The agent responsible for transmissible spongiform encephalopathies (TSEs) is thought to be a malfolded, protease-resistant version (PrPres) of the normal cellular prion protein (PrP). The interspecies transmission of bovine spongiform encephalopathy (BSE) to mice was studied. Although all of the mice injected with homogenate from BSE-infected cattle brain exhibited neurological symptoms and neuronal death, more than 55 percent had no detectable PrPres. During serial passage, PrPres appeared after the agent became adapted to the new host. Thus, PrPres may be involved in species adaptation, but a further unidentified agent may actually transmit BSE.

Prions are thought to consist of infectious proteins that cause transmissible spongiform encephalopathies. According to overwhelming evidence, the pathogenic prion protein PrPSc converts its host encoded isoform PrPC into insoluble aggregates of PrPSc, concomitant with pathological modifications (for review, see refs. 1-3). Although the physiological role of PrPC is poorly understood, studies with PrP knockout mice demonstrated that PrPC is required for the development of prion diseases. Using the yeast two-hybrid technology in Saccharomyces cerevisiae, we identified the 37-kDa laminin receptor precursor (LRP) as interacting with the cellular prion protein PrPC. Mapping analysis of the LRP-PrP interaction site in S. cerevisiae revealed that PrP and laminin share the same binding domain (amino acids 161 to 180) on LRP. The LRP-PrP interaction was confirmed in vivo in insect (Sf9) and mammalian cells (COS-7). The LRP level was increased in scrapie-infected murine N2a cells and in brain and spleen of scrapie-infected mice. In contrast, the LRP concentration was not significantly altered in these organs from mice infected with the bovine spongiform encephalopathic agent (BSE), which have a lower PrPSc accumulation. LRP levels, however, were dramatically increased in brain and pancreas, slightly increased in the spleen and not altered in the liver of crapie-infected hamsters. These data show that enhanced LRP concentrations are correlated with PrPSc accumulation in organs from mice and hamsters. The laminin receptor precursor, which is highly conserved among mammals and is located on the cell surface, may act as a receptor or co-receptor for the prion protein on mammalian cells.

IntroductionWe recently identi®ed the 37-kDa laminin receptor precursor (LRP) as an interactor for the prion protein (PrP) (Rieger et al., 1997; for reviews see Rieger et al., 1999;Gauczynski et al., 2001a). Employing a series of neuronal and non-neuronal cells, we proved that the 37-kDa LRP/67-kDa high-af®nity laminin receptor (LR) acts as the receptor for the cellular PrP (Gauczynski et al., 2001b). In the present manuscript we used the yeast twohybrid system and cell-binding studies on neuronal as well as non-neuronal cells involving the Semliki Forest virus (SFV) system (for reviews see Liljestrom and Garoff, 1991;Tubulekas et al., 1997) to identify domains on the PrP and the LRP involved in the PrP±LRP interaction on the cell surface. We identi®ed two binding domains for LRP on PrP termed PrPLRPbd1 and PrPLRPbd2. The ®rst one binds directly to LRP, whereas the second one depends on the presence of heparan sulfate proteoglycans (HSPGs) on the cell surface. The yeast two-hybrid system and cell-binding assays on wild-type and mutant HSPGde®cient Chinese hamster ovary (CHO) cells also identi®ed two binding domains for PrP on LRP.The relationship between 37-kDa LRP and 67-kDa LR is not yet fully understood and has been explained with homodimerization of 37-kDa LRP (Landowski et al., 1995) or an additional factor, such as a polypeptide (Castronovo et al., 1991), which might bind to 37-kDa LRP to form the 67-kDa form of the receptor. The 67-kDa heterodimer might be stabilized by hydrophobic interactions mediated by fatty acids such as palmitate, oleate and stearate bound to 37-kDa LRP and to a galectin-3 (gal-3) cross reacting polypeptide (Landowski et al., 1995;Buto et al., 1998). However, we recently proved that the b-galactoside lectin gal-3 is not present on the surface of neuronal or non-neuronal cells used for PrP-binding/ internalization studies (Gauczynski et al., 2001b) and antigal-3 antibodies failed to compete for the 37-kDa LRP/67-kDa LR-mediated binding and internalization of the cellular PrP (Gauczynski et al., 2001b), suggesting that gal-3 is not a partner of the 37-kDa LRP in this context. In this study we investigated by a yeast two-hybrid system analysis whether gal-3 interacts with 37-kDa LRP and/or the cellular PrP. In addition, we investigated whether 37-kDa LRP interacts with itself in the yeast two-hybrid and analysed the monomer/dimer status of the receptor by sizeexclusion chromatography. Both PrP (Gabizon et al., 1993;Caughey et al., 1994;Chen,S.G. et al., 1995;Brimacombe et al., 1999) and the 37-kDa/67-kDa LR (Guo et al., 1992;Kazmin et al., 2000) bind to heparan sulfates. HSPGs are required for the binding of the ®broblast growth factor (FGF) to its FGFR receptor (Yayon et al., 1991;Spivak et al., 1994;Venkataraman et al., 1999) and act as initial attachment receptors for bacteria (Chen,T. et al., 1995) and viruses including alphaviruses (Byrnes and Grif®n, 1998), human immunode®ciency virus (HIV) type 1 (Mondor et al., 1998) and vaccinia virus (Chung et al., 1998). Heparan sulfates are...