Cell Localization and Redistribution of the 67 kD Laminin Receptor and α6βl Integrin Subunits in Response to Laminin Stimulation: An Immunogold Electron Microscopy Study

The 67 kDa laminin receptor (67LR), one of several cell surface laminin-binding proteins, is involved in the interactions between cancer cells and laminin during tumor invasion and metastasis. A 37 kDa polypeptide (37LRP), previously identified as the 67LR precursor, is abundantly present in the cytoplasm and has been implicated in polysome formation. To better understand the cellular localization of the 67LR and its precursor, transmission electron microscopic studies of human melanoma A2058 cells were carried out using immunogold labeling and a variety of antibodies: (a) affinity purified antibodies directed against 37LRP cDNA-derived synthetic peptides; (b) anti-67LR monoclonal antibodies raised against intact human small cell lung carcinoma cells; and (c) monoclonal antibodies against the subunits of the integrin laminin receptor, α6β1. Double-labeling immunocyto-chemistry revealed that anti-67LR monoclonal antibodies as well as anti-37 LRP antibodies recognized antigens that were localized in the cytoplasm in electron dense structures. As expected, cell membrane labeling was also observed. Surprisingly, α6 and β1 integrin subunits were detected in the same cytoplasmic structures positive for the 67LR and the 37LRP. After addition of soluble laminin to A2058 cells in suspension, the number of labeled cytoplasmic structures increased especially in the vicinity of the plasma membrane, and were exported onto the cell surface. Neither fibronectin nor BSA induced such an effect. The data demonstrate that the 67 LR and the 37 LRP antibodies detect colocalized antigens that are in cytoplasmic structures with α6β1 integrin. These laminin binding protein rich structures could potentially form a supply of receptors that are exported to the surface upon exposure of the cells to laminin, with a consequent increase in the number of binding sites for the ligand. This system could define a mechanism through which cancer cells modulate their interaction with laminin.     

Molecular properties of poly(RGD) and its binding capacities to metastatic melanoma cells.

We examined the binding capacity of anti-metastatic polypeptide containing repetitive Arg-Gly-Asp(RGD) sequence derived from cell binding site of fibronectin, poly(RGD), to the surface of tumor cells. Poly(RGD) competitively inhibited the binding of radiolabeled fibronectin to the cell surface more potently than oligo(RGD) or RGD tripeptide on a molar basis. Compared on a weight basis to oligo(RGD) or RGD peptide, poly(RGD) was more active than the oligo- and monomeric peptide at inhibiting tumor cell adhesion to immobilized fibronectin. The secondary structure of poly(RGD) was predicted to be a beta-turn from the data of CD spectra and its amino acid sequence. These findings suggest that poly(RGD)-mediated inhibition of cell adhesion is due to its potent binding capacity to fibronectin receptors on cell surface probably through its conformational properties.     

The RGD containing site of the mouse laminin A chain is active for cell attachment, spreading, migration and neurite outgrowth

The laminin A chain has been sequenced by cDNA cloning and was found to contain an RGD sequence. Synthetic peptides containing the RGD sequence and flanking amino acids were active in mediating cell adhesion, spreading, migration, and neurite outgrowth. Furthermore, endothelial cell attachment to a laminin substrate was inhibited by an RGD-containing synthetic peptide. Antisera against the integrin (fibronectin) receptor, and monoclonal antibody to the integrin, VLA-6, inhibited cell interaction with laminin, as well as with peptides containing an RGD sequence. These results suggest that the RGD containing site of laminin is active and interacts with the integrin family of receptors in certain cells.     

Structure-function studies of the functional and binding epitope of the human 37 kDa laminin receptor precursor protein.

Expression of the 37 kDa laminin receptor precursor protein (37LRP) correlates directly with increased invasiveness and the metastatic potential of tumors. The 37LRP matures to a 67 kDa protein which facilitates the binding of cancer cells to basement membranes. The palindrome peptide sequence LMWWML, corresponding to the 173-178-residue stretch of the human 37LRP sequence, has been identified as the laminin-1-binding site. Peptides from 37LRP of species that contain this palindrome-bind laminin-1 with high affinity. Nuclear magnetic resonance (NMR) conformational studies have been undertaken on a synthetic 15-residue peptide (KGAHSVGLMWWMLAR) containing the palindrome to establish the structural basis of this activity. To further correlate the structural data with laminin-1-binding function, analogous structural studies were conducted for a similar peptide (RGKHSIGLIWYLLAR) lacking the palindrome, originating from 37LRP sequence of Saccharomyces cerevisiae and exhibiting low laminin-1-binding affinity. Finally, in vitro cell invasion assays were performed to investigate the possibility that the laminin-1-binding affinity of the peptides influences their inhibitory activity.     

Analysis of oligo-arginine cell-permeable peptides uptake by prostate cells

Recently, we have shown that oligo-arginine peptide (i.e., R11), a unique cell-permeable peptide (CPP), can be used as an imaging probe for prostate cancer detection. In this study, the mechanism(s) of oligo-arginine peptide in prostate cells was further analyzed. The length of the oligo-arginine peptide appears to be critical for the efficiency of uptake by prostate cells: poly (11)-arginine (R11) > poly (9)-arginine (R9) > poly (13)-arginine peptide (R13). The uptake of R11 peptide by prostate cells is mediated by macropinocytosis as evidenced by the fact that uptake can be blocked by a macropinocytosis inhibitor. However, the use of an inhibitor for carbohydrate chain elongation of glycosaminoglycan or inhibitors for carbohydrate synthesis of glycoprotein via either O-link or N-link showed minimal effects on R11 uptake. Nevertheless, pentosan sulfate (PentS) or dextran sulfate (DS) exhibited the highest inhibitory effect on R11 uptake in several prostate cells treated with various soluble glycosaminoglycans (GAGs) or anionic polymers. It is known that laminin receptor has been characterized as a PentS binding partner. Knocking down 37LRP (laminin receptor precursor) expression in prostate cells showed a reduction in their ability to uptake R11 peptides. In conclusion, laminin receptor is one of the initial binding site(s) responsible for R11 peptide uptake in prostate cells.     

A novel integrin specificity exemplified by binding of the alpha v beta 5 integrin to the basic domain of the HIV Tat protein and vitronectin

Several studies have addressed the interaction of the HIV Tat protein with the cell surface. Our analysis of the cell attachment-promoting activity of Tat and peptides derived from it revealed that the basic domain of Tat, not the arg-gly-asp (RGD) sequence, is required for cell attachment to Tat. Affinity chromatography with Tat peptides and immunoprecipitation with various anti-integrin antibodies suggest that the vitronectin-binding integrin, alpha v beta 5, is the cell surface protein that binds to the basic domain of Tat. The Tat basic domain contains the sequence RKKRRQRRR. A related sequence, KKQRFRHRNRKG, present in the heparin-binding domain of an alpha v beta 5 ligand, vitronectin, also bound alpha v beta 5 in affinity chromatography and, in combination with an RGD peptide, was an inhibitor of cell attachment to vitronectin. The alpha v beta 5 interaction with these peptides was not solely due to high content of basic amino acids in the ligand sequences; alpha v beta 5 did not bind substantially to peptides consisting entirely of arginine or lysine, whereas a beta 1 integrin did bind to these peptides. The interaction of alpha v beta 5 with Tat is atypical for integrins in that the binding to Tat is divalent cation independent, whereas the binding of the same integrin to an RGD- containing peptide or to vitronectin requires divalent cations. These data define an auxiliary integrin binding specificity for basic amino acid sequences. These basic domain binding sites may function synergistically with the binding sites that recognize RGD or equivalent sequences.     

Re-examination of CD91 function in GRP94 (glycoprotein 96) surface binding, uptake, and peptide cross-presentation

GRP94 (gp96)-peptide complexes can be internalized by APCs and their associated peptides cross-presented to yield activation of CD8(+) T cells. Investigations into the identity (or identities) of GRP94 surface receptors have yielded conflicting results, particularly with respect to CD91 (LRP1), which has been proposed to be essential for GRP94 recognition and uptake. To assess CD91 function in GRP94 surface binding and endocytosis, these parameters were examined in mouse embryonic fibroblast (MEF) cell lines whose expression of CD91 was either reduced via RNA interference or eliminated by genetic disruption of the CD91 locus. Reduction or loss of CD91 expression abrogated the binding and uptake of receptor-associated protein, an established CD91 ligand. Surface binding and uptake of an N-terminal domain of GRP94 (GRP94.NTD) was unaffected. GRP94.NTD surface binding was markedly suppressed after treatment of MEF cell lines with heparin, sodium chlorate, or heparinase II, demonstrating that heparin sulfate proteoglycans can function in GRP94.NTD surface binding. The role of CD91 in the cross-presentation of GRP94-associated peptides was examined in the DC2.4 dendritic cell line. In DC2.4 cells, which express CD91, GRP94.NTD-peptide cross-presentation was insensitive to the CD91 ligands receptor-associated protein or activated α(2)-macroglobulin and occurred primarily via a fluid-phase, rather than receptor-mediated, uptake pathway. These data clarify conflicting data on CD91 function in GRP94 surface binding, endocytosis, and peptide cross-presentation and identify a role for heparin sulfate proteoglycans in GRP94 surface binding.     

Effects of modifications of the RGD sequence and its context on recognition by the fibronectin receptor

The receptor for fibronectin is a member of the integrin superfamily of cell surface adhesion receptors, many of which recognize the sequence RGD in their ligands. We have developed sensitive enzyme-linked and radioreceptor assays to examine the ligand specificity of the fibronectin receptor. The fibronectin receptor bound only to fibronectin of the various Arg-Gly-Asp (RGD)-containing proteins tested. The smallest amount of receptor detectable in the assay was about 10 ng. Mn2+ enhanced the binding of the receptor to fibronectin 3-10-fold as compared to Ca2+ and Mg2+. Scatchard analysis of the saturation plot from the radioreceptor assay gave a dissociation constant (Kd) of 3 x 10(-8) M for the binding of fibronectin receptor to fibronectin in the presence of Mn2+. Inhibition experiments showed that the affinities of the ligands for the receptor decreased in the order of fibronectin approximately 110-kDa fibronectin fragment greater than GRGDSP peptide greater than 11.5-kDa fragment. Peptides not containing an RGD were several hundred to several thousand-fold less inhibitory than GRGDSP. These included the closely related peptides GRADSP and GRGESP, as well as three peptides containing the reverse sequence DGR. A peptide from the fibrinogen gamma-chain, KQAGDV, which had about 0.5% of the inhibitory activity of the standard GRGDSP peptide, was the most active peptide not containing an RGD. These results document the exquisite specificity of the fibronectin receptor for the RGD sequence.     

RGD and YIGSR synthetic peptides facilitate cellular adhesion identical to that of laminin and fibronectin but alter the physiology of neonatal cardiac myocytes

In the mammalian heart, the extracellular matrix plays an important role in regulating cell behavior and adaptation to mechanical stress. In cell culture, a significant number of cells detach in response to mechanical stimulation, limiting the scope of such studies. We describe a method to adhere the synthetic peptides RGD (fibronectin) and YIGSR (laminin) onto silicone for culturing primary cardiac cells and studying responses to mechanical stimulation. We first examined cardiac cells on stationary surfaces and observed the same degree of cellular adhesion to the synthetic peptides as their respective native proteins. However, the number of striated myocytes on the peptide surfaces was significantly reduced. Focal adhesion kinase (FAK) protein was reduced by 50% in cardiac cells cultured on YIGSR peptide compared with laminin, even though ₁-integrin was unchanged. Connexin43 phosphorylation increased in cells adhered to RGD and YIGSR peptides. We then subjected the cardiac cells to cyclic strain at 20% maximum strain (1 Hz) for 48 h. After this period, cell attachment on laminin was reduced to 50% compared with the unstretched condition. However, in cells cultured on the synthetic peptides, there was no significant difference in cell adherence after stretch. On YIGSR peptide, myosin protein was decreased by 50% after mechanical stimulation. However, total myosin was unchanged in cells stretched on laminin. These results suggest that RGD and YIGSR peptides promote the same degree of cellular adhesion as their native proteins; however, they are unable to promote the signaling required for normal FAK expression and complete sarcomere formation in cardiac myocytes. cell adhesion; connexin43; focal adhesion kinase; surface chemistry     

The 37-kDa/67-kDa laminin receptor acts as the cell-surface receptor for the cellular prion protein.

Recently, we identified the 37-kDa laminin receptor precursor (LRP) as an interactor for the prion protein (PrP). Here, we show the presence of the 37-kDa LRP and its mature 67-kDa form termed high-affinity laminin receptor (LR) in plasma membrane fractions of N2a cells, whereas only the 37-kDa LRP was detected in baby hamster kidney (BHK) cells. PrP co-localizes with LRP/LR on the surface of N2a cells and Semliki Forest virus (SFV) RNA transfected BHK cells. Cell-binding assays reveal the LRP/LR-dependent binding of cellular PrP by neuronal and non-neuronal cells. Hyperexpression of LRP on the surface of BHK cells results in the binding of exogenous PrP. Cell binding is similar in PrP(+/+) and PrP(0/0) primary neurons, demonstrating that PrP does not act as a co-receptor of LRP/LR. LRP/LR-dependent internalization of PrP is blocked at 4 degrees C. Secretion of an LRP mutant lacking the transmembrane domain (aa 86-101) from BHK cells abolishes PrP binding and internalization. Our results show that LRP/LR acts as the receptor for cellular PrP on the surface of mammalian cells.     

Production and properties of a bifunctional fusion protein that mediates attachment of vero cells to cellulosic matrices

The sequence Arg-Gly-Asp (RGD) in extracellular matrix proteins such as fibronectin, collagen, and laminin mediates cell attachment by interacting with proteins of the integrin family of cell surface receptors. A gene fusion encoding the RGD-containing peptide, fused to the C-terminus of a cellulose-binding domain (CBD/RGD), was expressed in Escherichia coli. Cultures produced up to 50 mg of CBD/RGD per liter, most of which was extracellular. It was purified from the culture supernatant by affinity chromatography on cellulose. CBD/RGD promoted the attachment of green monkey Vero cells to polystyrene and cellulose acetate. Attachment was inhibited by small synthetic peptides containing the RGD sequence. CBD/RGD was as effective as collagen in promoting the attachment of Vero cells to Cellsnowtrade mark microcarriers. (c) 1995 John Wiley & Sons, Inc.     

Biosynthesis of the 67 kDa high affinity laminin receptor.

High affinity interactions between cells and laminin are mediated, at least in part, by the 67 kDa laminin receptor (67 LR). A 37 kDa nascent polypeptide (37 LRP), predicted by a full length cDNA clone and obtained by in vitro translation of hybrid-selected laminin receptor mRNA, has been immunologically identified in cancer cell extracts as the putative precursor of the 67 LR. In this study, we used affinity purified antibodies developed against cDNA-deduced 37 LRP synthetic peptides in pulse chase experiments and demonstrated a precursor-product relationship between the 37 LRP and the 67 LR. Immunoblot, pulse chase and immunofluorescence experiments showed that transient transfection of the full length 37 LRP cDNA clone induced a dramatic increase in the synthesis of the 37 LRP but not of the mature 67 LR. We propose that the 67 LR results from the association of two gene products: the 37 LRP and a polypeptide yet to be identified.     

Interaction of human lactoferrin with cell adhesion molecules through RGD motif elucidated by lactoferrin-binding epitopes

Lactoferrin (LF) is an iron-binding secretory protein, which is distributed in the secondary granules of polynuclear lymphocytes as well as in the milk produced by female mammals. Although it has multiple functions, for example antimicrobial, immunomodulatory, antiviral, and anti-tumor metastasis activities, the receptors responsible for these activities are not fully understood. In this study, the binding epitopes for human LF were first isolated from a hexameric random peptide library displayed on T7 phage. Interestingly, two of the four isolated peptides had a representative cell adhesion motif, Arg-Gly-Asp (RGD), implying that human LF interacts with proteins with the RGD motif. We found that human LF bound to the RGD-containing human extracellular matrix proteins, fibronectin and vitronectin. Furthermore, human LF inhibited cell adhesion to these matrix proteins in a concentration-dependent manner but not to the RGD-independent cell adhesion molecule like laminin or collagen. These results indicate that a function of human LF is to block the various interactions between the cell surface and adhesion molecules. This may explain the multifunctionality of LF.     

Subcellular localization of prion proteins and the 37 kDa/67 kDa laminin receptor fused to fluorescent proteins

The 37 kDa/67 kDa laminin receptor LRP/LR acts as a receptor for both PrPc and PrPSc at the cell surface. Here, we further analyzed the subcellular localization of fluorescent labeled prion protein (PrP) and laminin receptor (LRP/LR) molecules. We show that EGFP-PrP is localized at the cell surface and in a perinuclear compartment, respectively. In contrast, a DsRed-DeltaSP-PrP mutant lacking the signal peptide is almost exclusively found in the nucleus but does not colocalize with heterochromatin. Interestingly, LRP-DsRed efficiently colocalizes with EGFP-PrP in the perinuclear compartment and LRP-ECFP partly colocalizes with DsRed-DeltaSP-PrP in the nucleus, respectively. We conclude that the interactions of PrP and LRP/LR are not restricted to the cell surface but occur also in intracellular compartments suggesting a putative role of LRP/LR in the trafficking of PrP molecules.     

Evidence for the presence of an integrin cell adhesion receptor on the oolemma of unfertilized human oocytes.

The Arg-Gly-Asp peptide (RGD), contained in several extracellular matrix proteins such as fibronectin, laminin, vitronectin, and collagen, is a tripeptide that plays a role as a recognition sequence in many cell-to-cell and cell-to-matrix adhesion mechanisms, through its interaction with several receptors of the integrin family. We previously described the ability of the oolemma of hamster oocytes to bind GRGDTP coupled to the surface of activated immunobeads and demonstrated that RGD-containing oligopeptides inhibit the adhesion of human and hamster spermatozoa to zona-free hamster oocytes and their subsequent penetration. In the present experiments, we show, utilizing immunobeads coated with an RGD-containing peptide (PepTiteTM 2000), that the oolemma of unfertilized human eggs is also able to recognize this adhesion sequence. The binding of PepTiteTM 2000-coated immunobeads to the oolemma was inhibited by the oligopeptide GRGDTP as well as by fibronectin and laminin. When immunobeads were prepared with a PepTiteTM concentration of 10 micrograms/ml, GRGDTP 150 micrograms/ml, laminin 80 micrograms/ml, and fibronectin 60 micrograms/ml inhibited bead rosetting on the egg surface. These data suggest that a specific binding moiety for RGD is present on the human egg surface. The binding of fibronectin to the oolemma was also demonstrated by the rosetting of immunobeads coupled with antifibronectin antibody to human oocytes after their exposure to 1 mg/ml free fibronectin. Such binding of fibronectin to the oolemma could be inhibited by coincubation with a monoclonal antibody directed against the cell adhesion fragment of fibronectin.(ABSTRACT TRUNCATED AT 250 WORDS)     

37-kDa laminin receptor precursor mediates GnRH-II-induced MMP-2 expression and invasiveness in ovarian cancer cells

GnRH-II enhances ovarian cancer cell invasion in an autocrine manner. We have now found that GnRH-II increases 37-kDa laminin receptor precursor (LRP) production in GnRH receptor (GnRHR)-positive OVCAR-3 and CaOV-3 ovarian cancer cells, while small interfering RNA (siRNA)-mediated depletion of GnRH-II or GnRHR mRNA abrogates this. The invasiveness of ovarian cancer cells is also reduced >85% by siRNA-mediated knockdown of LRP levels and >50% by pretreatment of Matrigel with a synthetic peptide that blocks interactions between laminin and the 67-kDa nonintegrin laminin receptor which comprises two LRP subunits. Conversely, overexpressing LRP in CaOV-3 cells increases their invasiveness 5-fold, while overexpressing LRP with a nonfunctional laminin-binding site does not. Depletion of LRP by siRNA treatment reduces CaOV-3 cell attachment to laminin-coated plates by ～80% but only reduces their binding to Matrigel by ～20%. Thus, while LRP influences CaOV-3 cell adhesion to laminin, LRP must act in other ways to enhance invasion. Matrix metalloproteinases (MMPs) are key mediators of invasion, and LRP siRNA treatment of OVCAR-3 and CaOV-3 cells inhibits MMP-2 but not MMP-9 mRNA levels. Overexpressing LRP in these cells increases MMP-2 production specifically, while a laminin-binding deficient LRP does not. Importantly, LRP siRNA treatment abolishes GnRH-II-induced MMP-2 production, and invasion in OVCAR-3 and CaOV-3 cells, which was also seen after MMP-2 siRNA treatment. These results suggest that GnRH-II-induced LRP expression increases the amount of the 67-kDa nonintegrin laminin receptor, which appears to interact with laminin in the extracellular matrix to promote MMP-2 expression and enhance ovarian cancer cell invasion.     

Characterization of the interaction between lysyl-tRNA synthetase and laminin receptor by NMR

Lysyl-tRNA synthetase (KRS) interacts with the laminin receptor (LR/RPSA) and enhances laminin-induced cell migration in cancer metastasis. In this nuclear magnetic resonance (NMR)-based study, we show that the anticodon-binding domain of KRS binds directly to the C-terminal region of 37LRP, and the previously found inhibitors BC-K-01 and BC-K-YH16899 interfere with KRS–37LRP binding. In addition, the anticodon-binding domain of KRS binds to laminin, observed by NMR and SPR. These results provide crucial insights into the structural characteristics of the KRS–LR interaction on the cell surface.     

The 67-kDa laminin receptor originated from a ribosomal protein that acquired a dual function during evolution

The 67-kDa laminin receptor (67LR) is a nonintegrin cell surface receptor that mediates high-affinity interactions between cells and laminin. Overexpression of this protein in tumor cells has been related to tumor invasion and metastasis. Thus far, only a full-length gene encoding a 37-kDa precursor protein (37LRP) has been isolated. The finding that the cDNA for the 37LRP is virtually identical to a cDNA encoding the ribosomal protein p40 has suggested that 37LRP is actually a component of the translational machinery, with no laminin-binding activity. On the other hand, a peptide of 20 amino acids deduced from the sequence of 37LR/p40 was shown to exhibit high laminin-binding activity. The evolutionary relationship between 23 sequences of 37LRP/p40 proteins was analyzed. This phylogenetic analysis indicated that all of the protein sequences derive from orthologous genes and that the 37LRP is indeed a ribosomal protein that acquired the novel function of laminin receptor during evolution. The evolutionary analysis of the sequence identified as the laminin-binding site in the human protein suggested that the acquisition of the laminin-binding capability is linked to the palindromic sequence LMWWML, which appeared during evolution concomitantly with laminin.      

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.     

A trans-dominant negative 37kDa/67kDa laminin receptor mutant impairs PrP(Sc) propagation in scrapie-infected neuronal cells

The 37kDa/67kDa laminin receptor (LRP/LR) has been identified as a cell surface receptor for cellular and infectious prion proteins. Here, we show that an N-terminally truncated LRP mutant encompassing the extracellular domain of the LRP/LR (LRP102-295::FLAG) reduces the binding of recombinant cellular huPrP to mouse neuroblastoma cells, and infectious moPrP27-30 to BHK cells, and interferes with the PrP(Sc) propagation in scrapie-infected neuroblastoma cells (N2aSc(+)). A cell-free binding assay demonstrated the direct binding of the LRP102-295::FLAG mutant to both PrP(c) and PrP(Sc). These results, together with the finding that endogenous LRP levels remain unaffected by the expression of the mutant, indicate that the secreted LRP102-295::FLAG mutant may act in a trans-dominant negative manner as a decoy by trapping PrP molecules. The LRP mutant might represent a potential therapeutic tool for the treatment of TSEs.