insights into the physiological function of cellular prion protein

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Braz J Med Biol Res 34(5) 2001

Cellular prion physiological function

Insights into the physiological functionof cellular prion protein

1Centro de Tratamento e Pesquisa, Hospital do Câncer,2Instituto Ludwig de Pesquisa sobre o Câncer, and3Departamento de Bioquímica, Instituto de Química,Universidade de São Paulo, São Paulo, SP, Brasil

V.R. Martins1,A.F. Mercadante2,3,

A.L.B. Cabral2,3,A.R.O. Freitas2,3 and

R.M.R.P.S. Castro1

Abstract

Prions have been extensively studied since they represent a new classof infectious agents in which a protein, PrPsc (prion scrapie), appearsto be the sole component of the infectious particle. They are respon-sible for transmissible spongiform encephalopathies, which affectboth humans and animals. The mechanism of disease propagation iswell understood and involves the interaction of PrPsc with its cellularisoform (PrPc) and subsequently abnormal structural conversion ofthe latter. PrPc is a glycoprotein anchored on the cell surface by aglycosylphosphatidylinositol moiety and expressed in most cell typesbut mainly in neurons. Prion diseases have been associated with theaccumulation of the abnormally folded protein and its neurotoxiceffects; however, it is not known if PrPc loss of function is animportant component. New efforts are addressing this question andtrying to characterize the physiological function of PrPc. At least fourdifferent mouse strains in which the PrP gene was ablated weregenerated and the results regarding their phenotype are controversial.Localization of PrPc on the cell membrane makes it a potentialcandidate for a ligand uptake, cell adhesion and recognition moleculeor a membrane signaling molecule. Recent data have shown a poten-tial role for PrPc in the metabolism of copper and moreover that thismetal stimulates PrPc endocytosis. Our group has recently demon-strated that PrPc is a high affinity laminin ligand and that this interac-tion mediates neuronal cell adhesion and neurite extension and main-tenance. Moreover, PrPc-caveolin-1 dependent coupling seems totrigger the tyrosine kinase Fyn activation. These data provide the firstevidence for PrPc involvement in signal transduction.

CorrespondenceV.R. Martins

Rua Antônio Prudente, 109, 4º andar

01509-010 São Paulo, SP

Brasil

Presented at

SIMEC 2000 - International

Symposium on Extracellular

Matrix, Angra dos Reis, RJ,

Brazil, September 24-27, 2000.

Publication supported by FAPESP.

Received November 9, 2000

Accepted February 13, 2001

Key words· PrPc· Cellular function· Transmissible spongiform

encephalopathies· Laminin· Signal transduction· Copper

Introduction

The cellular prion protein was first iden-tified in experiments conducted in an at-tempt to find the exogenous nucleic acidcomponent of the infectious agent respon-sible for neurodegenerative diseases called

transmissible spongiform encephalopathies(TSE). This agent was partially purified fromthe brain of affected animals. An insolubleprotein of 33-35 kDa designated PrPsc (prionscrapie) which generates a 27-30-kDa formafter protease treatment was identified as themajor component of the agent. Amino acid

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sequencing of the amino-terminal region fromthe purified protein allowed the synthesis ofan isocoding mixture of nucleotides that wassubsequently used to identify prion cDNAclones. The amino-terminal peptide was alsoused to produce polyclonal antibodies. Thesereagents allowed the identification of achromosomal gene and a host cellular pro-tein, PrPc, expressed in a variety of neuronaland nonneuronal tissues independently ofthe infection by scrapie or any other TSEagent (1).

The participation of PrPc is absolutelynecessary for infection since animals in whichthe PrP gene has been ablated are totallyresistant to the infection (2). PrPc and PrPschave the same amino acid composition, al-though the a-helix content of PrPc is about40%, with less than 10% ß-sheet conforma-tion. In contrast, PrPsc shows about 50% ofits structure as a ß-sheet. Therefore, it wasproposed that gain of infectivity is a conse-quence of conformational modification ofPrPc by PrPsc (1).

PrPc protein is highly conserved amongspecies; the similarity is about 85 to 97%among mammals (3) and the comparisonbetween primates and humans showed aminoacid identity ranging from 92.9 to 99.6% (4).PrPc has also been described in chickens (3)and turtles (5). The entire open reading frameof all known PrP genes is located in a singleexon which codes for a protein of approxi-mately 250 amino acids (3). A signal se-quence of 22 amino acids is present at theamino-terminal (6) and a 23-amino acid sig-nal sequence encoding for attachment to aglycosylphosphatidylinositol (GPI) anchorsat the carboxy-terminal sequence (7).

The cellular prion protein has some char-acteristics that make it a very interestingmolecule and its conservation among spe-cies strongly suggests its relevance in physi-ological processes. Moreover, the role ofPrPc in TSE is viewed as gain of functiondue to accumulation of a new PrPc isoform,PrPsc. However, it is still possible that prion

diseases could be mediated, at least in part,by loss of function (8,9).

During the last few years several researchgroups have been working with differentmodels aiming to understand the physiologi-cal function of PrPc. Since this protein is acell surface molecule its role could be re-lated to ligand uptake, cell adhesion andrecognition or cell signaling (10). Herein weintend to present an overview of the possiblefunctions of cellular prion protein.

Generation of PrP-deleted animals

The generation of animals in which thegene that codes for a protein of interest isdeleted, is a very interesting approach tostudy the unknown function of this gene incellular or animal physiology.

Charles Weismann�s group generated thefirst PrPc gene (Prnp)-deleted mouse (PrP-/-

Zrch) in 1992 by homologous recombina-tion replacing the Prnp open reading framewith the neomycin phosphotransferase geneunder control of the herpes simplex virusthymidine kinase promoter (11). They per-formed learning, immunologic and anatomi-cal tests in order to determine if PrPc isessential in some of these processes. Thestructure of PrP-/- mouse brain was normaland no detectable effect on the level of lym-phocyte surface MHC class I and II antigenmarkers was observed. Behavior analysiswith these mice included three tests: swim-ming navigation to find a submerged plat-form, Y-maze discrimination and two-wayavoidance with shock sensitivity. Normaland PrP-/- mice showed a poor but overallsignificant learning performance, which istypical for many mouse strains, but no sig-nificant differences between them were ob-served. These results were somehow disap-pointing since due to the high conservationof PrP among species and its brain expres-sion, the research community was expectingimportant physiological deficiencies in thoseanimals. However, at least two possibilities

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can be proposed: the resulting defect is sosubtle that a selective disadvantage mayemerge only after many generations, or theprotein function is redundant (11).

One year later, the same group observedthat these mice devoid of PrP are completelyprotected against scrapie disease, at least upto 13 months after inoculation. Moreover,even heterozygous Prnp+/- mice are partiallyprotected, inasmuch as 9 of 10 scrapie-in-oculated animals showed signs of scrapieonly 253-322 days after inoculation but arestill alive after 322 days, while all Prnp+/+

controls died within about 180 days (2).These observations definitely prove that thedevelopment of scrapie symptoms and pa-thology is strictly dependent on the presenceof PrPc and also that the expression level ofPrPc is inversely related to incubation timeand disease progression.

A second Prnp-/- line was created (Npu)two years later by a different targeting strat-egy but again it was impossible to obtaininformation about the normal function ofPrPc since the line showed normal develop-ment (12).

Later, the application of specific behav-ioral tests characterized some abnormalitiesimputable to the lack of PrPc in Zrch animals(the first generated PrPc-/- mice). Tobler andco-workers (13) described an alteration incircadian rhythms which correlated the cir-cadian regulation function of PrPc with fatalfamilial insomnia (a hereditary disease asso-ciated with specific mutations of the Prnpgene). A weakened GABA-A receptor-me-diated fast inhibition and impaired long-termpotentiation (14) were also described andcould be involved in the epileptiform activ-ity seen in Creutzfeldt-Jakob disease. Pru-siner�s group (15) did not reproduce theseresults; however, we observed that thesePrnp-/- mice have an increased sensitivity toseizures in four different epileptogenicalmodels (16).

In an attempt to analyze different behav-ioral tasks of mice devoid of PrPc our group

reported an increased locomotor activity inPrnp-/- Zrch mice, but normal inhibitoryavoidance learning and anxiety (17).

In 1996 Sakaguchi and co-workers (18)developed a third line of mice homozygousfor a disrupted Prnp gene (Ngsk) and ob-served that these animals grew normally af-ter birth, but at about 70 weeks of age allbegan to show progressive symptoms ofataxia. The brain of mice with neurologicalsymptoms presented considerable atrophyof the cerebellum due to an extensive loss ofPurkinje cells.

One year later another Prnp-/- mouse linewas generated (Rcm0) and also developed alate onset fatal ataxia (19). Thus, it wassuggested that PrPc has a role in the long-term survival of Purkinje neurons. However,it was puzzling why Ngsk and Rcm0 micedeveloped a fatal ataxia while two otherlines of Prnp-/- mice did not exhibit anyextensive CNS dysfunction.

In order to determine the role of PrPc inthis phenotype, Moore�s group (19) ana-lyzed PrPc-related genes. Since most of theserelated genes are localized in clusters theysequenced large cosmid clones containingthe Prnp gene and found a novel PrP-likegene named Doppel (German for double andalso meaning downstream prion-like pro-tein). The coding region for Doppel (Prnd) islocated 16 kb downstream from the Prnpgene and two major transcripts of 1.7 and 2.7kb as well as an unusual chimeric transcript,generated by intergenic splicing with Prnp,are produced. Interestingly, the chimeric tran-script is up-regulated in the Ngsk and Rcm0strains of PrP-/- mice that developed ataxiabut not in Zrch or Npu strains with a normalphenotype. The authors suggest that Doppeloverexpression may provoke neurodegen-eration (19).

The construction of a mouse with a priontransgene rescues the ataxia and Purkinjecell degeneration phenotype in Ngsk Prnp-/-

mice (20), suggesting that PrPc and Doppelproteins might compete for a common re-

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ceptor protein (19). A few years ago ourgroup described a receptor for prion protein(21) and we speculated that PrPc binding tothis receptor should participate in PrPc inter-nalization and signaling. The evaluation ofPrPc, Doppel, a common ligand and theirinteraction with other proteins might allowthe characterization of PrPc physiologicalfunction. This subject will be discussed laterin this article.

PrPc as a copper uptake protein

There is increasing evidence supportinga functional role for PrPc in copper metabo-lism. Several studies have indicated that PrPccan bind copper. First, it was possible toisolate PrPc from hamster brain on a copperaffinity column (22). Moreover, purified re-combinant PrPc, as well as synthetic PrPc-derived peptides, bind copper ions with mi-cromolar affinity through four histidine-con-taining peptide repeats in the amino-termi-nal half of the protein (23-25). The repeatedregion contains five or six octapeptide tan-dem motifs of the general form P(H/G)GGGWGQ and is highly conserved amongmammalian PrPc, while chicken PrPc has asimilar region of eight hexapeptide tandemrepeats (3).

Copper may contribute to PrPc confor-mation since the highly flexible amino-ter-minus of recombinant PrPc is more struc-tured in the presence of copper (26). More-over, spectroscopic data have suggested abridged arrangement of coordinating histi-dine imidazole nitrogens binding four Cu2+

per PrPc molecule. This proposed coordina-tion accounts for the cooperative binding ofcopper by PrPc (25).

Copper is an essential metal, which playsa fundamental role in the biochemistry of allaerobic organisms and is also required forthe catalytic activity of several enzymes ofinterest to neurobiology (27). Free or incor-rectly bound Cu2+ can catalyze the genera-tion of damaging radicals such as hydroxyl

radicals (28). Specific mechanisms haveevolved in an appropriate compartmentali-zation and trafficking of this metal, avoidingoxidative stress (27).

Several groups have investigated thephysiological meaning of the associationbetween PrPc and copper. Brown and co-workers (24) reported that the copper con-tent of membrane-enriched brain extractsfrom PrP-/- mice is 10-15-fold lower than inwild-type controls while no significant dif-ference was observed for other metals. Theseresults suggested that PrPc is a major cop-per-binding protein in brain membrane frac-tions and controls the activity of other mem-brane-associated copper-binding proteins.The same group, using cerebellar cell cul-tures from mice expressing different levelsof PrPc, demonstrated that cells with highlevels of PrPc have an increasing resistanceto oxidative stress compared to PrP-/- cells(29,30).

The ability of PrPc to bind copper maymodulate the activity of the major cellularantioxidant enzyme Cu/Zn superoxide dis-mutase (SOD-1) and consequently cellularresistance to oxidative stress. Western andNorthern-blot analysis indicated that miceeither lacking or overexpressing PrPc hadlevels of Cu/Zn SOD protein and mRNAequivalent to those expressed in wild-typemice. However, increasing levels of PrPcexpression were linked to increased levels ofCu/Zn SOD activity (29,30). SOD-1 activityfrom cultured cerebellar neurons was ap-proximately 50% the normal level in PrPcnull mice and was elevated by 20% in trans-genic mice overexpressing PrPc. In addition,experiments using cells metabolically labeledwith radioactive copper have shown that Cu/Zn SOD immunoprecipitated from cells over-expressing PrPc has higher levels of radioac-tivity when compared to PrPc-deficient cells(30). These observations suggest that PrPcmay play some role in the delivery of copperto cuproenzymes such as SOD-1.

Morphological and biochemical investi-

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gations of different PrPc-transgenic mouselines provide strong evidence for a predomi-nantly synaptic location of PrPc (31). A re-duction in copper concentration in synapto-somal preparations of PrP-/- mice has beenobserved (24,31), indicating that PrPc is in-volved in synaptic copper homeostasis. Infact, electrophysiological studies have sug-gested that PrPc may regulate synaptic trans-mission by modulating copper content in thesynaptic cleft (24,31).

More recently, a study using mass spec-trometry methodology (32) failed to find anydifference in the amount of ionic copper insubcellular fractions from brains of micewith different PrPc expression levels. Theyalso showed that the enzymatic activity ofSOD-1 and cytochrome c oxidase in brainextracts are similar for these groups, as alsois the incorporation of copper into Cu/ZnSOD. The results of Waggoner et al. (32)differ from others (29,30) and suggest thatPrPc is not the primary carrier responsiblefor copper entry into the brain and does notplay a role in the specialized trafficking path-ways involved in delivery of copper to SOD-1 in this tissue.

The exact mechanism by which copperand PrPc are functionally related is not knownat present. PrPc, on the cell surface, mayfunction as a sink for chelation of extracellu-lar copper ions or as a carrier protein foruptake and delivery of these cations to intra-cellular targets. Pauly and Harris (10) havereported that copper stimulates endocytosisof PrPc from the cell surface via clathrin-coated pits. An attractive hypothesis to ex-plain this observation is that PrPc may bindcopper ions in the extracellular domain, de-liver them to endocytic compartments andtransfer these cations to other copper-carriercytosolic proteins. The binding of coppercould stimulate internalization of PrPc, al-tering its conformation and increasing itsaffinity for a putative endocytic transmem-brane receptor (10).

Another possibility is that bound copper

serves as an essential cofactor for an un-known enzymatic activity of PrPc. Indeed, ithas recently been shown that recombinantchicken and mouse PrPc, as well as PrPcimmunoprecipitated from mouse brain tis-sue, have SOD activity (33). These resultssuggest that PrPc has an enzymatic functiondependent on copper incorporation and indi-cate that it could have a direct role in cellularresistance to oxidative stress.

Disturbances in copper homeostasis lead-ing to CNS dysfunction are well documentedin humans and animals. Some neurodegen-erative diseases such as Menkes� syndrome,Wilson�s disease, amyotrophic lateral scle-rosis and Alzheimer�s disease are linked toaltered copper transport and homeostasis(27). The evidence that PrPc has a role incopper metabolism may be important in un-derstanding the pathogenesis of prion dis-eases, since loss of this copper-related func-tion (as a result of conversion to PrPsc)could help to explain some features of thesedisorders. Interestingly, early studies haverevealed that cuprizone, a copper-chelatingagent, induces neuropathological changes inmice similar to those found in prion diseases(27), suggesting a role for copper in thesedisorders.

PrPc-binding proteins

The identification of PrPc-binding pro-teins can provide insights into the functionof PrPc and the molecular mechanisms in-volved in prion diseases. There are a numberof structural features within PrPc that mightallow it to interact with other proteins. Twopotential sites for binding are an amphi-pathic helix near the middle of the moleculethat in other proteins has been implicated inprotein-protein interaction and the GPI an-chor which may internalize and deliver sig-nals (34). Many studies have been conductedin recent years to probe the interaction of PrPwith other molecules.

PrPc binds to a family of heparin-like

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compounds; this interaction can influencethe intracellular fate of the prion protein andinhibits the conversion of PrPc to PrPsc (35).Heparin is a sulfated polyanion closely re-lated to cellular glycosaminoglycans, whichin turn are associated with PrPsc in amyloidplaques. Therefore, it was proposed that thesemolecules act by directly competing with thebinding of PrPc or PrPsc to cellular gly-cosaminoglycans. A recent publication char-acterized the sulfated polyanion-bindingproperties of recombinant PrPc protein us-ing surface plasmon resonance and showedthat the PrPc affinity for polyanions is paral-lel to their anti-scrapie formation potency(36).

PrPc also associates with PrPsc and thisinteraction is more efficient when the twoisoforms have the same sequence, explain-ing the �species barrier� for prion transmis-sion. Telling and co-workers (37) suggestedthat PrPc binds a species-specific macro-molecule designated protein X which mightfunction as a molecular chaperone in theformation of PrPsc.

Using a yeast two-hybrid system, Eden-hofer and co-workers (38) identified the heatshock protein 60, a cellular chaperone, as aspecific ligand for PrPc. The interaction sitewas mapped between amino acids 180 and210 of the PrPc protein. In a recent report(39) BiP, another chaperone, was describedto be associated with PrPc. This protein re-mains associated with PrPc mutants for anextended period of time. BiP normally inter-acts with misfolded or unassembled pro-teins, mediating their retrograde transloca-tion for proteosomal degradation. The au-thors presumed that the impairment of theendosomal-lysosomal degradation leads tothe accumulation of PrPsc.

Oesch and co-workers (34) also foundthat PrPc binds to glial fibrillary acidic pro-tein (GFAP); however, studies with null micefor the GFAP gene revealed that this proteinis not essential for TSE development (40).

In a study using a soluble tagged PrPc

probe to screen an expression mouse braincDNA library, six potential PrPc-binding pro-teins were identified (41). Four of them arecoded by novel cDNAs, one is Nrf2 (NF-E2related factor 2) and the last is apolipopro-tein 1 (Aplp1), which plays a role in thepathogenesis of Alzheimer�s disease. Theauthors suggested that Aplp1 and PrP maypossibly interact on the surface of neuronalcells or in the vicinity of the plasma mem-brane, but the role of this interaction in thedevelopment of prion or Alzheimer�s dis-eases remains to be clarified.

Bcl-2, an anti-apoptotic protein, was alsoreported to be associated with PrPc (42),with the binding site being located in thecarboxy-terminal region of Bcl-2 which in-cludes the transmembrane region. The au-thors suggested that Bcl-2 may act as a chap-erone and induce conformational modifica-tions in PrPc.

Another protein characterized as a PrPcligand is the 37-kDa laminin receptor pre-cursor. This protein interacts with PrPc invitro and in vivo and is overexpressed inorgans that accumulate PrPsc (43), suggest-ing that it could be a receptor or co-receptorfor the prion protein in mammalian cells.

Our group has described the interactionof PrPc with a 66-kDa membrane proteinboth in vivo and in vitro, and antiserumagainst this ligand inhibits the toxicity of aprion-derived peptide towards neuronal cellsin culture (21). The protein has been isolatedon two-dimensional gels and its sequencingis underway. We will discuss the possiblerole of this protein in PrPc internalizationand signaling in the last section of this ar-ticle.

It is remarkable that, as previously de-scribed, PrPc binds to a large number ofproteins; however, the physiological rel-evance of these interactions remains to beestablished.

We have recently characterized a specif-ic high affinity binding between PrPc andlaminin, an extracellular matrix protein.

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Moreover, we provide for the first time con-sistent data regarding the physiological roleof a PrPc association with another protein(44,45). These data will be discussed below.

PrPc in neuronal survival anddifferentiation

One of the proteins that associate withPrPc is laminin (44), a fact consistent with apossible PrPc function as a cell adhesion andrecognition molecule. Laminin is an 800-kDa heterotrimeric glycoprotein consistingof two short and one long polypeptide chains,predominantly found in basement mem-branes and known to play a pivotal role incell proliferation, differentiation, migrationand death (46). The cellular responses trig-gered by laminin are mediated by its interac-tion with cell membrane receptors. The bestknown receptors are integrins but non-inte-grin laminin receptors have also been de-scribed (47).

In the CNS, laminin has likewise beenshown to mediate neuronal differentiationthrough its interaction with integrins. Thisinteraction is characterized by neurite for-mation and extension, migration of neuronsboth in vitro and in vivo (48), neuronal aswell as axonal regeneration (48), and pre-vention of neuronal death after kainic acidinjections (49). In the last model, tissue-typeplasminogen activator was found to act byconverting plasminogen into plasmin, whichsubsequently degrades laminin. Even thoughthe cell receptor involved was not identified,it was clear that detachment of neurons froma laminin substrate was the determinant eventfor neuronal death, characteristic of seizuremodels.

Our group has observed that PrPc-/- miceare more sensitive to seizures caused bythree convulsant agents including kainic acid,suggesting that the absence of PrPc rendersanimals more susceptible to neuronal deathdue to laminin degradation (16). Moreover,a recent study (50) using cells from PrPc-/-

mice has shown that PrPc prevents serumdeprivation-dependent apoptosis of neuronsin culture and has suggested that PrPc mightbe similarly involved in neurite extension bythese cells.

We have established the PrPc-lamininconnection showing that PrPc is a specific,high affinity, saturable receptor for lamininand the binding site resides at the carboxy-terminal decapeptide (RNIAEIIKDI) of thelaminin g-1 chain (44). Indeed, neurite ex-tension observed in primary cultures of hip-pocampal neurons in the presence of intactlaminin-1 was quite sensitive to anti-PrPcantibodies, whereas that elicited by the car-boxy-terminal peptide was completely in-hibited by such antibodies. Furthermore, theneuritogenesis elicited by intact laminin wassubstantially decreased and was not inhib-ited by anti-PrPc antibodies when cells werederived from PrPc-/- mice, whereas no neuri-togenesis could be elicited from such neu-rons by the carboxy-terminal peptide alone.

Very recently, we described the impor-tance of PrPc-laminin interaction for neu-ronal cell adhesion. Indeed, using chro-mophore-assisted laser microscopy we con-firmed the importance of this interaction forneurite extension and also showed its in-volvement in neurite maintenance (45).

The mapping of the decapeptideRNIAEIIKDI as the PrPc-binding site in thelaminin molecule is particularly importantsince the g-1 chain is the most conserved inall laminin types (48). Therefore, these datasupport the notion that PrPc-laminin interac-tion could be important in a variety of tissuesin which both PrPc and different lamininisoforms are expressed.

In fact, PrPc is present on the surface oflymphocytes and its expression is increasedwhen cells are activated by concanavalin A.Blockage of PrPc with specific antibodiessuppresses mitogen-induced activation (51),suggesting that PrPc could participate in lym-phocyte activation. On the other hand, lami-nin inhibits the proliferation of lymphocytes

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stimulated by concanavalin A (52). Whetherthe PrPc-laminin interaction participates inthis event remains to be evaluated.

PrPc and signal transduction

Due to its cell membrane localization,PrPc could participate in cell signaling path-ways and some progress has been made inthe identification and characterization of thesignaling involving PrPsc molecules.

It was described (53) that bradykinin-stimulated calcium responses in scrapie-in-fected cells was reduced by 30 to 50% com-pared with uninfected cells. The authors sug-gested that prion infection compromises cal-cium channel function.

After Forloni and co-workers (54) re-ported that a 21-amino acid fragment of theprion protein (PrP 106-126) could be toxicwhen chronically exposed to primary rathippocampal cultures, some results aboutsignal transduction were generated. This pep-tide forms ion-permeable channels in planarlipid bilayer membranes and these channelsare freely permeable to the most commonphysiological ions like Ca2+ and Na+ (55).Moreover, it was also observed that the neu-rotoxic peptide increases intracellular freecalcium concentration in cultured microgliafrom wild-type and PrP-/- mice (56). Later,this peptide was found to be involved in theactivation of tyrosine kinases Lyn and Syk,initiating a signaling cascade that results in atransient release of intracellular calcium andactivation of classical protein kinase C andthe calcium-sensitive tyrosine kinase PYK2.Activation of MAP kinases ERK-1 and ERK-2 follows as a subsequent downstream sig-naling event. An important point of the workof Combs et al. (57) is the demonstration thatthe signaling response elicited by neurotoxicpeptide induces the production of neuro-toxic products.

Nevertheless, important questions are stillunanswered. How is the observed signal trig-gered? What is the nature of the signaling

triggered by PrPc? What kind of moleculesare involved in this event?

In an attempt to answer these questionsour group has been working on the identifi-cation and characterization of a putative re-ceptor for PrPc. We used the complementaryhydropathy theory to predict a hypotheticalpeptide complementary to the human prionregion from amino acid 114 to 126 (21)previously described to be neurotoxic in pri-mary neuronal cultures (54) and responsiblefor PrPc internalization (58). Antiserumraised against the prion complementary pep-tide 114-126 recognized a 66-kDa mem-brane protein that binds PrPc both in vitroand in vivo. Furthermore, the complemen-tary peptide as well as antiserum against itinhibited the toxicity of a prion-derived pep-tide towards neuronal cells in culture.

Shmerling and co-workers (59) reportedin 1998 that mice with PrP lacking residues32-121 or 32-134 but not those deleted from32 to 106 presented severe ataxia and neu-ronal death limited to the granular layer ofthe cerebellum as early as 1-3 months afterbirth. Interestingly, the deleted PrPc regioninvolved in the disease maps on the pre-dicted binding site for the putative receptordescribed by us (21), suggesting that PrPc-receptor interaction should be important forthe normal function of PrPc. It is tempting tospeculate that the PrPc region from aminoacids 106-126 contains the binding site forthe receptor association and signal transduc-tion. Since PrPc-/- animals do not developthe disease a PrP-like molecule might bind tothe receptor and transduce similar signals.Moreover, the truncated PrPc proteins donot act as dominant negative molecules forthe receptor since the defect was completelyabolished by introducing one copy of a wild-type PrP gene (59).

Very recent data have shown that PrPctriggers cell signaling increasing the phos-phorylation levels of the tyrosine kinase Fyn,and caveolin-1 was characterized as the in-termediate factor between PrPc on the outer

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membrane and the intracellular protein Fyn.However, the nature of the PrPc ligand re-sponsible for the signal generation was notidentified (60).

The two PrPc ligands characterized byus, the 66-kDa receptor (21) and laminin,seem to transduce signals through cAMP(Freitas AF, Martins VR and Brentani RR,unpublished results) and calcium (Lee KS,Prado MA, Brentani RR and Martins VR,

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insights into the physiological function of cellular prion protein

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