Monoclonal Antibody DL11C8 Identifies ADAM23 as a Component of Lipid Raft Microdomains.

A disintegrin and metalloprotease protein 23 (ADAM23) is a transmembrane type I glycoprotein involved with the development and maintenance of the nervous system, including neurite outgrowth, neuronal adhesion and differentiation and regulation of synaptic transmission. In addition, ADAM23 seems to participate in immune response and tumor establishment through interaction with different members of integrin receptors. Here, we describe a novel monoclonal antibody (DL11C8) that specifically recognizes the cysteine-rich domain of both pre-protein (100 kDa) and mature (70 kDa) forms of ADAM23 from different species, including human, rodents and avian orthologs. Using this antibody, we detected both forms of ADAM23 on the cell surface of three neuronal cell lineages (Neuro-2a, SH-SY5Y and CHLA-20), with a higher relative content of ADAM23100 kDa. Furthermore, we demonstrate for the first time that a catalytically inactive member of the ADAM family is present in the membrane signaling platforms, namely lipid rafts. Indeed, the mature ADAM2370 kDa partitions between raft and non-raft membrane domains, while the pro-protein ADAM23100 kDa is mainly expressed in non-raft domains. These membranous distributions were observed in both different brain regions homogenates and primary cultured neurons lysates from mouse cortex and cerebellum. Taken together, these findings point out ADAM23 as a lipid raft molecular component.

STI1 antagonizes cytoskeleton collapse mediated by small GTPase Rnd1 and regulates neurite growth.

Rnd proteins comprise a branch of the Rho family of small GTP-binding proteins, which have been implicated in rearrangements of the actin cytoskeleton and microtubule dynamics. Particularly in the nervous system, Rnd family proteins regulate neurite formation, dendrite development and axonal branching. A secreted form of the co-chaperone Stress-Inducible Protein 1 (STI1) has been described as a prion protein partner that is involved in several processes of the nervous system, such as neurite outgrowth, neuroprotection, astrocyte development, and the self-renewal of neural progenitor cells. We show that cytoplasmic STI1 directly interacts with the GTPase Rnd1. This interaction is specific for the Rnd1 member of the Rnd family. In the COS collapse assay, overexpression of STI1 prevents Rnd1-plexin-A1-mediated cytoskeleton retraction. In PC-12 cells, overexpression of STI1 enhances neurite outgrowth in cellular processes initially established by Rnd1. Therefore, we propose that STI1 participates in Rnd1-induced signal transduction pathways that are involved in the dynamics of the actin cytoskeleton.

Characterization of a specific interaction between ADAM23 and cellular prion protein.

ADAMs are transmembrane proteins implicated in several biological functions, including cytokine and growth factor shedding, fertilization, muscle and nervous system development. Here, we show for the first time that ADAM23, which is predominantly expressed in the central nervous system, co-localizes with cellular prion protein (PrP(C)) at plasma membrane of mouse hippocampal neurons and neuroblastoma cells. Co-immunoprecipitation and pull-down assay showed a physical interaction between ADAM23 and both recombinant and endogenous PrP(C). Glycosylation seems to be not relevant to the observed interaction since both ADAM23 and PrP(C) recombinant proteins expressed in bacteria or extracted from eukaryotic cells treated with tunicamycin are still able to bind each other. In vitro binding assays also suggested that the disintegrin domain of ADAM23 is able to interact directly with PrP(C). Taken together, these findings point out PrP(C) as a novel molecular partner for ADAM23 in the nervous systems.

The Odorant Signaling Pathway

Through the sense of smell mammals can obtain information about food, danger, sexual partners and predators. Two main different types of signals can be recognized by the olfactory system: volatile odorants, which are detected by the olfactory sensory neurons of the nose; and pheromones, which are detected by the vomeronasal neurons of the accessory olfactory system, or vomeronasal organ. These sensory neurons express respectively hundreds of odorant and pheromone receptors, which belong to the superfamily of G protein-coupled receptors. We review the general organization of the main and accessory olfactory systems, the structures of the receptor families in each of these organs and their signaling pathways.

Guanosine promotes B16F10 melanoma cell differentiation through PKC-ERK 1/2 pathway.

Malignant melanoma is one of the most lethal cancers. Nowadays, several anti-melanoma therapies have been employed. However, the poor prognosis and/or the increased toxicity of those treatments clearly demonstrate the requirement of searching for new drugs or novel combined chemotherapeutic protocols, contemplating both effectiveness and low toxicity. Guanosine (Guo) has been used in combination with acriflavina to potentiate the latter's antitumor activity, through still unknown mechanisms. Here, we show that Guo induces B16F10 melanoma cell differentiation, attested by growth arrest, dendrite-like outgrowth and increased melanogenesis, and also reduced motility. A sustained ERK 1/2 phosphorylation was observed after Guo treatment and ERK inhibition led to blockage of dendritogenesis. Intracellular cyclic AMP was not involved in ERK activation, since its levels remained unchanged. Protein kinase C (PKC), in contrast to phospholipase C (PLC), inhibition completely prevented ERK activation. While the classical melanoma differentiation agent forskolin activates cAMP-PKA-Raf-MEK-ERK pathway in B16F10 cells, here we suggest that a cAMP-independent, PKC-ERK axis is involved in Guo-induced B16F10 differentiation. Altogether, our results show that Guo acts as a differentiating agent, with cytostatic rather than cytotoxic properties, leading to a decreased melanoma malignancy. Thus, we propose that Guo may be envisaged in combination with lower doses of conventional anti-melanoma drugs, in an attempt to prevent or diminish their adverse effects.

Cellular prion protein interaction with vitronectin supports axonal growth and is compensated by integrins.

The physiological functions of the cellular prion protein, PrP(C), as a cell surface pleiotropic receptor are under debate. We report that PrP(C) interacts with vitronectin but not with fibronectin or collagen. The binding sites mediating this PrP(C)-vitronectin interaction were mapped to residues 105-119 of PrP(C) and the residues 307-320 of vitronectin. The two proteins were co-localized in embryonic dorsal root ganglia from wild-type mice. Vitronectin addition to cultured dorsal root ganglia induced axonal growth, which could be mimicked by vitronectin peptide 307-320 and abrogated by anti-PrP(C) antibodies. Full-length vitronectin, but not the vitronectin peptide 307-320, induced axonal growth of dorsal root neurons from two strains of PrP(C)-null mice. Functional assays demonstrated that relative to wild-type cells, PrP(C)-null dorsal root neurons were more responsive to the Arg-Gly-Asp peptide (an integrin-binding site), and exhibited greater alphavbeta3 activity. Our findings indicate that PrP(C) plays an important role in axonal growth, and this function may be rescued in PrP(C)-knockout animals by integrin compensatory mechanisms.

Ric-8B promotes functional expression of odorant receptors.

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 Galphaolf, promotes efficient heterologous expression of ORs. Our results also show that Ric-8B enhances accumulation of Galphaolf at the cell periphery, indicating that it promotes functional OR expression by improving the efficiency of OR coupling to Galphaolf. Expression systems containing Galphaolf and Ric-8B should contribute to the functional characterization of ORs.

Ric-8B, an olfactory putative GTP exchange factor, amplifies signal transduction through the olfactory-specific G-protein Galphaolf.

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 Galphaolf. We found that Ric-8B (for resistant to inhibitors of cholinesterase), a putative GTP exchange factor, is able to interact with Galphaolf. Like Galphaolf, 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 Galphaolf strongly indicate that Ric-8B is a functional partner for Galphaolf. Finally, we show that Ric-8B is able to potentiate Galphaolf-dependent cAMP accumulation in human embryonic kidney 293 cells and therefore may be an important component for odorant signal transduction.

Stress-inducible protein 1 is a cell surface ligand for cellular prion that triggers neuroprotection.

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 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 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.