Prions are composed of an isoform of a normal sialoglycoprotein called PrPc, whose physiological role has been under investigation, with focus on the screening for ligands. Our group described a membrane 66 kDa PrPc‐binding protein with the aid of antibodies against a peptide deduced by complementary hydropathy. Using these antibodies in western blots from two‐dimensional protein gels followed by sequencing the specific spot, we have now identified the molecule as stress‐inducible protein 1 (STI1). We show that this protein is also found at the cell membrane besides the cytoplasm. Both proteins interact in a specific and high affinity manner with a K d of 10−7 M. The interaction sites were mapped to amino acids 113–128 from PrPc and 230–245 from STI1. Cell surface binding and pull‐down experiments showed that recombinant PrPc binds to cellular STI1, and co‐immunoprecipitation assays strongly suggest that both proteins are associated in vivo . Moreover, PrPc interaction with either STI1 or with the peptide we found that represents the binding domain in STI1 induce neuropro tective signals that rescue cells from apoptosis.

Understanding the physiological function of the cellular prion (PrP^c) depends on the investigation of PrP^c-interacting proteins. Stress-inducible protein 1 (STI1) is a specific PrP^cligand that promotes neuroprotection of retinal neurons through cAMP-dependent protein kinase A (PKA). Here, we examined the signaling pathways and functional consequences of the PrP^cinteraction with STI1 in hippocampal neurons. Both PrP^cand STI1 are abundantly expressed and highly colocalized in the hippocampusin situ, indicating that they can interactin vivo. Recombinant STI1 (His₆-STI1) added to hippocampal cultures interacts with PrP^cat the neuronal surface and elicits neuritogenesis in wild-type neurons but not in PrP^c-null cells. This effect was abolished by antibodies against either PrP^cor STI1 and was dependent on the STI1 domain that binds PrP^c. Binding of these proteins induced the phosphorylation/activation of the mitogen-activated protein kinase, which was essential for STI1-promoted neuritogenesis. His₆-STI1, but not its counterpart lacking the PrP^cbinding site, prevented cell death via PKA activation. These results demonstrate that two parallel effects of the PrP^c–STI1 interaction, neuritogenesis and neuroprotection, are mediated by distinct signaling pathways.

The prion protein (PrP ^C ) is highly expressed in the nervous system, and its abnormal con‐former is associated with prion diseases. PrP ^C is anchored to cell membranes by glycosylphosphatidylinositol, and transmembrane proteins are likely required for PrP ^C ‐mediated intracellular signaling. Binding of laminin (Ln) to PrP ^C modulates neuronal plasticity and memory. We addressed signaling pathways triggered by PrP ^C ‐Ln interaction in order to identify transmembrane proteins involved in the transduction of PrP ^C ‐Ln signals. The Ln γl‐chain peptide, which contains the Ln binding site for PrP ^C , induced neuritogenesis through activation of phos‐pholipase C (PLC), Ca ²⁺ mobilization from intracellular stores, and protein kinase C and extracellular signalregulated kinase (ERK1/2) activation in primary cultures of neurons from wild‐type, but not PrP ^C ‐null mice. Phage display, coimmunoprecipitation, and colocalization experiments showed that group I metabotropic glutamate receptors (mGluRl/5) associate with PrP ^C . Expression of either mGluRl or mGluR5 in HEK293 cells reconstituted the signaling pathways mediated by PrP ^C ‐Ln γl peptide interaction. Specific inhibitors of these receptors impaired PrP ^C ‐Ln γl peptide‐induced signaling and neuri‐togenesis. These data show that group I mGluRs are involved in the transduction of cellular signals triggered by PrP ^C ‐Ln, and they support the notion that PrP ^C participates in the assembly of multiprotein complexes with physiological functions on neurons.—Beraldo, F. H., Arantes, C. P., Santos, T. G., Machado, C. F. _, Roffe, M., Hajj, G. N., Lee, K. S., Magalhães, A. C., Caetano, F. A., Mancini, G. L., Lopes, M. H., Amãrico, T. A., Magdesian, M. H., Ferguson, S. S. G., Linden, R., Prado, M. A. M., Martins, V. R. Metabotropic glutamate receptors transduce signals for neurite outgrowth after binding of the prion protein to laminin γl chain. FASEB J. 25, 265–279 (20ll). www.fasebj.org

Prion protein (PrP^C), when associated with the secreted form of the stress-inducible protein 1 (STI1), plays an important role in neural survival, neuritogenesis, and memory formation. However, the role of the PrP^C-STI1 complex in the physiology of neural progenitor/stem cells is unknown. In this article, we observed that neurospheres cultured from fetal forebrain of wild-type (Prnp^+/+) and PrP^C-null (Prnp^0/0) mice were maintained for several passages without the loss of self-renewal or multipotentiality, as assessed by their continued capacity to generate neurons, astrocytes, and oligodendrocytes. The homogeneous expression and colocalization of STI1 and PrP^C suggest that they may associate and function as a complex in neurosphere-derived stem cells. The formation of neurospheres from Prnp^0/0 mice was reduced significantly when compared with their wild-type counterparts. In addition, blockade of secreted STI1, and its cell surface ligand, PrP^C, with specific antibodies, impaired Prnp^+/+ neurosphere formation without further impairing the formation of Prnp^0/0 neurospheres. Alternatively, neurosphere formation was enhanced by recombinant STI1 application in cells expressing PrP^C but not in cells from Prnp^0/0 mice. The STI1-PrP^C interaction was able to stimulate cell proliferation in the neurosphere-forming assay, while no effect on cell survival or the expression of neural markers was observed. These data suggest that the STI1-PrP^C complex may play a critical role in neural progenitor/stem cells self-renewal via the modulation of cell proliferation, leading to the control of the stemness capacity of these cells during nervous system development. STEM CELLS 2011;29:1126–1136