Intracellular retention of thyroglobulin in the absence of the low-density lipoprotein receptor-associated protein (RAP) is likely due to premature binding to megalin in the biosynthetic pathway.

OBJECTIVE: The low-density lipoprotein receptor associated protein (RAP) is expressed by thyroid epithelial cells (TEC) in a TSH-dependent manner. In the thyroid RAP functions as a molecular chaperone for the thyroglobulin (Tg) endocytic receptor megalin/LRP2, which is retained intracellularly in RAP KO mice rather than being expressed on the apical membrane of TEC, its usual location. RAP binds also to Tg, which is also retained intracellularly in RAP KO mice, thereby suggesting a role of RAP in Tg secretion. Here we investigated whether Tg intracellular retention in the absence of RAP is due to premature Tg-megalin interactions during the biosynthetic pathway or to a direct action of RAP on Tg secretion. METHODS: We performed immunoprecipitation experiments in thyroid extracts from RAP KO and WT mice. In addition, we investigated Tg secretion in COS-7 cells co-transfected with human RAP (hRAP) and mouse Tg (mTg). RESULTS: An anti-megalin megalin precipitated greater amounts of Tg in thyroid extracts from RAP KO than from WT mice, suggesting increased intracellular interactions between megalin and Tg in the absence of RAP. COS-7 cells transiently transfected with hRAP, mTg or both, expressed the two proteins accordingly. RAP was found almost exclusively in cell extracts, whereas Tg was found both in extracts and media, as expected from the knowledge that RAP is ER-resident and that Tg is secreted. Regardless of whether cells were transfected with mTg alone or were co-transfected with hRAP, similar proportions of the total Tg synthesized were detected in cell extracts and media. CONCLUSIONS: The intracellular retention of Tg in the absence of RAP is likely due to its premature interaction with megalin, whereas RAP does not seem to affect Tg secretion directly.

Role of megalin and the soluble form of its ligand RAP in Cd-metallothionein endocytosis and Cd-metallothionein-induced nephrotoxicity in vivo.

Orally administered Cd is predominantly distributed to the intestine, and the majority of this mucosal Cd is bound to metallothionein (MT). MT attenuates heavy metal-induced cytotoxicity by sequestering these metals and lowering their intracellular concentrations. In addition, MT acts as an extracellular transporter of orally administered Cd to the kidney. Because of its low molecular weight, the Cd-MT complex is freely filtered at the glomerulus, and the filtered Cd-MT is then incorporated into renal proximal tubular cells. Megalin, a multiligand endocytic receptor (also known as low-density lipoprotein receptor-related protein 2 or Lrp2), acts as the receptor for Cd-MT in a renal proximal tubular cell model. Here, we used the soluble form of 39-kDa receptor-associated protein (sRAP; also known as Lrpap1), a ligand of megalin, to inhibit megalin function, and then analyzed the effect of megalin loss on Cd-MT distribution and Cd-MT-induced nephrotoxicity in an animal model. Administration of sRAP to mice caused acute loss of megalin function by removing megalin in the brush border membrane. The pre-injection of sRAP decreased renal Cd content and decreased Cd-MT-induced kidney damage. Our results demonstrate that sRAP reduces Cd-MT-induced kidney toxicity in vivo.

The hemopexin domain of matrix metalloproteinase-9 activates cell signaling and promotes migration of schwann cells by binding to low-density lipoprotein receptor-related protein.

Low-density lipoprotein receptor-related protein (LRP-1) is an endocytic receptor for diverse proteins, including matrix metalloproteinase-9 (MMP-9), and a cell-signaling receptor. In the peripheral nervous system (PNS), LRP-1 is robustly expressed by Schwann cells only after injury. Herein, we demonstrate that MMP-9 activates extracellular-signal-regulated kinase (ERK1/2) and Akt in Schwann cells in culture. MMP-9 also promotes Schwann cell migration. These activities require LRP-1. MMP-9-induced cell signaling and migration were blocked by inhibiting MMP-9-binding to LRP-1 with receptor-associated protein (RAP) or by LRP-1 gene silencing. The effects of MMP-9 on Schwann cell migration also were inhibited by blocking the cell-signaling response. An antibody targeting the hemopexin domain of MMP-9, which mediates the interaction with LRP-1, blocked MMP-9-induced cell signaling and migration. Furthermore, a novel glutathione-S-transferase fusion protein (MMP-9-PEX), which includes only the hemopexin domain of MMP-9, replicated the activities of intact MMP-9, activating Schwann cell signaling and migration by an LRP-1-dependent pathway. Constitutively active MEK1 promoted Schwann cell migration; in these cells, MMP-9-PEX had no further effect, indicating that ERK1/2 activation is sufficient to explain the effects of MMP-9-PEX on Schwann cell migration. Injection of MMP-9-PEX into sciatic nerves, 24 h after crush injury, robustly increased phosphorylation of ERK1/2 and Akt. This response was inhibited by RAP. MMP-9-PEX failed to activate cell signaling in uninjured nerves, consistent with the observation that Schwann cells express LRP-1 at significant levels only after nerve injury. These results establish LRP-1 as a cell-signaling receptor for MMP-9, which may be significant in regulating Schwann cell migration and physiology in PNS injury.

Receptor-associated protein (RAP) plays a central role in modulating Abeta deposition in APP/PS1 transgenic mice.

BACKGROUND: Receptor associated protein (RAP) functions in the endoplasmic reticulum (ER) to assist in the maturation of several membrane receptor proteins, including low density lipoprotein receptor-related protein (LRP) and lipoprotein receptor 11 (SorLA/LR11). Previous studies in cell and mouse model systems have demonstrated that these proteins play roles in the metabolism of the amyloid precursor protein (APP), including processes involved in the generation, catabolism and deposition of beta-amyloid (Abeta) peptides. METHODOLOGY/PRINCIPAL FINDINGS: Mice transgenic for mutant APPswe and mutant presenilin 1 (PS1dE9) were mated to mice with homozygous deletion of RAP. Unexpectedly, mice that were homozygous null for RAP and transgenic for APPswe/PS1dE9 showed high post-natal mortality, necessitating a shift in focus to examine the levels of amyloid deposition in APPswe/PS1dE9 that were hemizygous null for RAP. Immunoblot analysis confirmed 50% reductions in the levels of RAP with modest reductions in the levels of proteins dependent upon RAP for maturation [LRP trend towards a 20% reduction ; SorLA/LR11 statistically significant 15% reduction (p<0.05)]. Changes in the levels of these proteins in the brains of [APPswe/PS1dE9](+/-)/RAP(+/-) mice correlated with 30-40% increases in amyloid deposition by 9 months of age. CONCLUSIONS/SIGNIFICANCE: Partial reductions in the ER chaperone RAP enhance amyloid deposition in the APPswe/PS1dE9 model of Alzheimer amyloidosis. Partial reductions in RAP also affect the maturation of LRP and SorLA/LR11, which are each involved in several different aspects of APP processing and Abeta catabolism. Together, these findings suggest a central role for RAP in Alzheimer amyloidogenesis.

Binding of alpha2ML1 to the low density lipoprotein receptor-related protein 1 (LRP1) reveals a new role for LRP1 in the human epidermis.

BACKGROUND: The multifunctional receptor LRP1 has been shown to bind and internalize a large number of protein ligands with biological importance such as the pan-protease inhibitor alpha2-macroglobulin (alpha2M). We recently identified Alpha2ML1, a new member of the alpha2M gene family, expressed in epidermis. alpha2ML1 might contribute to the regulation of desquamation through its inhibitory activity towards proteases of the chymotrypsin family, notably KLK7. The expression of LRP1 in epidermis as well as its ability to internalize alpha2ML1 was investigated. METHODS AND PRINCIPAL FINDINGS: In human epidermis, LRP1 is mainly expressed within the granular layer of the epidermis, which gathers the most differentiated keratinocytes, as shown by immunohistochemistry and immunofluorescence using two different antibodies. By using various experimental approaches, we show that the receptor binding domain of alpha2ML1 (RBDl) is specifically internalized into the macrophage-like cell line RAW and colocalizes with LRP1 upon internalization. Coimmunoprecipitation assays demonstrate that RBDl binds LRP1 at the cell surface. Addition of RAP, a universal inhibitor of ligand binding to LRP1, prevents RBDl binding at the cell surface as well as internalization into RAW cells. Silencing Lrp1 expression with specific siRNA strongly reduces RBDl internalization. CONCLUSIONS AND SIGNIFICANCE: Keratinocytes of the upper differentiated layers of epidermis express LRP1 as well as alpha2ML1. Our study also reveals that alpha2ML1 is a new ligand for LRP1. Our findings are consistent with endocytosis by LRP1 of complexes formed between alpha2ML1 and proteases. LRP1 may thus control desquamation by regulating the biodisponibility of extracellular proteases.

Phylogenetic conservation of glycoprotein 96 ability to interact with CD91 and facilitate antigen cross-presentation.

Although the ability of gp96 to activate APCs and generate CD8 CTLs against peptides they chaperone through interaction with the endocytic receptors CD91 is supported by solid evidence, its biological relevance in immune surveillance is debated. We have used an evolutionary approach to determine whether gp96 interacts with receptors expressed on APCs and promotes MHC class I cross-presentation of minor histocompatibility Ags (H-Ags) to CTLs in the frog Xenopus. We show that in Xenopus gp96 binds the CD91 homolog at the surface of peritoneal leukocytes, and that this binding is inhibited by molar excess of unlabeled gp96 or the CD91 ligand alpha2-macroglobulin, by anti-CD91 Ab and by the specific CD91 antagonist receptor-associated protein. Surface binding followed by internalization of gp96 was confirmed by fluorescent microscopy. Furthermore, adoptive transfer of peritoneal leukocytes pulsed with as little as 800 ng of gp96 chaperoning minor H-Ags, but not minor H-Ag-free gp96, induces potent CD8 T cell infiltration and Ag-specific accelerated rejection of minor H-locus disparate skin grafts. Inhibition of gp96-CD91 interaction by pretreatment with anti-CD91 Ab and receptor-associated protein impairs both CD8 T cell infiltration and acute skin graft rejection. These data provide evidence of the conserved ability of gp96 to facilitate cross-presentation of chaperoned Ags by interacting with CD91. The persistence of this biological process for >350 million years that separate mammals and amphibians from a common ancestor strongly supports the proposition that gp96 and CD91 are critically involved in immune surveillance.

Intraperitoneal administration of recombinant receptor-associated protein causes phosphaturia via an alteration in subcellular distribution of the renal sodium phosphate co-transporter.

Megalin is a multifunctional endocytic receptor that is expressed in renal proximal tubules and plays critical roles in the renal uptake of various proteins. It was hypothesized that megalin-dependent endocytosis might play a role in renal phosphate reabsorption. For addressing the short-term effects of altered megalin function, a recombinant protein for the soluble form of 39-kD receptor-associated protein (RAP) was administered intraperitoneally to 7-wk-old mice. Histidine (His)-tagged soluble RAP (amino acids 39 to 356) lacking the amino-terminal signal peptide and the carboxy-terminal endoplasmic reticulum retention signal was prepared by bacterial expression (designated His-sRAP). After the direct interaction between His-sRAP and megalin was confirmed, mice were given a single intraperitoneal administration of His-sRAP (3.5 mg/dose). Immunostaining and Western blot analyses demonstrated the uptake of His-sRAP and the accelerated internalization of megalin in proximal tubular cells 1 h after administration. In addition, internalization of the type II sodium/phosphate co-transporter (NaPi-II) was observed. The effects of three sequential administrations of His-sRAP (3.5 mg/dose, three doses at 4-h intervals) then were examined, and increased urinary excretion of low molecular weight proteins, including vitamin D-binding protein, was found, which is consistent with findings reported for megalin-deficient mice. It is interesting that urinary excretion of phosphate was also increased, and the protein level of NaPi-II in the brush border membrane was decreased. Serum concentration of 25-hydroxyvitamin D was decreased, whereas the plasma level of intact parathyroid hormone was not altered by the administration of His-sRAP. The results suggest that the His-sRAP-induced acceleration of megalin-mediated endocytosis caused phosphaturia via altered subcellular distribution of NaPi-II.

LDL receptor cooperates with LDL receptor-related protein in regulating plasma levels of coagulation factor VIII in vivo.

Low-density lipoprotein (LDL) receptor (LDLR) and LDLR-related protein (LRP) are members of the LDLR family of endocytic receptors. LRP recognizes a wide spectrum of structurally and functionally unrelated ligands, including coagulation factor VIII (FVIII). In contrast, the ligand specificity of LDLR is restricted to apolipoproteins E and B-100. Ligand binding to the LDLR family is inhibited by receptor-associated protein (RAP). We have previously reported that, apart from LRP, other RAP-sensitive mechanisms contribute to the regulation of FVIII in vivo. In the present study, we showed that the extracellular ligand-binding domain of LDLR interacts with FVIII in vitro and that binding was inhibited by RAP. The physiologic relevance of the FVIII-LDLR interaction was addressed using mouse models of LDLR or hepatic LRP deficiency. In the absence of hepatic LRP, LDLR played a dominant role in the regulation and clearance of FVIII in vivo. Furthermore, FVIII clearance was accelerated after adenovirus-mediated gene transfer of LDLR. The role of LDLR in FVIII catabolism was not secondary to increased plasma lipoproteins or to changes in lipoprotein profiles. We propose that LDLR acts in concert with LRP in regulating plasma levels of FVIII in vivo. This represents a previously unrecognized link between LDLR and hemostasis.

Efficient transfer of receptor-associated protein (RAP) across the blood-brain barrier.

We have sought to identify a high-capacity transport system that mediates transcytosis of proteins from the blood to the brain. The 39 kDa receptor-associated protein (RAP) functions as a specialized endoplasmic reticulum chaperone assisting in the folding and trafficking of members of the low-density lipoprotein (LDL) receptor family. RAP efficiently binds to these receptors and antagonizes binding of other ligands. Previous studies have shown that two large members of the LDL receptor family, LDL receptor-related protein 1 (LRP1) and LDL receptor-related protein 2 (LRP2 or megalin), possess the ability to mediate transcytosis of ligands across the brain capillary endothelium. Here, we tested whether blood-borne RAP crosses the blood-brain barrier (BBB) by LRP1- or megalin-mediated transport by studying the pharmacokinetics of [125I]-RAP transport into the brain in intact mice and across cell monolayers in vitro. Our results show that [125I]-RAP is relatively stable in blood for 30 minutes and has a mean influx constant of 0.62+/-0.08 microl/g-minute from blood to brain. In situ brain perfusion in blood-free buffer shows that transport of [125I]-RAP across the BBB is a saturable process. Capillary depletion of brain homogenates indicates that 70% of [125I]-RAP is localized in the parenchyma rather than in the vasculature of the brain. Results of transport in stably transfected MDCK cells are consistent with the hypothesis that megalin mediates most of the apical-to-basolateral transport across polarized epithelial cells. The inhibition of [125I]-RAP influx by excess RAP and the involvement of megalin indicate the presence of a saturable transport system at the BBB. The higher permeability of RAP compared with that of melanotransferrin and transferrin show that the LRP receptor is a high capacity transport system. These studies suggest that RAP may provide a novel means of protein-based drug delivery to the brain.

A CD91-positive subset of CD11c+ blood dendritic cells: characterization of the APC that functions to enhance adaptive immune responses against CD91-targeted antigens.

Dendritic cells (DC) and other APCs rely on a number of specialized receptors to facilitate the uptake and intracellular accumulation of Ags. In this capacity, APCs use receptor-mediated endocytosis to enhance Ag presentation and the stimulation of Ag-specific T cells. Studies have demonstrated that the targeted delivery of Ags in vivo to CD91/the low-density lipoprotein receptor-related protein (CD91/LRP) induces enhanced activation of the adaptive immune system. However, the APC that mediates these augmented, Ag-specific responses remains to be characterized. In this study, we show that a subset of CD11c(+) lineage-negative (lin(-)) DC expresses the scavenger receptor CD91/LRP and that these rare APC are primarily responsible for the T cell activation that occurs following CD91/LRP-mediated Ag uptake in whole blood. The targeting of Ags to CD91/LRP results in enhanced receptor-mediated uptake within both lin(-) DCs and monocytes, and this uptake results in markedly increased T cell activation. Finally, purified cellular populations were used to demonstrate that CD11c(+) lin(-) DC, but not monocytes, are capable of stimulating T cell activation following CD91/LRP-mediated Ag uptake. Therefore, CD11c(+) lin(-) DC use CD91/LRP to facilitate the uptake and subsequent presentation of an array of Ags complexed within the CD91/LRP ligand, the activated form of alpha2-macroglobulin (alpha2M*).

The low density lipoprotein receptor-related protein complexes with cell surface heparan sulfate proteoglycans to regulate proteoglycan-mediated lipoprotein catabolism.

It has been proposed that clearance of cholesterol-enriched very low density lipoprotein (VLDL) particles occurs through a multistep process beginning with their initial binding to cell-surface heparan sulfate proteoglycans (HSPG), followed by their uptake into cells by a receptor-mediated process that utilizes members of the low density lipoprotein receptor (LDLR) family, including the low density lipoprotein receptor-related protein (LRP). We have further explored the relationship between HSPG binding of VLDL and its subsequent internalization by focusing on the LRP pathway using a cell line deficient in LDLR. In this study, we show that LRP and HSPG are part of a co-immunoprecipitable complex at the cell surface demonstrating a novel association for these two cell surface receptors. Cell surface binding assays show that this complex can be disrupted by an LRP-specific ligand binding antagonist, which in turn leads to increased VLDL binding and degradation. The increase in VLDL binding results from an increase in the availability of HSPG sites as treatment with heparinase or competitors of glycosaminoglycan chain addition eliminated the augmented binding. From these results we propose a model whereby LRP regulates the availability of VLDL binding sites at the cell surface by complexing with HSPG. Once HSPG dissociates from LRP, it is then able to bind and internalize VLDL independent of LRP endocytic activity. We conclude that HSPG and LRP together participate in VLDL clearance by means of a synergistic relationship.

Impaired thyroglobulin (Tg) secretion by FRTL-5 cells transfected with soluble receptor associated protein (RAP): evidence for a role of RAP in the Tg biosynthetic pathway.

Secretion of thyroglobulin (Tg) by thyrocytes requires several endoplasmic reticulum (ER)-resident molecular chaperones. The receptor-associated protein (RAP), a known molecular chaperone, binds to Tg in thyroid cells shortly after biosynthesis. Here we investigated whether RAP is involved in Tg secretion by FRTL-5 cells. For this purpose, we studied Tg secretion by FRTL-5 cells transfected with a soluble RAP chimera, as a mean for interfering with endogenous RAP. We used a RAP-human IgG Fc (RAP-Ig) chimeric cDNA, which was designed in order to exclude the ER retention sequence of RAP and to allow generation of a secreted form of RAP. FRTL-5 cells were transiently transfected with the RAP-Ig cDNA or, as control, with a CD8-Ig cDNA. Media were collected at 24, 48 and 72 h after transfection. Secretion of fusion proteins and of Tg in the media was measured by ELISA. As expected, under standard culture conditions, RAP was not secreted into the media by FRTL-5 cells, even though it could be detected by Western blotting in cell extracts. In transfection experiments, fusion proteins were present in the media of FRTL-5 cells transfected with either RAP-Ig or CD8-Ig, indicating that transfection was successful. Although Tg was found in the media of FRTL-5 cells transfected with either CD8-Ig or RAP-Ig, a lower amount was found in cells transfected with RAP-Ig. Therefore, we concluded that RAP is involved in Tg secretion by FRTL-5 cells suggesting that RAP may function as a Tg molecular chaperone.

Low-density lipoprotein receptor-related protein mediates the endocytosis of anionic liposomes in neurons.

We have recently demonstrated that anionic liposomes efficiently introduce foreign DNA into postmitotic neurons and other cell types (Lakkaraju, A., Dubinsky, J. M., Low, W. C., and Rahman, Y.-E. (2001) J. Biol. Chem. 276, 32000-32007). To investigate the mechanism of liposome uptake, we followed the internalization of anionic liposome-encapsulated Cy3-labeled oligonucleotides (AL-Cy3ONs) by hippocampal neurons using confocal microscopy. Uptake of AL-Cy3ONs was widespread and time- and temperature-dependent, indicative of receptor-mediated endocytosis. The low-density lipoprotein receptor-related protein (LRP) was crucial for anionic liposome endocytosis because the receptor-associated protein or an anti-LRP antibody inhibited internalization, and fibroblasts lacking LRP did not internalize AL-Cy3ONs. Using selective endocytosis inhibitors, we found that liposome endocytosis and intracellular transport required clathrin, dynamin, an intact cytoskeletal network, and phosphatidylinositol 3-kinase activity. Cy3ONs did not significantly colocalize with recycling endosomal/lysosomal markers and entered neuronal nuclei within 1-3 h of incubation. Approximately 50% of the internalized liposomal phospholipids were recycled back to the cell surface, in keeping with the fluidity of their acyl chains. Liposome endocytosis did not require heparan sulfate proteoglycans or cause calcium influx into neurons. Thus, constitutive endocytosis of anionic liposomes by LRP utilizes only one component, in contrast to the more involved heparan sulfate proteoglycan-LRP pathway implicated in the pathogenesis of Alzheimer's disease.