RESUMO
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.
Assuntos
Proteoglicanas de Heparan Sulfato/metabolismo , Príons/metabolismo , Precursores de Proteínas/metabolismo , Receptores de Laminina/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Animais , Sítios de Ligação/fisiologia , Células CHO , Linhagem Celular , Cromatografia em Gel , Cricetinae , Galactosídeos/metabolismo , Glutationa Transferase/genética , Proteoglicanas de Heparan Sulfato/genética , Humanos , Camundongos , Oligopeptídeos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Peptídeos/genética , Príons/genética , Ligação Proteica/fisiologia , Precursores de Proteínas/genética , Estrutura Terciária de Proteína/fisiologia , Receptores de Laminina/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Vírus da Floresta de Semliki/genéticaRESUMO
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.