contributed equally to this work Prions are composed of an isoform of a normal sialoglycoprotein called PrP c , whose physiological role has been under investigation, with focus on the screening for ligands. Our group described a membrane 66 kDa PrP c -binding protein with the aid of antibodies against a peptide deduced by complementary hydropathy. Using these antibodies in western blots from twodimensional protein gels followed by sequencing the speci®c spot, we have now identi®ed 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 speci®c and high af®nity manner with a K d of 10 ±7 M. The interaction sites were mapped to amino acids 113±128 from PrP c and 230±245 from STI1. Cell surface binding and pull-down experiments showed that recombinant PrP c binds to cellular STI1, and co-immunoprecipitation assays strongly suggest that both proteins are associated in vivo. Moreover, PrP c interaction with either STI1 or with the peptide we found that represents the binding domain in STI1 induce neuroprotective signals that rescue cells from apoptosis.

No abstract

Prions, the etiological agents for infectious degenerative encephalopathies, act by entering the cell and inducing conformational changes in PrPC (a normal cell membrane sialoglycoprotein), which result in cell death. A specific cell-surface receptor to mediate PrPC and prion endocytosis has been predicted. Complementary hydropathy let us generate a hypothetical peptide mimicking the receptor binding site. Antibodies raised against this peptide stain the surface of mouse neurons and recognize a 66-kDa membrane protein that binds PrPC both in vitro and in vivo. Furthermore, both the complementary prion peptide and antiserum against it inhibit the toxicity of a prion-derived peptide toward neuronal cells in culture. Such reagents might therefore have therapeutic applications.

Odorants are detected by a large family of odorant receptors (ORs) expressed in the nose. The information provided by the ORs is transmitted to specific regions of the brain, leading to odorant perception. The determination of the odorant specificities of the different ORs will contribute to the understanding of how odorants are discriminated by the olfactory system. However, to date only a few ORs have been linked to odorants they recognize, because ORs are poorly expressed on the cell surface of heterologous cells. Here we show that Ric-8B, a putative guanine nucleotide exchange factor for G␣olf, promotes efficient heterologous expression of ORs. Our results also show that Ric-8B enhances accumulation of G␣olf at the cell periphery, indicating that it promotes functional OR expression by improving the efficiency of OR coupling to G␣olf. Expression systems containing G␣olf and Ric-8B should contribute to the functional characterization of ORs.guanine nucleotide exchange factor ͉ olfactory receptor ͉ synembryn ͉ G protein ␣ olf ͉ heterologous expression M ammals can discriminate a vast number of odorants with remarkable sensitivity and accuracy. Odorants are first detected by a large family of odorant receptors (ORs) expressed in the cilia of the olfactory sensory neurons (OSNs) of the nose (1). The information provided by the different ORs is then transmitted to the olfactory bulb of the brain, which in turn relays information to the olfactory cortex. A series of experiments indicated that the information provided by the different ORs in the nose is organized into a sensory map in the olfactory bulb (2-4). In the olfactory cortex, which receives input from the bulb, there is another map of OR inputs, different from the map in the bulb (5).The discrimination of odorants presumably derives from the different ligand specificities of the ORs. Therefore, the determination of the odorant specificities of individual ORs should provide information regarding how odor identities are encoded. However, so far only Ϸ30 ORs have been linked to odorants they recognized (6-9). The major reason for this fact is that the functional expression of ORs in heterologous cell types has proven difficult, mostly because ORs cannot reach the plasma membrane (10-12). To circumvent this problem, we previously used a combination of Ca 2ϩ imaging and single-cell RT-PCR to identify the ORs expressed by olfactory neurons that responded to different aliphatic odorants (6). By using this approach we identified 13 different ORs that responded to various aliphatic alcohols and acids. Our results revealed that the olfactory system uses combinatorial receptor codes to encode odorant identities.The complete repertoires of mouse and human OR genes have recently been determined from the corresponding genome sequences (13-19). All together, 1,200 intact OR genes were identified in the mouse, and Ϸ388 intact genes were identified in humans (20). Gene expression studies confirmed that a large fraction of the mouse OR genes is expressed in the olfactory ep...

Prions, the etiological agents for infectious degenerative encephalopathies, act by inducing structural modifications in the cellular prion protein (PrPc). Recently, we demonstrated that PrPc binds laminin (LN) and that this interaction is important for the neuritogenesis of cultured hippocampal neurons. Here we have used the PC-12 cell model to explore the biological role of LN^PrPc interaction. Antibodies against PrPc inhibit cell adhesion to LN-coated culture plaques. Furthermore, chromophore-assisted laser inactivation of cell surface PrPc perturbs LN-induced differentiation and promotes retraction of mature neurites. These results point out to the importance of PrPc as a cell surface ligand for LN. ß

The olfactory system is able to detect a large number of chemical structures with a remarkable sensitivity and specificity. Odorants are first detected by odorant receptors present in the cilia of olfactory neurons. The activated receptors couple to an olfactory-specific G-protein (Golf), which activates adenylyl cyclase III to produce cAMP. Increased cAMP levels activate cyclic nucleotide-gated channels, causing cell membrane depolarization. Here we used yeast two-hybrid to search for potential regulators for G␣olf. We found that Ric-8B (for resistant to inhibitors of cholinesterase), a putative GTP exchange factor, is able to interact with G␣olf. Like G␣olf, Ric-8B is predominantly expressed in the mature olfactory sensory neurons and also in a few regions in the brain. The highly restricted and colocalized expression patterns of Ric-8B and G␣olf strongly indicate that Ric-8B is a functional partner for G␣olf. Finally, we show that Ric-8B is able to potentiate G␣olf-dependent cAMP accumulation in human embryonic kidney 293 cells and therefore may be an important component for odorant signal transduction.

. (1992). Acetylcholine synthesis and release is enhanced by dibutyryl cyclic AMP in a neuronal cell line derived from mouse septum. J. Neurosci. 12, 793-799. Brentani, R. R. (1988

Prions have been extensively studied since they represent a new class of infectious agents in which a protein, PrPsc (prion scrapie), appears to be the sole component of the infectious particle. They are responsible for transmissible spongiform encephalopathies, which affect both humans and animals. The mechanism of disease propagation is well understood and involves the interaction of PrPsc with its cellular isoform (PrPc) and subsequently abnormal structural conversion of the latter. PrPc is a glycoprotein anchored on the cell surface by a glycosylphosphatidylinositol moiety and expressed in most cell types but mainly in neurons. Prion diseases have been associated with the accumulation of the abnormally folded protein and its neurotoxic effects; however, it is not known if PrPc loss of function is an important component. New efforts are addressing this question and trying to characterize the physiological function of PrPc. At least four different mouse strains in which the PrP gene was ablated were generated and the results regarding their phenotype are controversial. Localization of PrPc on the cell membrane makes it a potential candidate for a ligand uptake, cell adhesion and recognition molecule or a membrane signaling molecule. Recent data have shown a potential role for PrPc in the metabolism of copper and moreover that this metal stimulates PrPc endocytosis. Our group has recently demonstrated that PrPc is a high affinity laminin ligand and that this interaction mediates neuronal cell adhesion and neurite extension and maintenance. Moreover, PrPc-caveolin-1 dependent coupling seems to trigger the tyrosine kinase Fyn activation. These data provide the first evidence for PrPc involvement in signal transduction.