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.     

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.     

Different isoforms of the non-integrin laminin receptor are present in mouse brain and bind PrP

The prion protein (PrP) plays a central role in prion diseases, and identifying its cellular receptor appears to be of crucial interest. We previously showed in the yeast two-hybrid system that PrP interacts with the 37 kDa precursor (LRP) of the high affinity 67 kDa laminin receptor (LR), which acts as the cellular receptor of PrP in cellular models. However, among the various isoforms of the receptor that have been identified so far, those which are present in the central nervous system and which bind PrP are still unknown. In this study, we have purified mouse brain fractions enriched in the laminin receptor and have performed overlay assays in order to identify those isoforms that interact with the prion protein. We demonstrate (i) the presence, in mouse brain, of several isoforms of the LRP/LR corresponding to different maturation states of the receptor (44, 60, 67 and 220 kDa) and (ii) the binding of all of these isoforms to PrP. Our data strongly support a physiological role of the laminin receptor/PrP interaction in the brain and highlight its relevance for transmissible spongiform encephalopathies.     

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.     

Interaction of Doppel with the full-length laminin receptor precursor protein

Doppel (Dpl) is a homolog of normal cellular prion protein (PrPc) with unknown functions. Ectopic expression of Dpl in the central nervous system (CNS) causes neurotoxicity and this effect is rescued by the expression of PrPc. However, the molecular basis for the protective effect of PrPc remains unclear. Using a yeast two-hybrid system, we showed that Dpl binds the full-length 37-kDa laminin receptor precursor protein (LRP), one of the receptors of PrPc. The interaction was also validated by immunoprecipitation and immunoblotting using transfected cell lines and in vivo derived tissues. Further mapping experiments showed that although the middle fragment containing residues 100-220 of LRP was able to interact with Dpl, deletion of the N-terminal domain of the full-length LRP abolished its interaction with Dpl. These results suggest that while both PrPc and Dpl interact with LRP, the domains that are involved in the binding are not the same. Our results may have implications for the molecular mechanisms of Dpl-PrPc antagonism and physiological roles of Dpl.     

In Vitro Inhibition of Angiogenesis by Antibodies Directed against the 37kDa/67kDa Laminin Receptor

The 37kDa/67kDa laminin receptor (LRP/LR) is a central receptor mediating interactions between tumour cells and the basement membrane and is thereby a key player in adhesion and invasion, essential processes in metastatic cancer. To affect continued tumour growth, tumours induce angiogenesis for the constant delivery of nutrients and oxygen. This study aims to determine the blocking effect of the anti-LRP/LR specific antibody, W3 on the angiogenic potential of HUVE (human umbilical vein endothelial) cells. Flow cytometric analysis revealed that 97% of HUVE cells display cell surface LRP/LR. An angiogenesis assay was conducted employing HUVE cells seeded on the basement membrane reconstituent Matrigel™ supplemented with the pro-angiogenic factor vascular endothelial growth factor (VEGF). Post 18h incubation at 37°C tubular structures, namely tube lengths were assessed. Treatment of established tubular structures with 100 µg/ml anti-LRP/LR specific antibody completely blocked angiogenesis. Our findings suggest a central role of the 37kDa/67kDa LRP/LR in tube formation and recommends anti-LRP/LR specific antibodies as potential therapeutic tools for treatment of tumour angiogenesis.     

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.     

Anti-LRP/LR Antibody W3 Hampers Peripheral PrPSc Propagation in Scrapie Infected Mice

We identified the 37kDa/67kDa laminin receptor (LRP/LR) as a cell surface receptor for the cellular prion protein (PrP^c) and the infectious prion protein (PrP^Sc). Recently, we showed that anti-LRP/LR antibody W3 cured scrapie infected N2a cells. Here, we demonstrate that W3 delivered by passive immunotransfer into C57BL/6 mice reduced the PrP^Sc content in the spleen significantly by 66%, demonstrating an impairment of the peripheral PrP^Sc propagation. In addition, we observed a 1.8-fold increase in survival of anti-LRP/LR antibody W3 treated mice (mean survival of 31 days) compared to preimmune serum treated control animals (mean survival of 17 days). We conclude that the significant effect of anti-LRP/LR antibody W3 on the reduction of peripheral PrP^Sc propagation might be due to the blockage of the prion receptor LRP/LR which is required, as previously shown in vitro, for PrP^Sc propagation in vivo.Key Words: 37kDa/67kDa laminin receptor, LRP/LR, prion, PrP, TSE-therapy     

Identification of interaction domains of the prion protein with its 37-kDa/67-kDa laminin receptor.

Cell-binding and internalization studies on neuronal and non-neuronal cells have demonstrated that the 37-kDa/67-kDa laminin receptor (LRP/LR) acts as the receptor for the cellular prion protein (PrP). Here we identify direct and heparan sulfate proteoglycan (HSPG)-dependent interaction sites mediating the binding of the cellular PrP to its receptor, which we demonstrated in vitro on recombinant proteins. Mapping analyses in the yeast two-hybrid system and cell-binding assays identified PrPLRPbd1 [amino acids (aa) 144-179] as a direct and PrPLRPbd2 (aa 53-93) as an indirect HSPG-dependent laminin receptor precursor (LRP)-binding site on PrP. The yeast two-hybrid system localized the direct PrP-binding domain on LRP between aa 161 and 179. Expression of an LRP mutant lacking the direct PrP-binding domain in wild-type and mutant HSPG-deficient Chinese hamster ovary cells by the Semliki Forest virus system demonstrates a second HSPG-dependent PrP-binding site on LRP. Considering the absence of LRP homodimerization and the direct and indirect LRP-PrP interaction sites, we propose a comprehensive model for the LRP-PrP-HSPG complex.     

The 67 kDa laminin receptor: structure, function and role in disease

The 67LR (67 kDa laminin receptor) is a cell-surface receptor with high affinity for its primary ligand. Its role as a laminin receptor makes it an important molecule both in cell adhesion to the basement membrane and in signalling transduction following this binding event. The protein also plays critical roles in the metastasis of tumour cells. Isolation of the protein from either normal or cancerous cells results in a product with an approx. molecular mass of 67 kDa. This protein is believed to be derived from a smaller precursor, the 37LRP (37 kDa laminin receptor precursor). However, the precise mechanism by which cytoplasmic 37LRP becomes cell-membrane-embedded 67LR is unclear. The process may involve post-translational fatty acylation of the protein combined with either homo- or hetero-dimerization, possibly with a galectin-3-epitope-containing partner. Furthermore, it has become clear that acting as a receptor for laminin is not the only function of this protein. 67LR also acts as a receptor for viruses, such as Sindbis virus and dengue virus, and is involved with internalization of the prion protein. Interestingly, unmodified 37LRP is a ribosomal component and homologues of this protein are found in all five kingdoms. In addition, it appears to be strongly associated with histones in the eukaryotic cell nucleus, although the precise role of these interactions is not clear. Here we review the current understanding of the structure and function of this molecule, as well as highlighting areas requiring further research.     

Knock-Down of the 37kDa/67kDa Laminin Receptor LRP/LR Impedes Telomerase Activity

Cancer has become a major problem worldwide due to its increasing incidence and mortality rates. Both the 37kDa/67kDa laminin receptor (LRP/LR) and telomerase are overexpressed in cancer cells. LRP/LR enhances the invasiveness of cancer cells thereby promoting metastasis, supporting angiogenesis and hampering apoptosis. An essential component of telomerase, hTERT is overexpressed in 85–90% of most cancers. hTERT expression and increased telomerase activity are associated with tumor progression. As LRP/LR and hTERT both play a role in cancer progression, we investigated a possible correlation between LRP/LR and telomerase. LRP/LR and hTERT co-localized in the perinuclear compartment of tumorigenic breast cancer (MDA_MB231) cells and non-tumorigenic human embryonic kidney (HEK293) cells. FLAG^® Co-immunoprecipitation assays confirmed an interaction between LRP/LR and hTERT. In addition, flow cytometry revealed that both cell lines displayed high cell surface and intracellular LRP/LR and hTERT levels. Knock-down of LRP/LR by RNAi technology significantly reduced telomerase activity. These results suggest for the first time a novel function of LRP/LR in contributing to telomerase activity. siRNAs targeting LRP/LR may act as a potential alternative therapeutic tool for cancer treatment by (i) blocking metastasis (ii) promoting angiogenesis (iii) inducing apoptosis and (iv) impeding telomerase activity.     

The human "prion-like" protein Doppel is expressed in both Sertoli cells and spermatozoa

The prion-like Doppel protein (Dpl) has many biochemical and structural properties in common with the cellular prion protein (PrP(c)), and the physiological role of neither protein is known. Experimental data suggest either direct or indirect interaction between the two proteins. In this study, we investigated the expression pattern and biochemical characteristics of Dpl in human tissues and in Chinese hamster ovary cells transfected with wild-type or variant human Dpl gene constructs. Human Dpl appears to be a glycosylphosphatidylinositol-anchored glycoprotein with N- and O-linked sugars. It was found on Sertoli cells in the testis, on the flagella of epididymal and mature spermatozoa, and in seminal plasma. Dpl coexists only with N-terminally truncated isoforms of PrP(c) on mature spermatozoa. The localization of human Dpl on both Sertoli cells (somatic cells) and spermatozoa (germinal cells) strongly suggests that this protein may play a major role in human male fertility. Finally, our data indicate that spermatozoa are thus an interesting model for studies of the potential interaction between Dpl and PrP(c).     

Copper(II) binding to the human Doppel protein may mark its functional diversity from the prion protein

Doppel (Dpl) is the first described homologue of the prion protein, the main constituent of the agent responsible for prion diseases. The cellular prion protein (PrP(C)) is predominantly present in the central nervous system. Although its role is not yet completely clarified, PrP(C) seems to be involved in Cu(2+) recycling from synaptic clefts and in preventing neuronal oxidative damage. Conversely, Dpl is expressed in heart and testis and has been shown to regulate male fertility by intervening in gametogenesis and sperm-egg interactions. Therefore, despite a high sequence homology and a similar three-dimensional fold, the functions of PrP(C) and Dpl appear unrelated. Here we show by electron paramagnetic resonance and fluorescence spectroscopy that the in vitro binding of copper(II) to human recombinant Dpl occurs with a different pattern from that observed for recombinant PrP. At physiological pH values, two copper(II)-binding sites with different affinities were found in Dpl. At lower pH values, two additional copper(II)-binding sites can be identified as follows: one complex is present only at pH 4, and the other is observed in the pH range 5-6. As derived from the electron paramagnetic resonance characteristics, all Dpl-copper(II) complexes have a different coordination sphere from those present in PrP. Furthermore, in contrast to the effect shown previously for PrP(C), addition of Cu(2+) to Dpl-expressing cells does not cause Dpl internalization. These results suggest that binding of the ion to PrP(C) and Dpl may contribute to the different functional roles ascribed to these highly homologous proteins.     

Cloning and characterization of full-length coding sequence (CDS) of the ovine 37/67-kDa laminin receptor (<Emphasis Type="Italic">RPSA</Emphasis>)

The 37-kDa Laminin Receptor Precursor (LRP)/67-kDa Laminin Receptor (LR), also known as ribosomal protein SA (RPSA), had been identified as a putative cell surface receptor for prions. Herein, we isolated the full-length coding sequence (CDS) of the ovine 37/67-kDa LRP/LR gene and submitted it to the GenBank under accession number EF649775. The open reading frame (ORF) of the 37/67-kDa LRP/LR CDS is 885 bp in length, containing six exons encoding a protein of 295 amino acids. The nucleotide sequence presented here is well coincided with the whole ovine genome of the 37/67-kDa LRP/LR previously published. Moreover, we identified four novel single nucleotide polymorphism sites (SNPs) at position 324 in exon 4, positions at 809, 875, and 881 in exon 7, respectively. Further, based on the deduced amino acid sequence alignment of the 37/67-kDa LRP/LR from human, cattle, mice, pig, chicken, and sheep, we also identified three polymorphic amino acid sites (PAAs) at residues 241, 272, and a novel site at residue 270 in the putative indirect prion protein (PrP) interaction region (180–285) on 37/67-kDa LRP/LR. Prediction of protein secondary structure further indicated that PAAs at residues 241, 270 and 272 may cause protein conformation changes as predicted, which may affect on the binding with prion protein. In addition, multiple-tissues RT-PCR results revealed that 37/67-kDa LRP/LR mRNA is expressed in all the 11 selected ovine tissues.     

Serum withdrawal-induced apoptosis in ZrchI prion protein (PrP) gene-deficient neuronal cell line is suppressed by PrP, independent of Doppel

Previous studies have shown that cellular prion protein (PrP(C)) plays anti-apoptotic and antioxidative role against cell death induced by serum-deprivation (SDP) in an immortalized prion protein gene-deficient neuronal cell line derived from Rikn prion protein (PrP) gene-deficient (Prnp(-/-)) mice, which ectopically produce excess Doppel (Dpl) (PrP-like glycoprotein). To investigate whether PrP(C) inhibits apoptotic neuronal cell death without Dpl, an immortalized cell line was established from the brain of ZrchI Prnp(-/-) mice, which do not show ectopic expression of Dpl. The results using a ZrchI neuronal Prnp(-/-) cell line (NpL2) showed that PrP(C) potently inhibited SDP-induced apoptotic cell death. Furthermore, PrP(C) expression enhanced the superoxide dismutase (SOD) activity in NpL2 cells. These results indicate that Dpl production did not affect anti-apoptotic and anti-oxidative functions of PrP, suggesting that PrP(C) may be directly correlated with protection against oxidative stress.     

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.     

Prion proteins and infertility: insight from mouse models

A wealth of evidence points to an abnormal form of the prion protein called PrP(Sc) as the transmissible agent responsible for prion diseases. However, the physiological function of its normal conformer, the cellular prion protein (PrP(C)), is still unknown. Recently, a homologue of PrP(C) was discovered and denoted Doppel (Dpl). In contrast to PrP, mice deficient for Dpl suffer from an important pathological phenotype: male sterility. This phenotype shifts the attention from the brain, where most of the investigations on Dpl have been performed, to testis, raising hope to resolve the long lasting search of PrP(C) function.     

Tissue- and cell type-specific modification of prion protein (PrP)-like protein Doppel, which affects PrP endoproteolysis

A prion protein (PrP)-like protein, Doppel (Dpl) is a homologue of cellular PrP (PrP^C). Immunoblotting revealed heterogeneous glycosylation patterns of Dpl and PrP^C in several cell lines and tissues, including brain and testis. To investigate whether the glycosylation and modification of Dpl and PrP^C could influence each other, PrP gene (Prnp)-deficient neuronal cells, transfected with Prnp and/or the Dpl gene (Prnd), were analyzed by deglycosylation with peptide N-glycosidase F. The modification of Dpl was not influenced by PrP^C, whereas an N-terminally truncated fragment of PrP^C was reduced by Dpl expression. These results indicated that Dpl was glycosylated in a cell type- and tissue-specific manner regardless of PrP^C, while PrP^C endoproteolysis was modulated by Dpl expression.     

Fusion of Doppel to octapeptide repeat and N-terminal half of hydrophobic region of prion protein confers resistance to serum deprivation

Our previous studies have shown an essential role played by the octapeptide repeat region (OR) and the N-terminal half of hydrophobic region (HR) in the anti-apoptotic activity of prion protein (PrP). As PrP-like protein Doppel (Dpl), which structurally resembles an N-terminally truncated PrP, did not show any anti-apoptotic activity, we examined apoptosis of HpL3-4 cells expressing Dpl fused to various lengths of the N-terminal region of PrP to investigate whether the PrP/Dpl fusion proteins retain anti-apoptotic function. HpL3-4 cells expressing Dpl fused to PrP(1-124) with the OR and N-terminal half of HR of PrP showed anti-apoptotic function, whereas Dpl fused to PrP(1-95) with OR did not rescue cells from apoptotic cell death induced by serum deprivation. These results indicate that the OR and N-terminal half of HR of PrP retains anti-apoptotic activity similar to full-length PrP.     

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.