The low-density lipoprotein receptor-related protein 1 (LRP1) mediates the endocytosis of the cellular prion protein

PrP(C) (cellular prion protein) is located at the surface of neuronal cells in detergent-insoluble lipid rafts, yet is internalized by clathrin-dependent endocytosis. As PrP(C) is glycosyl-phosphatidylinositol-anchored, it requires a transmembrane adaptor protein to connect it to the clathrin endocytosis machinery. Using receptor-associated protein and small interfering RNA against particular LDL (low-density lipoprotein) family members, in combination with immunofluorescence microscopy and surface biotinylation assays, we show that the transmembrane LRP1 (LDL receptor-related protein 1) is required for the Cu(2+)-mediated endocytosis of PrP(C) in neuronal cells. We show also that another LRP1 ligand that can cause neurodegenerative disease, the Alzheimer's amyloid precursor protein, does not modulate the endocytosis of PrP(C).     

Identification of the low density lipoprotein receptor-related protein (LRP) as an endocytic receptor for thrombospondin-1

Thrombospondin-1 (TSP1) has potent biological effects on vasculature smooth muscle cells (SMCs) and endothelial cells. The regulation of extracellular accumulation of TSP1 is mediated by a previously obscure process of endocytosis which leads to its lysosomal degradation. Since members of the low density lipoprotein receptor (LDLR) family have been found to mediate endocytosis which leads to degradation of a diverse array of ligands, we evaluated their possible role in the uptake and degradation of TSP1 by vascular SMCs, endothelial-cells and fibroblasts. 125I-TSP1 was found to be internalized and degraded lysosomally by all these cell types. Both the internalization and degradation of 125I-TSP1 could be inhibited by a specific antagonist of the LDLR family, the 39-kD receptor-associated protein (RAP). Antibodies to the LDLR-related protein (LRP) completely blocked the uptake and degradation of 125I-TSP1 in SMCs and fibroblasts but not endothelial cells. Solid-phase binding assays confirmed that LRP bound to TSP1 and that the interaction was of high affinity (Kd = 5 nM). Neither RAP nor LRP antibodies inhibited the binding of 125I-TSP1 to surfaces of SMCs. However, cell surface binding, as well as, endocytosis and degradation could be blocked by heparin or by pre- treatment of the cells with either heparitinase, chondroitinase or beta- D-xyloside. The data indicates that cell surface proteoglycans are involved in the LRP-mediated clearance of TSP1. A model for the clearance of TSP1 by these cells is that TSP1 bound to proteoglycans is presented to LRP for endocytosis. In endothelial cells, however, the internalization of TSP1 was not mediated by LRP but since RAP inhibited TSP1 uptake and degradation, we postulate that another member of the LDLR family is likely to be involved.     

Low density receptor-related protein 1 (LRP1) promotes anti-inflammatory phenotype in murine macrophages

We have previously reported that apolipoprotein E (apoE), a protein component of very-low-density lipoproteins (VLDL) and high-density lipoproteins and a potent plasma-borne atheroprotective factor, exerts anti-inflammatory activity in macrophages by switching the activation profile from M1 (“classic”) to M2 (“alternative”) in a process involving signaling via low-density lipoprotein receptor (LDLR) family members including the VLDL receptor (VLDLR) or apoE receptor-2 (apoER2). The present study was undertaken to investigate whether LDLR-related protein 1 (LRP-1), another member of the LDLR family and a ubiquitously expressed multifunctional cell surface receptor, modulates M1→M2 conversion in murine macrophages. We investigate bone marrow or peritoneal macrophages isolated from wild-type C57/Bl6 mice or mice with conditional inactivation of the LRP-1 gene in the myeloid lineage for the expression of polarization markers. Our results suggest that the deficiency of LRP-1 down-regulates M2 marker expression in macrophages, while enhancing the macrophage response to M1 stimuli. To our knowledge, this is the first demonstration that LRP-1 affects macrophage polarization and promotes the development of an anti-inflammatory M2 functional phenotype.     

The low density lipoprotein receptor-related protein (LRP) is a novel beta-secretase (BACE1) substrate

BACE is a transmembrane protease with beta-secretase activity that cleaves the amyloid precursor protein (APP). After BACE cleavage, APP becomes a substrate for gamma-secretase, leading to release of amyloid-beta peptide (Abeta), which accumulates in senile plaques in Alzheimer disease. APP and BACE are co-internalized from the cell surface to early endosomes. APP is also known to interact at the cell surface and be internalized by the low density lipoprotein receptor-related protein (LRP), a multifunctional endocytic and signaling receptor. Using a new fluorescence resonance energy transfer (FRET)-based assay of protein proximity, fluorescence lifetime imaging (FLIM), and co-immunoprecipitation we demonstrate that the light chain of LRP interacts with BACE on the cell surface in association with lipid rafts. Surprisingly, the BACE-LRP interaction leads to an increase in LRP C-terminal fragment, release of secreted LRP in the media and subsequent release of the LRP intracellular domain from the membrane. Taken together, these data suggest that there is a close interaction between BACE and LRP on the cell surface, and that LRP is a novel BACE substrate.     

Structure of the low-density lipoprotein receptor-related protein (LRP) promoter

The low-density Lipoprotein receptor-related protein (LRP) is a 4544-amino-acid membrane protein which closely resembles the LDL receptor in its arrangement of cysteine-rich motifs. Binding studies have suggested that one function of the molecule is as a receptor for ligands containing apolipoprotein E. We present here the sequence and structure of the promoter region of the LRP. These data show that the LRP contains no sterol regulatory element, and is not down-regulated by sterols like the LDL receptor. This lends further support to the identity of the LRP as a chylomicron remnant receptor.     

Low density lipoprotein receptor-related protein (LRP) interacts with presenilin 1 and is a competitive substrate of the amyloid precursor protein (APP) for gamma-secretase

Presenilin 1 (PS1) is a critical component of the gamma-secretase complex, which is involved in the cleavage of several substrates including the amyloid precursor protein (APP) and the Notch receptor. Recently, the low density receptor-related protein (LRP) has been shown to be cleaved by a gamma-secretase-like activity. We postulated that LRP may interact with PS1 and tested its role as a competitive substrate for gamma-secretase. In this report we show that LRP colocalizes and interacts with endogenous PS1 using coimmunoprecipitation and fluorescence lifetime imaging microscopy. In addition, we found that gamma-secretase active site inhibitors do not disrupt the interaction between LRP and PS1, suggesting that the substrate associates with a gamma-secretase docking site located in close proximity to PS1. This is analogous to APP-gamma-secretase interactions. Finally, we show that LRP competes with APP for gamma-secretase activity. Overexpression of a truncated LRP construct consisting of the C terminus, the transmembrane domain, and a short extracellular portion leads to a reduction in the levels of the Abeta40, Abeta42, and p3 peptides without changing the total level of APP expression. In addition, transfection with the beta-chain of LRP causes an increase in uncleaved APP C-terminal fragments and a concomitant decrease in the signaling effects of the APP intracellular domain. In conclusion, LRP is a PS1 interactor and can compete with APP for gamma-secretase enzymatic activity.     

Platelet-derived growth factor (PDGF)-induced tyrosine phosphorylation of the low density lipoprotein receptor-related protein (LRP). Evidence for integrated co-receptor function betwenn LRP and the PDGF

The low density lipoprotein receptor-related protein (LRP) functions in the catabolism of numerous ligands including proteinases, proteinase inhibitor complexes, and lipoproteins. In the current study we provide evidence indicating an expanded role for LRP in modulating cellular signaling events. Our results show that platelet-derived growth factor (PDGF) BB induces a transient tyrosine phosphorylation of the LRP cytoplasmic domain in a process dependent on PDGF receptor activation and c-Src family kinase activity. Other growth factors, including basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor-1, were unable to mediate tyrosine phosphorylation of LRP. The basis for this selectivity may result from the ability of LRP to bind PDGFBB, because surface plasmon resonance experiments demonstrated that only PDGF, and not basic fibroblast growth factor, epidermal growth factor, or insulin-like growth factor-1, bound to purified LRP immobilized on a sensor chip. The use of LRP mini-receptor mutants as well as in vitro phosphorylation studies demonstrated that the tyrosine located within the second NPXY motif found in the LRP cytoplasmic domain is the primary site of tyrosine phosphorylation by Src and Src family kinases. Co-immunoprecipitation experiments revealed that PDGF-mediated tyrosine phosphorylation of LRPs cytoplasmic domain results in increased association of the adaptor protein Shc with LRP and that Shc recognizes the second NPXY motif within LRPs cytoplasmic domain. In the accompanying paper, Boucher et al. (Boucher, P., Liu, P. V., Gotthardt, M., Hiesberger, T., Anderson, R. G. W., and Herz, J. (2002) J. Biol. Chem. 275, 15507-15513) reveal that LRP is found in caveolae along with the PDGF receptor. Together, these studies suggest that LRP functions as a co-receptor that modulates signal transduction pathways initiated by the PDGF receptor.     

Low density lipoprotein receptor-related protein (LRP) is required for lactoferrin-enhanced collagen gel contractile activity of human fibroblasts

Fibroblasts plated on a type I collagen gel can reduce the size of the gel in a way that mimics the reorganization of the collagen matrix that accompanies the wound healing process. We demonstrated previously that lactoferrin (Lf) specifically binds to WI-38 human fibroblasts and enhances their collagen gel contractile activity. The effect of Lf correlated with the phosphorylation of myosin light chain (MLC), suggesting that Lf promotes fibroblast contractile activity by regulating MLC phosphorylation. We found here that the binding of Lf to WI-38 cells was inhibited by recombinant receptor-associated protein (RAP), a universal competitor for ligand binding to LRP (LDL receptor-related protein), and RAP can also promote the collagen gel contractile activity. These observations suggest that LRP is a receptor that mediates the Lf-induced enhancement of collagen gel contractile activity in WI-38 fibroblasts. To confirm the hypothesis, we utilized LRP antisense oligonucleotide, which was modified by morpholino linkage. Suppression of LRP expression abrogated the Lf-induced enhancement the contractile activity in fibroblasts. Treatment of fibroblasts with Lf enhanced the phosphorylation of ERK1/2 and the activation of MLC kinase (MLCK). These effects were attenuated by suppression of LRP expression. These findings suggest that LRP is involved in the Lf-enhanced collagen gel contractile activity of WI-38 fibroblasts by converting the Lf binding signal into the activation of ERK1/2 and MLCK.     

Low density lipoprotein receptor-related protein mediates endocytosis of monoclonal antibodies in cultured cells and rabbit liver

Monoclonal antibodies that bound to the external domain of the rabbit low density lipoprotein receptor-related protein (LRP) were taken into rabbit fibroblasts by receptor-mediated endocytosis. Uptake occurred in fibroblasts from Watanabe-heritable hyperlipidemic rabbits, which lack low density lipoprotein receptors, as well as in normal rabbit fibroblasts. The fate of the internalized antibodies differed, depending on the domain of LRP that was recognized. LRP is synthesized as a single polypeptide chain that is cleaved to form a heterodimer of two noncovalently bound proteins, 1) a 515-kDa subunit that contains the binding domain, and 2) an 85-kDa subunit that contains the membrane-spanning region and cytoplasmic tail. A monoclonal antibody directed against the 515-kDa subunit (anti-LRP 515) rapidly dissociated from LRP at pH 5.2. After uptake by cells this antibody dissociated from the receptor and was degraded in lysosomes. A second antibody directed against the external portion of the 85-kDa subunit (anti-LRP 85) failed to dissociate at acid pH. After uptake by cells this antibody was not degraded, but instead was released from the cells in an acid-precipitable form. When administered intravenously to rabbits, both 125I-labeled antibodies were rapidly cleared from the circulation, 75-95% of the uptake occurring in the liver. The anti-LRP 515 antibody was degraded and acid-soluble products appeared in the plasma. No significant acid-soluble products appeared when the anti-LRP-85 antibody was infused. We conclude that LRP can carry out receptor-mediated endocytosis and that its ligand-binding domain, like the similar domain of the low density lipoprotein receptor, undergoes an acid-dependent conformational change that ejects ligands within the endosome. We also conclude that in the body this endocytotic function is expressed primarily in the liver. Both of these conclusions lend support to the hypothesis that LRP may function in humans and animals as a receptor for apolipoprotein E-enriched lipoproteins, such as chylomicron remnants.     

Platelet-derived growth factor receptor-beta (PDGFR-beta) activation promotes its association with the low density lipoprotein receptor-related protein (LRP). Evidence for co-receptor function

Activation of the platelet-derived growth factor receptor-beta (PDGFR-beta) leads to tyrosine phosphorylation of the cytoplasmic domain of LRP and alters its association with adaptor and signaling proteins, such as Shc. The mechanism of the PDGF-induced LRP tyrosine phosphorylation is not well understood, especially since PDGF not only activates PDGF receptor but also binds directly to LRP. To gain insight into this mechanism, we used a chimeric receptor in which the ligand binding domain of the PDGFR-beta was replaced with that from the macrophage colony-stimulating factor (M-CSF) receptor, a highly related receptor tyrosine kinase of the same subfamily, but with different ligand specificity. Activation of the chimeric receptor upon the addition of M-CSF readily mediated the tyrosine phosphorylation of LRP. Since M-CSF is not recognized by LRP, these results indicated that growth factor binding to LRP is not necessary for this phosphorylation event. Using a panel of cytoplasmic domain mutants of the chimeric M-CSF/PDGFR-beta, we confirmed that the kinase domain of PDGFR-beta is absolutely required for LRP tyrosine phosphorylation but that PDGFR-beta-mediated activation of phosphatidylinositol 3-kinase, RasGAP, SHP-2, phospholipase C-gamma, and Src are not necessary for LRP tyrosine phosphorylation. To identify the cellular compartment where LRP and the PDGFR-beta may interact, we employed immunofluorescence and immunogold electron microscopy. In WI-38 fibroblasts, these two receptors co-localized in coated pits and endosomal compartments following PDGF stimulation. Further, phosphorylated forms of the PDGFR-beta co-immunoprecipitated with LRP following PDGF treatment. Together, these studies revealed close association between activated PDGFR-beta and LRP, suggesting that LRP functions as a co-receptor capable of modulating the signal transduction pathways initiated by the PDGF receptor from endosomes.     

Differential RNA interference: replacement of endogenous with recombinant low density lipoprotein receptor-related protein (LRP)

The interpretation of experiments involving the overexpression of a recombinant cDNA is often hampered by the interference of mRNA expression from the endogenous gene locus. Unless cell lines from naturally occurring mutations or knockout mice are available, difficult and time-consuming gene targeting techniques are required to inhibit endogenous gene expression. Using a method we refer to as "differential RNA interference" we demonstrate that RNA interference can be used to selectively suppress endogenous gene expression without affecting the expression of a co-transfected recombinant version of the same protein. Functional analyses of recombinant low density lipoprotein receptor-related protein (LRP) to study its involvement in lipid metabolism have been shown to be extremely difficult due to its large cDNA and the unavailability of suitable LRP-deficient cell lines. We constructed an expression vector containing the full-length coding sequence of human LRP fused to EGFP and a vector expressing small hairpin RNA directed against the 3'-untranslated region of the wild-type human LRP mRNA (LRP-shRNA). When overexpressed, EGFP-tagged LRP colocalizes with endogenous LRP and stimulates the uptake of LRP ligands. Overexpression of LRP-shRNA vectors significantly inhibits LRP expression, as judged by quantitative RT-PCR, Western blot and immunofluorescence analysis, and it dramatically decreases receptor-associated protein (RAP) uptake. Finally, co-transfection of EGFP-LRP and LRP-shRNA vectors demonstrates selective inhibition of endogenous LRP expression without affecting simultaneous expression of recombinant LRP protein. Thus, utilization of "differential RNA interference" provides a new experimental approach to selectively study the function of any recombinant protein in any given cell line without interference of endogenous protein expression.     

Sequences from the low density lipoprotein receptor-related protein (LRP) cytoplasmic domain enhance amyloid beta protein production via the beta-secretase pathway without altering amyloid precursor protein/LRP nuclear signaling

Increasing evidence suggests that the low density lipoprotein receptor-related protein (LRP) affects the processing of amyloid precursor protein (APP) and amyloid beta (Abeta) protein production as well as mediates the clearance of Abeta from the brain. Recent studies indicate that the cytoplasmic domain of LRP is critical for this modulation of APP processing requiring perhaps a complex between APP, the adaptor protein FE65, and LRP. In this study, we expressed a small LRP domain consisting of the C-terminal 97 amino acids of the cytoplasmic domain, or LRP-soluble tail (LRP-ST), in CHO cells to test the hypothesis that the APP.LRP complex can be disrupted. We anticipated that LRP-ST would inhibit the normal interaction between LRP and APP and therefore perturb APP processing to resemble a LRP-deficient state. Surprisingly, CHO cells expressing LRP-ST demonstrated an increase in both sAPP secretion and Abeta production compared with control CHO cells in a manner reminiscent of the cellular effects of the APP "Swedish mutation." The increase in sAPP secretion consisted mainly of sAPPbeta, consistent with the increase in Abeta release. Further, this effect is LRP-independent, as the same alterations remained when LRP-ST was expressed in LRP-deficient cells but not when the construct was membrane-anchored. Finally, deletion experiments suggested that the last 50 amino acid residues of LRP-ST contain the important domain for altering APP processing and Abeta production. These observations indicate that there are cellular pathways that may suppress Abeta generation but that can be altered to facilitate Abeta production.     

Low density lipoprotein (LDL) receptor-related protein 1B impairs urokinase receptor regeneration on the cell surface and inhibits cell migration

The low density lipoprotein (LDL) receptor-related protein 1B (LRP1B) is a newly identified member of the LDL receptor family and is closely related to LRP. It was discovered as a putative tumor suppressor and is frequently inactivated in lung cancer cells. In the present study, we used an LRP1B minireceptor (mLRP1B4), which mimics the function and trafficking of LRP1B, to explore the roles of LRP1B on the plasminogen activation system. We found that mLRP1B4 and urokinase plasminogen activator receptor (uPAR) form immunoprecipitable complexes on the cell surface in the presence of complexes of uPA and its inhibitor, plasminogen activator inhibitor type-1 (PAI-1). However, compared with cells expressing the analogous LRP minireceptor (mLRP4), cells expressing mLRP1B4 display a substantially slower rate of uPA.PAI-1 complex internalization. Expression of mLRP1B4, or an mLRP4 mutant deficient in endocytosis, leads to an accumulation of uPAR at the cell surface and increased cell-associated uPA and PAI-1 when compared with cells expressing mLRP4. In addition, we found that expression of mLRP1B or the mLRP4 endocytosis mutant impairs the regeneration of unoccupied uPAR on the cell surface and that this correlates with a diminished rate of cell migration. Taken together, these results demonstrate that LRP1B can function as a negative regulator of uPAR regeneration and cell migration.     

Both platelet-derived growth factor receptor (PDGFR)-alpha and PDGFR-beta promote murine fibroblast cell migration

Cell motility plays a critical role for many physiological and pathological processes including wound healing, fibrosis, angiogenesis, and tumor metastasis. Platelet-derived growth factor (PDGF) is among the most potent stimuli for mesenchymal cell migration. The PDGF B-chain homodimer PDGF BB activates both alpha- and beta-receptor subunits (alpha-PDGFR and beta-PDGFR), and promotes cell migration in many cell types including fibroblasts and smooth muscle cells. PDGF-A chain homodimer PDGF AA activates alpha-PDGFR only, and its role for cell migration is still debatable. PDGF BB, but not PDGF AA, induces smooth muscle cell migration. Interestingly, alpha-PDGFR was shown to antagonize beta-PDGFR-induced smooth muscle cell migration. In the present study, we investigated the role of alpha-PDGFR and beta-PDGFR in PDGF-mediated cell migration of murine fibroblasts (NIH 3T3). Unlike smooth muscle cells, both PDGF AA and PDGF BB promoted NIH 3T3 cell migration. The effect of PDGF BB activation of beta-PDGFR alone for cell migration was examined using previously established NIH 3T3 clones in which alpha-PDGFR signaling is inhibited by a dominant-negative alpha-PDGFR, or an antisense construct of alpha-PDGFR. PDGF BB activation of beta-PDGFR alone was sufficient to induce cell migration, but the efficiency was significantly lower compared to PDGF activation of both receptors. These results showed that both alpha- and beta-PDGFRs promote fibroblast cell migration and their effects are additive. Taken together, we propose that cell-type specific alpha-PDGFR signaling is critical for regulation of mesenchymal cell migration in response to PDGF isoform, whereas beta-PDGFR mainly promotes cell migration.     

Low density lipoprotein receptor-related protein-1 promotes beta1 integrin maturation and transport to the cell surface

Low density lipoprotein receptor-related protein-1 (LRP-1) mediates the endocytosis of multiple plasma membrane proteins and thereby models the composition of the cell surface. LRP-1 also functions as a catabolic receptor for fibronectin, limiting fibronectin accumulation in association with cells. The goal of the present study was to determine whether LRP-1 regulates cell surface levels of the beta(1) integrin subunit. We hypothesized that LRP-1 may down-regulate cell surface beta(1) by promoting its internalization; however, unexpectedly, LRP-1 expression was associated with a substantial increase in cell surface beta(1) integrin in two separate cell lines, murine embryonic fibroblasts (MEFs) and CHO cells. The total amount of beta(1) integrin was unchanged because LRP-1-deficient cells retained increased amounts of beta(1) in the endoplasmic reticulum (ER). Expression of human LRP-1 in LRP-1-deficient MEFs reversed the shift in subcellular beta(1) integrin distribution. Metabolic labeling experiments demonstrated that the precursor form of newly synthesized beta(1) integrin (p105) is converted into mature beta(1) (p125) more slowly in LRP-1-deficient cells. Although low levels of cell surface beta(1) integrin, in LRP-1-deficient MEFs, were associated with decreased adhesion to fibronectin, the subcellular distribution of beta(1) integrin was most profoundly dependent on LRP-1 only after the cell cultures became confluent. A mutagen-treated CHO cell line, in which LRP-1 is expressed but retained in the secretory pathway, also demonstrated nearly complete ER retention of beta(1) integrin. These studies support a model in which LRP-1 either directly or indirectly promotes maturation of beta(1) integrin precursor and thereby increases the level of beta(1) integrin at the cell surface.     

Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP).

Sphingolipid activator proteins SAP-A, -B, -C and -D (also called saposins) are generated by proteolytic processing from a 73 kDa precursor and function as obligatory activators of lysosomal enzymes involved in glycosphingolipid metabolism. Although the SAP precursor can be recognized by the mannose-6-phosphate (M-6-P) receptor and shuttled directly from the secretory pathway to the lysosome, a substantial fraction of newly synthesized precursor is secreted from the cell where it may participate in sphingolipid transport and signaling events. Re-uptake of the secreted precursor is mediated by high-affinity cell surface receptors that are apparently distinct from the M-6-P receptor. We found that the low density lipoprotein receptor-related protein (LRP), a multifunctional endocytic receptor that is expressed on most cells, can mediate cellular uptake and lysosomal delivery of SAP precursor. Additional in vivo experiments in mice revealed that the mannose receptor system on macrophages also participates in precursor internalization. We conclude that SAP precursor gains entry into cells by at least three independent receptor mechanisms including the M-6-P receptor, the mannose receptor and LRP.     

The low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor is a receptor for connective tissue growth factor

Connective tissue growth factor (CTGF) expression is regulated by transforming growth factor-beta (TGF-beta) and strong up-regulation occurs during wound healing; in situ hybridization data indicate that there are high levels of CTGF expression in fibrotic lesions. Recently the binding parameters of CTGF to both high and lower affinity cell surface binding components have been characterized. Affinity cross-linking and SDS-polyacrylamide gel electrophoresis analysis demonstrated the binding of CTGF to a cell surface protein with a mass of approximately 620 kDa. We report here the purification of this protein by affinity chromatography on CTGF coupled to Sepharose and sequence information obtained by mass spectroscopy. The binding protein was identified as the multiligand receptor, low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor (LRP). The identification of LRP as a receptor for CTGF was validated by several studies: 1) binding competition with many ligands that bind to LRP, including receptor-associated protein; 2) immunoprecipitation of CTGF-receptor complex with LRP antibodies; and 3) cells that are genetically deficient for LRP were unable to bind CTGF. Last, CTGF is rapidly internalized and degraded and this process is LRP-dependent. In summary, our data indicate that LRP is a receptor for CTGF, and may play an important role in mediating CTGF biology.