of LDL protein, labeled with (125)I, and cholesteryl esters traced with [(3)H]cholesteryl oleoyl ether, was compared. LpL mediated greater uptake of [(3)H]cholesteryl oleoyl ether than (125)I-LDL protein, a result that indicated selective lipid uptake. Lipid enrichment of cells was confirmed by measuring cellular cholesterol mass. LpL-mediated LDL selective uptake was not affected by the LpL inhibitor tetrahydrolipstatin but was nearly abolished by heparin, monoclonal anti-LpL antibodies, or chlorate treatment of cells and was not found using proteoglycan-deficient Chinese hamster ovary cells. Selective uptake from HDL, but not LDL, was 2-3-fold greater in scavenger receptor class B type I overexpressing cells (SR-BI cells) than compared control cells. LpL, however, induced similar increases in selective uptake from LDL and HDL in either control or SR-BI cells, indicative of the SR-BI-independent pathway. This was further supported by ability of LpL to promote selective uptake from LDL in human embryonal kidney 293 cells, cells that do not express SR-BI. In Chinese hamster ovary cell lines that overexpress LpL, we also found that selective uptake from LDL was induced by both endogenous and exogenous LpL. Transgenic mice that overexpress human LpL via a muscle creatine kinase promoter had more LDL selective uptake in muscle than did wild type mice. In summary LpL stimulates selective uptake of cholesteryl esters from LDL via pathways that are distinct from SR-BI. Moreover this process also occurs in vivo in tissues where abundant LpL is present.     

Role of cholesteryl ester transfer protein in selective uptake of high density lipoprotein cholesteryl esters by adipocytes

Previous reports attributed cholesteryl ester transfer protein (CETP)-mediated HDL cholesteryl ester (CE) selective uptake to the CETP-mediated transfer of CE from HDL to newly secreted apolipoprotein B-containing lipoproteins, which are then internalized by the LDL receptor (LDL-R). CETP has also been implicated in the remodeling of HDL, which renders it a better substrate for selective uptake by scavenger receptor class B type I (SR-BI). However, CETP-mediated selective uptake of HDL3-derived CE was not diminished in LDL-R null adipocytes, SR-BI null adipocytes, or in the presence of the receptor-associated protein. We found that monensin treatment or energy depletion of the SW872 liposarcoma cells with 2-deoxyglucose and NaN3 had no effect on CETP-mediated selective uptake, demonstrating that endocytosis is not required. This is supported by data indicating that CETP transfers CE into a compartment from which it can be extracted by unlabeled HDL. CETP could also mediate the selective uptake of HDL3-derived triacylglycerol (TG) and phospholipid (PL). The CETP-specific kinetics for TG and CE uptake were similar, and both reached saturation at approximately 5 microg/ml HDL. In contrast, CETP-specific PL uptake did not attain saturation at 5 microg/ml HDL and was approximately 6-fold greater than the uptake of CE. We propose two possible mechanisms to account for the role of CETP in selective uptake.     

The second and fourth cluster of class A cysteine-rich repeats of the low density lipoprotein receptor-related protein share ligand-binding properties.

The low density lipoprotein receptor-related protein (LRP) is a multifunctional endocytic cell-surface receptor that binds and internalizes a diverse array of ligands. The receptor contains four putative ligand-binding domains, generally referred to as clusters I, II, III, and IV. In this study, soluble recombinant receptor fragments, representing each of the four individual clusters, were used to map the binding sites of a set of structurally and functionally distinct ligands. Using surface plasmon resonance, we studied the binding of these fragments to methylamine-activated alpha(2)-macroglobulin, pro-urokinase-type plasminogen activator, tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1, t-PA.plasminogen activator inhibitor-1 complexes, lipoprotein lipase, apolipoprotein E, tissue factor pathway inhibitor, lactoferrin, the light chain of blood coagulation factor VIII, and the intracellular chaperone receptor-associated protein (RAP). No binding of the cluster I fragment to any of the tested ligands was observed. The cluster III fragment only bound to the anti-LRP monoclonal antibody alpha(2)MRalpha3 and weakly to RAP. Except for t-PA, we found that each of the ligands tested binds both to cluster II and to cluster IV. The affinity rate constants of ligand binding to clusters II and IV and to LRP were measured, showing that clusters II and IV display only minor differences in ligand-binding kinetics. Furthermore, we demonstrate that the subdomains C3-C7 of cluster II are essential for binding of ligands and that this segment partially overlaps with a RAP-binding site on cluster II. Finally, we show that one RAP molecule can bind to different clusters simultaneously, supporting a model in which RAP binding to LRP induces a conformational change in the receptor that is incompatible with ligand binding.     

The low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor binds and mediates catabolism of bovine milk lipoprotein lipase.

Lipoprotein lipase (LPL), the major lipolytic enzyme involved in the conversion of triglyceride-rich lipoproteins to remnants, was found to compete with binding of activated alpha 2-macroglobulin (alpha 2M*) to the low density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin receptor. Bovine milk LPL displaced both 125I-labeled alpha 2M* and 39-kDa alpha 2M receptor-associated protein (RAP) from the surface of cultured mutant fibroblasts lacking LDL receptors with apparent KI values at 4 degrees C of 6.8 and 30 nM, respectively. Furthermore, LPL inhibited the cellular degradation of 125I-alpha 2M* at 37 degrees C. Because both alpha 2M* and RAP interact with LRP, these data suggest that LPL binds specifically to this receptor. This was further supported by observing that an immunoaffinity-isolated polyclonal antibody against LRP blocked cellular degradation of 125I-LPL in a dose-dependent manner. In addition, 125I-LPL bound to highly purified LRP in a solid-phase assay with a KD of 18 nM, and this binding could be partially displaced with alpha 2M* (KI = 7 nM) and RAP (KI = 3 nM). Taken together, these data establish that LPL binds with high affinity to LRP and undergoes LRP-mediated cellular uptake. The implication of these findings for lipoprotein catabolism in vivo may be important if LRP binding is preserved when LPL is attached to lipoproteins. If so, LPL might facilitate LRP-mediated clearance of lipoproteins.     

Analysis of expression of genes involved in apolipoprotein E-based lipoprotein metabolism in pregnant mice deficient in the receptor-associated protein, the low density lipoprotein receptor, or apolipoprotein E.

Mice deficient in receptor-associated protein (RAP) were phenotypically normal, but in contrast to results previously reported in RAP(-/-) mice, nearly 50% of the offspring died at or shortly after birth. To attempt to determine the reason for this, we analyzed the regulation of expression of genes involved in apolipoprotein E (apoE)-based mechanisms in RAP-deficient mice and compared this to results in mice deficient in low density lipoprotein receptor (LDLR) or apoE. The major finding concerned a large increase in hepatic lipoprotein receptor-related protein (LRP) mRNA and LDLR mRNA levels in pregnant RAP knockout mice. This is in contrast to the down-regulation of LRP mRNA and LDLR mRNA, which is normally seen in wild-type mice. Also in LDLR knockout mice, a significant up-regulation in expression of LRP mRNA was demonstrated. In apoE knockout mice, hepatic LRP mRNA did not change significantly, while hepatic LDLR mRNA expression was increased. In placenta and uterus, the deficiency of RAP did not markedly affect the expression of LRP and LDLR. Lipoprotein lipase mRNA and apoE mRNA increased during pregnancy in all mice, independent of their genetic status. The current study does not directly explain the increased mortality of RAP(-/-) pups. The data demonstrate, however, important relative changes in expression of the genes analyzed, an indication that LRP and LDLR play an important role in lipid metabolism during pregnancy.     

Sortilin/neurotensin receptor-3 binds and mediates degradation of lipoprotein lipase

Lipoprotein lipase and the receptor-associated protein (RAP) bind to overlapping sites on the low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor (LRP). We have investigated if lipoprotein lipase interacts with the RAP binding but structurally distinct receptor sortilin/neurotensin receptor-3. We show, by chemical cross-linking and surface plasmon resonance analysis, that soluble sortilin binds lipoprotein lipase with an affinity similar to that of LRP. The binding was inhibited by heparin and RAP and by the newly discovered sortilin ligand neurotensin. In 35S-labeled 3T3-L1 adipocytes treated with the cross-linker dithiobis(succinimidyl propionate), lipoprotein lipase-containing complexes were isolated by anti-sortilin antibodies. To elucidate function in cells, sortilin-negative Chinese hamster ovary cells were transfected with full-length sortilin and shown to express about 8% of the receptors on the cell surface. These cells degraded 125I-labeled lipoprotein lipase much faster than the wild-type cells. The degradation was inhibited by unlabeled lipoprotein lipase, indicating a saturable pathway, and by RAP and heparin. Moreover, inhibition by the weak base chloroquine suggested that degradation occurs in an acidic vesicle compartment. The results demonstrate that sortilin is a multifunctional receptor that binds lipoprotein lipase and, when expressed on the cell surface, mediates its endocytosis and degradation.     

High density lipoprotein metabolism in low density lipoprotein receptor-deficient mice

The LDL receptor (LDLR) and scavenger receptor class B type I (SR-BI) play physiological roles in LDL and HDL metabolism in vivo. In this study, we explored HDL metabolism in LDLR-deficient mice in comparison with WT littermates. Murine HDL was radiolabeled in the protein (¹²⁵I) and in the cholesteryl ester (CE) moiety ([³H]). The metabolism of ¹²⁵I-/[³H]HDL was investigated in plasma and in tissues of mice and in murine hepatocytes. In WT mice, liver and adrenals selectively take up HDL-associated CE ([³H]). In contrast, in LDLR^−/− mice, selective HDL CE uptake is significantly reduced in liver and adrenals. In hepatocytes isolated from LDLR^−/− mice, selective HDL CE uptake is substantially diminished compared with WT liver cells. Hepatic and adrenal protein expression of lipoprotein receptors SR-BI, cluster of differentiation 36 (CD36), and LDL receptor-related protein 1 (LRP1) was analyzed by immunoblots. The respective protein levels were identical both in hepatic and adrenal membranes prepared from WT or from LDLR^−/− mice. In summary, an LDLR deficiency substantially decreases selective HDL CE uptake by liver and adrenals. This decrease is independent from regulation of receptor proteins like SR-BI, CD36, and LRP1. Thus, LDLR expression has a substantial impact on both HDL and LDL metabolism in mice.     

The apolipoprotein E-dependent low density lipoprotein cholesteryl ester selective uptake pathway in murine adrenocortical cells involves chondroitin sulfate proteoglycans and an alpha 2-macroglobulin receptor

Cells acquire lipoprotein cholesterol by receptor-mediated endocytosis and selective uptake pathways. In the latter case, lipoprotein cholesteryl ester (CE) is transferred to the plasma membrane without endocytosis and degradation of the lipoprotein particle. Previous studies with Y1/E/tet/2/3 murine adrenocortical cells that were engineered to express apolipoprotein (apo) E demonstrated that apoE expression enhances low density lipoprotein (LDL) CE uptake by both selective and endocytic pathways. The present experiments test the hypothesis that apoE-dependent LDL CE selective uptake is mediated by scavenger receptor, class B, type I (SR-BI). Surprisingly, SR-BI expression was not detected in the Y1/E/tet/2/3 clone of Y1 adrenocortical cells, indicating the presence of a distinct apoE-dependent pathway for LDL CE selective uptake. ApoE-dependent LDL CE selective uptake in Y1/E/tet/2/3 cells was inhibited by receptor-associated protein and by activated alpha(2)-macroglobulin (alpha(2)M), suggesting the participation of the LDL receptor-related protein/alpha(2)M receptor. Reagents that inhibited proteoglycan synthesis or removed cell surface chondroitin sulfate proteoglycan completely blocked apoE-dependent LDL CE selective uptake. None of these reagents inhibited SR-BI-mediated LDL CE selective uptake in the Y1-BS1 clone of Y1 cells in which LDL CE selective uptake is mediated by SR-BI. We conclude that LDL CE selective uptake in adrenocortical cells occurs via SR-BI-independent and SR-BI-dependent pathways. The SR-BI-independent pathway is an apoE-dependent process that involves both chondroitin sulfate proteoglycans and an alpha(2)M receptor.     

Opposing effects of apolipoproteins E and C on lipoprotein binding to low density lipoprotein receptor-related protein

The low density lipoprotein receptor-related protein (LRP) from rat liver membranes binds apoprotein E (apoE)-enriched rabbit beta-migrating very low density lipoproteins (beta-VLDL) in a ligand blotting assay on nitrocellulose membranes. Binding was markedly activated when the beta-VLDL was preincubated with recombinant human apoE-3, native human apoE-3 or E-4, or native rabbit apoE. Human apoE-2, which binds poorly (1-2% of apo E-3 binding) to low density lipoprotein receptors, was approximately 40% as effective as apoE-3 or apoE-4 in binding to LRP. Stimulation of apoE-dependent binding to LRP was blocked by the inclusion of a mixture of human apoC proteins, but not apoA-I or A-II, in the preincubation reaction. High concentrations of apoE did not overcome the apoC inhibition. The effects of apoE and apoC on the ligand blotting assay were paralleled by similar effects in the ability of beta-VLDL to stimulate cholesteryl ester synthesis in mutant human fibroblasts that lack low density lipoprotein receptors. These properties of LRP are consistent with the known effects of apoE and apoC on uptake of chylomicron and very low density lipoprotein remnants in the liver and raise the possibility that LRP functions as a receptor for apoE-enriched forms of these lipoproteins in intact animals.     

The insulin-stimulated cell surface presentation of low density lipoprotein receptor-related protein in 3T3-L1 adipocytes is sensitive to phosphatidylinositide 3-kinase inhibition

The regulation of low density lipoprotein receptor-related protein (LRP) activity by insulin was studied using 3T3-L1 adipocytes. The LRP mRNA and protein expression were independent of differentiation state of the cells and of insulin treatment. In differentiated cells, insulin treatment acutely stimulated the cell surface presentation of LRP (approximately 2-fold) as evidenced by methylamine-activated alpha(2)-macroglobulin binding and by biotinylation of cell surface LRP. The increased cell surface presentation was accompanied by a 39% decrease in LRP level in the low density microsomes. The magnitude of insulin-stimulated cell surface presentation of LRP was similar to that of transferrin receptor but was much less than that of GLUT4. Both the increases in LRP and GLUT4 cell surface presentation upon insulin treatment were abolished by inhibition of phosphatidylinositide 3-kinase. The increased cell surface presentation of LRP was associated with proportionally increased endocytic activity, and the internalization rate constant (K(e)) was not decreased by insulin treatment. Thus, insulin treatment most likely stimulates recycling of LRP from an endosomal pool to the plasma membrane, which is regulated in a phosphatidylinositide 3-kinase-dependent manner in 3T3-L1 adipocytes.     

The 39-kDa receptor-associated protein interacts with two members of the low density lipoprotein receptor family, alpha 2-macroglobulin receptor and glycoprotein 330.

The 39-kDa receptor-associated protein (RAP) binds to the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) and inhibits binding of ligands to this receptor. The in vivo function of RAP may be to regulate ligand binding and/or assist in the correct biosynthetic processing or trafficking of the alpha 2MR/LRP. Here we show that RAP binds another putative receptor, the kidney glycoprotein 330 (gp330). Gp330 is a high molecular weight glycoprotein that is structurally similar to both the alpha 2MR/LRP and low density lipoprotein receptor. The ability of RAP to bind to gp330 was demonstrated by ligand blotting and solid phase binding assays, which showed that RAP binds to gp330 with high affinity (Kd = 8 nM). Exploiting the interaction of gp330 and RAP, we purified gp330 by affinity chromatography with a column of RAP coupled to Sepharose. Gp330 preparations obtained by this procedure were notably more homogeneous than those obtained by conventional methods. Immunocytochemical staining of human kidney sections localized RAP to the brush-border epithelium of proximal tubules. The fact that gp330 is also primarily expressed by proximal tubule epithelial cells strengthens the likelihood that the interaction between gp330 and RAP occurs in vivo. The functional significance of RAP binding to gp330 may be to antagonize ligand binding as has been demonstrated for the alpha 2MR/LRP or to assist in the biosynthetic processing and/or trafficking of this receptor.     

Allosteric modulation of ligand binding to low density lipoprotein receptor-related protein by the receptor-associated protein requires critical lysine residues within its carboxyl-terminal domain

The low density lipoprotein receptor-related protein (LRP) is a large endocytic receptor that recognizes more than 30 different ligands and plays important roles in protease and lipoprotein catabolism. Ligand binding to newly synthesized LRP is modulated by the receptor-associated protein (RAP), an endoplasmic reticulum-resident protein that functions as a molecular chaperone and prevents ligands from associating with LRP via an allosteric-type mechanism. RAP is a multidomain protein that contains two independent LRP binding sites, one located at the amino-terminal portion of the molecule and the other at the carboxyl-terminal portion of the molecule. The objective of the present investigation was to gain insight into how these two regions of RAP interact with LRP and function to modulate its ligand binding properties. These objectives were accomplished by random mutagenesis of RAP, which identified two critical lysine residues, Lys-256 and Lys-270, within the carboxyl-terminal domain that are necessary for binding of this region of RAP to LRP and to heparin. RAP molecules in which either of these two lysine residues was mutated still bound LRP but with reduced affinity. Furthermore, the mutant RAPs were significantly impaired in their ability to inhibit alpha(2)M* binding to LRP via allosteric mechanisms. In contrast, the mutant RAP molecules were still effective at inhibiting uPA.PAI-1 binding to LRP. These results confirm that both LRP binding sites within RAP cooperate to inhibit ligand binding via an allosteric mechanism.     

The low density lipoprotein receptor-related protein mediates fibronectin catabolism and inhibits fibronectin accumulation on cell surfaces

Low density lipoprotein receptor-related protein (LRP) is a member of the low density lipoprotein receptor family, which functions as an endocytic receptor for diverse ligands. In this study, we demonstrate that murine embryonic fibroblasts (MEF-2 cells) and 13-5-1 Chinese hamster ovary cells, which are LRP-deficient, accumulate greatly increased levels of cell-surface fibronectin (Fn), compared with LRP-expressing MEF-1 and CHO-K1 cells. Increased Fn was also detected in conditioned medium from LRP-deficient MEF-2 cells; however, biosynthesis of Fn by MEF-1 and MEF-2 cells was not significantly different. When LRP-deficient cells were dissociated from monolayer culture, increased levels of Fn remained with the cells, as determined by cell-surface protein biotinylation, suggesting an intimate relationship with cell surface-binding sites. The LRP antagonist, receptor-associated protein (RAP), promoted Fn accumulation in association with MEF-1 cells, whereas expression of full-length LRP in MEF-2 cells substantially decreased Fn accumulation, confirming the role of LRP in this process. Purified LRP bound directly to immobilized Fn, and this interaction was inhibited by RAP. Furthermore, MEF-1 cells degraded (125)I-Fn at an increased rate, compared with MEF-2 cells. (125)I-Fn degradation by MEF-1 cells was inhibited by RAP. These results demonstrate that LRP functions as a catabolic receptor for Fn. The function of LRP in Fn degradation and the ability of LRP to regulate levels of other plasma membrane proteins represent possible mechanisms whereby LRP prevents Fn accumulation on cell surfaces.     

Mechanism of the cholesteryl ester transfer protein-mediated uptake of high density lipoprotein cholesteryl esters by Hep G2 cells

Plasma cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl esters (CE) between lipoproteins and was reported to also directly mediate the uptake of high density lipoprotein (HDL) CE by human Hep G2 cells and fibroblasts. The present study investigates that uptake and its relationship to a pathway for "selective uptake" of HDL CE that does not require CETP. HDL3 labeled in both the CE and apoprotein moieties was incubated with Hep G2 cells. During 4-h incubations, CE tracer was selectively taken up from doubly labeled HDL3 in excess of apoA-I tracer, and added CETP did not modify that uptake. However, during 18-20-h incubations, CETP stimulated the uptake of CE tracer more than 4-fold without modifying the uptake of apoA-I tracer. This suggested that secreted products, perhaps lipoproteins, might be required for the CETP effect. Four inhibitors of lipoprotein uptake via low density lipoprotein (LDL) receptors (heparin, monensin, an antibody against the LDL receptor, and antibodies against the receptor binding domains of apoB and apoE) effectively blocked the CETP stimulation of CE tracer uptake. Heparin caused an increase in CE tracer in a d less than 1.063 g/ml fraction of the medium that more than accounted for the heparin blockade of CETP-stimulated CE uptake. CETP did not affect the uptake of doubly labeled HDL3 by human fibroblasts, even at twice plasma levels of activity, and heparin did not modify uptake of HDL3 tracers. Thus the CETP effect on Hep G2 cells can be accounted for by transfer of HDL CE to secreted lipoproteins which are then retaken up, and there is no evidence for a direct effect of CETP on cellular uptake of HDL CE.     

RAP,a specialized chaperone,prevents ligand-induced ER retention and degradation of LDL receptor-related endocytic receptors.

The multifunctional low density lipoprotein (LDL) receptor-related protein (LRP) forms a complex with a receptor-associated protein (RAP) within the secretory pathway. RAP inhibits ligand binding to LRP and is required for normal functional expression of LRP in vivo, suggesting a physiological function as a specialized chaperone. We have used RAP-deficient mice, generated by gene targeting, to investigate the role of RAP in the biosynthesis and biological activity of LRP and other members of the LDL receptor gene family in various organs and in embryonic fibroblasts. Our results demonstrate that RAP is required for the proper folding and export of the receptors from the endoplasmic reticulum (ER) by preventing the premature binding of co-expressed ligands. Overexpression of apolipoprotein E (apoE), a high affinity ligand for LRP, results in dramatically reduced cellular LRP expression, an effect that is prevented by co-expression of RAP. RAP thus defines a novel class of molecular chaperones that selectively protect endocytic receptors by binding to newly synthesized receptor polypeptides, thereby preventing ligand-induced aggregation and subsequent degradation in the ER.     

39-kDa protein modulates binding of ligands to low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor.

A 39-kDa protein of unknown function has previously been reported to copurify with the low density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin receptor. In this study we demonstrate that a recombinant 39-kDa fusion protein can reversibly bind to the 515-kDa subunit of the LRP/alpha 2-macroglobulin receptor. This interaction inhibits the binding and uptake of the receptor's two known ligands: 1) beta-migrating very low density lipoproteins activated by enrichment with apoprotein E and 2) alpha 2-macroglobulin activated by incubation with plasma proteases or methylamine. A potential in vivo role of the 39-kDa protein is to modulate the uptake of apoE-enriched lipoproteins and activated alpha 2-macroglobulin in hepatic and extrahepatic tissues.     

Hepatic lipase promotes the selective uptake of high density lipoprotein-cholesteryl esters via the scavenger receptor B1.

Hepatic lipase (HL) plays a major role in high-density lipoprotein (HDL) metabolism both as a lipolytic enzyme and as a ligand. To investigate whether HL enhances the uptake of HDL-cholesteryl ester (CE) via the newly described scavenger receptor BI (SR-BI), we measured the effects of expressing HL and SR-BI on HDL-cell association as well as uptake of 125I-labeled apoA-I and [3H]CE-HDL, by embryonal kidney 293 cells. As expected, HDL cell association and CE selective uptake were increased in SR-BI transfected cells by 2- and 4-fold, respectively, compared to controls (P < 0.001). Cells transfected with HL alone or in combination with SR-BI expressed similar amounts of HL, 20% of which was bound to cell surface proteoglycans. HL alone increased HDL cell association by 2-fold but had no effect on HDL-CE uptake in 293 cells. However, in cells expressing SR-BI, HL further enhanced the selective uptake of CE from HDL by 3-fold (P < 0.001). To determine whether the lipolytic and/or ligand function of HL are required in this process, we generated a catalytically inactive form of HL (HL-145G). Cells co-transfected with HL-145G and SR-BI increased their HDL cell association and HDL-CE selective uptake by 1.4-fold compared to cells expressing SR-BI only (P < 0.03). Heparin abolished the effect of HL-145G on SR-BI-mediated HDL-CE selective uptake.Thus, the enhanced uptake of HDL-CE by HL is mediated by both its ligand role, which requires interaction with proteoglycans, and by lipolysis with subsequent HDL particle remodeling. These results establish HL as a major modulator of SR-BI mediated selective uptake of HDL-CE.     

Endocytosis of hepatic lipase and lipoprotein lipase into rat liver hepatocytes in vivo is mediated by the low density lipoprotein receptor-related protein

In isolated cell studies, the internalization and degradation of hepatic lipase (HL) has been linked to its binding to the low density lipoprotein receptor-related protein (LRP). We have utilized the receptor-associated protein (RAP), a universal inhibitor of high affinity ligand binding to LRP, to evaluate the participation of LRP in the endocytosis of HL and lipoprotein lipase (LPL). We isolated a total endosome fraction from rat livers after a 30-min infusion of recombinant RAP, administered as a glutathione S-transferase conjugate (GST-RAP). GST-RAP infusion had no effect on the concentration of HL in liver homogenates, but its concentration in blood plasma increased progressively by 20%, and enrichment over homogenate of HL in endosomes was reduced by 50% as compared with infusion of GST alone. The concentrations of LPL in liver and plasma were 1.4 and 0.5%, respectively, those of HL, but endosomal enrichment of the two enzymes was similar ( approximately 10-fold). GST-RAP infusion had no effect on the concentration of LPL in liver but increased its concentration in blood plasma by 250% and reduced its endosomal enrichment by 95% or greater. GST-RAP infusion also reduced endosomal enrichment of LRP by 40%, but enrichment of several other endocytic receptors was unaffected. Endosomal enrichment of several membrane trafficking proteins associated with the endocytic pathway in hepatocytes was unaffected by GST-RAP with the exception of early endosome endosome antigen 1, which was reduced by 85%. We conclude that HL is partially and LPL almost exclusively taken up into rat hepatocytes after binding to the endocytic receptor LRP.