Phosphoinositide-3-OH kinase (PI(3)K), activated through growth factor stimulation, generates a lipid second messenger, phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3). PtdIns(3,4,5)P3 is instrumental in signalling pathways that trigger cell activation, cytoskeletal rearrangement, survival and other reactions. However, some targets of PtdIns(3,4,5)P3 are yet to be discovered. We demonstrate that SWAP-70, a unique signalling protein, specifically binds PtdIns(3,4,5)P3. On stimulation by growth factors, cytoplasmic SWAP-70, which is dependent on PI(3)K but independent of Ras, moved to cell membrane rearrangements known as ruffles. However, mutant SWAP-70 lacking the ability to bind PtdIns(3,4,5)P3 blocked membrane ruffling induced by epidermal growth factor or platelet-derived growth factor. SWAP-70 shows low homology with Rac-guanine nucleotide exchange factors (GEFs), and catalyses PtdIns(3,4,5)P3-dependent guanine nucleotide exchange to Rac. SWAP-70-deficient fibroblasts showed impaired membrane ruffling after stimulation with epidermal growth factor, and failed to activate Rac fully. We conclude that SWAP-70 is a new type of Rac-GEF which, independently of Ras, transduces signals from tyrosine kinase receptors to Rac.

Insect Odorant Receptors (ORs) comprise an enormous protein family that translates environmental chemical signals into neuronal electrical activity. These heptahelical receptors are proposed to function as ligand-gated ion channels and/or to act metabotropically as G protein-coupled receptors (GPCRs). Resolving their signalling mechanism has been hampered by the lack of tertiary structural information and primary sequence similarity to other proteins. We use amino acid evolutionary covariation across these ORs to define restraints on structural proximity of residue pairs, which permit de novo generation of three-dimensional models. The validity of our analysis is supported by the location of functionally important residues in highly constrained regions of the protein. Importantly, insect OR models exhibit a distinct transmembrane domain packing arrangement to that of canonical GPCRs, establishing the structural unrelatedness of these receptor families. The evolutionary couplings and models predict odour binding and ion conduction domains, and provide a template for rationale structure-activity dissection.

IL‐6 induces differentiation of PC12 cells pretreated with nerve growth factor (NGF). We explored the signals required for neurite outgrowth of PC12 cells by using a series of mutants of a chimeric receptor consisting of the extracellular domain of the granulocyte‐colony stimulating factor (G‐CSF) receptor and the cytoplasmic domain of gp130, a signal‐transducing subunit of the IL‐6 receptor. The mutants incapable of activating the MAP kinase cascade failed to induce neurite outgrowth. Consistently, a MEK inhibitor, PD98059, inhibited neurite outgrowth, showing that activation of the MAP kinase cascade is essential for the differentiation of PC12 cells. In contrast, a mutation that abolished the ability to activate STAT3 did not inhibit, but rather stimulated neurite outgrowth. This mutant did not require NGF pretreatment for neurite outgrowth. Dominant‐negative STAT3s mimicked NGF pretreatment, and NGF suppressed the IL‐6‐induced activation of STAT3, supporting the idea that STAT3 might regulate the differentiation of PC12 cells negatively. These results suggest that neurite outgrowth of PC12 cells is regulated by the balance of MAP kinase and STAT3 signal transduction pathways, and that STAT3 activity can be regulated negatively by NGF.

It has been shown that inhibition of phosphatidylinositol (PI) 3-kinase blocks neurite outgrowth of PC12 cells stimulated with nerve growth factor. To further assess the role of PI 3-kinase, the active form of PI 3-kinase was expressed in PC12 cells by the adenovirus mediated introduction of a site-specific recombinase, Cre. After expression of the active PI 3-kinase, elevation of the levels of PI 3,4-diphosphate and PI 3,4,5-trisphosphate as well as formation of neurite-like processes was observed. The process formation was inhibited by wortmannin, a selective inhibitor of PI 3-kinase, which suggests that a high activity of PI 3-kinase was responsible for the formation of these processes. The processes lacked accumulation of F-actin and GAP43 at the growth cone, which suggests that the processes were incomplete compared with neurites. Instead, the bundling of microtubules was enhanced, which suggests that organization of the microtubules might be driving the process of elongation in the cells expressing the active PI 3-kinase. Induction of active PI 3-kinase resulted in activation of Jun N-terminal kinase but not of mitogenactivated protein kinase or protein kinase B/Rac protein kinase/Akt. These results suggest that PI 3-kinase is involved in neurite outgrowth in PC12 cells and that activation of Jun N-terminal kinase cascade may be involved in the cell response.The PC12, rat pheochromocytoma cell line provides a useful model system for the differentiation of neuronal cells. They respond to nerve growth factor (NGF) 1 with growth arrest and exhibit typical characteristics of neuronal cells (1). After stimulation with NGF, a number of signaling pathways are activated, including the Ras-MAP kinase, phospholipase C, and phosphatidylinositol (PI) 3-kinase cascades (2). In the setting of multiple activation of signaling pathways, it has been suggested that sustained activation of MAP kinase in particular is involved in the differentiation (3, 4). Indeed, constitutive activation of MAP kinase by activated Ras or MAP kinase kinase results in full differentiation of the cells (5-7). Besides this, we have shown that PI 3-kinase activity is required for neurite outgrowth in PC12 cells (8). The results suggested that the PI 3-kinase activity was required especially for the neurite elongation. The analysis of the levels of phosphoinositides in NGFtreated PC12 cells revealed that PI 3-kinase was strongly activated immediately after NGF treatment, and this activity declined rapidly. However, even long after the burst of PI 3-kinase activation, levels of the products of PI 3-kinase remained slightly higher than that of unstimulated cells (8). The PI 3-kinase activated by NGF stimulation consists of two subunits, p85 and p110 (9). The regulatory subunit, p85, contains one SH3 domain and two SH2 domains, which may be involved in interaction with other proteins. The p85 subunit binds to p110 through the region between the two SH2 domains of p85 (iSH2) (10, 11). P110 is the catalytic subunit, and p85 binding is necessary to ac...

PIP 3 BP is a phosphatidylinositol 3,4,5-trisphosphatebinding protein (PIP 3 BP) abundant in brain, containing a zinc finger motif and two pleckstrin homology (PH) domains. Staining of rat brain cells with anti-PIP 3 BP antibody and determination of localization of PIP 3 BP fused to the green fluorescent protein (GFP-PIP 3 BP) revealed that PIP 3 BP was targeted to the nucleus. Targeting was dependent on a putative nuclear localization signal in PIP 3 BP. Generation of PIP 3 in the nucleus was detected in H 2 O 2 -treated 293T cells, nerve growth factor (NGF)-treated PC12 cells, and platelet-derived growth factor (PDGF)-treated NIH 3T3 cells. Translocation of phosphatidylinositol 3-kinase (PI 3-kinase) to the nucleus and enhanced activity of PI 3-kinase in the nucleus fraction were observed after H 2 O 2 treatment of 293T cells, suggesting that PI 3-kinase can be activated in the nucleus as well as in the membrane after appropriate stimulation of the cells. Co-expression of the constitutively active PI 3-kinase with PIP 3 BP resulted in exportation of the protein from the nucleus to the cytoplasm, suggesting that PIP 3 BP can function as a PIP 3 -binding protein in the intact cells. These results imply that there may be an unknown function of PI 3-kinase in the nucleus.Phosphatidylinositol 3-kinase 1 is an enzyme that is activated immediately after growth factor or differentiation factor stimulation of the cells (1) and that generates second messengers, phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) and phosphatidylinositol 3,4-bisphosphate (PI 3,4-P 2 ) (2-5). These 3Ј-phosphorylated phosphoinositides can activate serine, threonine kinases such as PKB/Akt, PKCs, and PDKs (6 -9). They are also suggested to be involved in other events such as rearrangement of cytoskeleton and vesicle transport because these phenomena are sensitive to the PI 3-kinase inhibitors and dominant negative mutants of PI 3-kinase (10). Recently, it was reported that the 3Ј-phosphorylated phosphoinositides can activate guanine nucleotide exchanging factors of Rac and Arf, small G proteins involved in actin rearrangement and vesicle transport, respectively (11,12). Therefore, G proteins as well as kinases are downstream of PI 3-kinase.We have identified PIP 3 BP as a PIP 3 -binding protein, using a PIP 3 analogue column (13). It is abundant in brain, implying that it may be involved in the function of nerve systems. PIP 3 BP binds to PIP 3 but not to PI 3,4-P 2 or phosphatidylinositol 4,5-bisphosphate (PI 4,5-P 2 ). It has a zinc finger motif homologous to that of Arf-GTPase activating protein (GAP) and two PH domains. Both PH domains are shown to be involved in binding to PIP 3 . Another PIP 3 -binding protein, centaurin ␣, is highly homologous to PIP 3 BP (14). No GAP activity to Arf has been detected in either protein. Although the binding of centaurin ␣ and PIP 3 BP to PIP 3 was specific, the role of the protein is unclear. To address this question, we determined the intracellular localization by immunological techniques, using mono...

Musk odors have been used widely for fragrance and medicine for Ͼ2000 years because of their fascinating scent and physiological effects. Therefore, fragrance manufacturers have been eager to develop high-quality musk compounds that are safe and easily synthesized. We recently identified muscone-responsive olfactory receptors (ORs) MOR215-1 and OR5AN1 in mice and humans, respectively (Shirasu et al., 2014). In this study, we identified musk ORs that are evolutionarily closely related to MOR215-1 or OR5AN1 in various primates and investigated structure-activity relationships for various musk odorants and related compounds. We found that each species has one or two functional musk ORs that exhibit specific ligand spectra to musk compounds. Some of them, including the human OR5AN1, responded to nitro musks with chemical properties distinct from muscone. The ligand specificity of OR5AN1 reflects the perception of musk odors in humans. Genetic deletion of MOR215-1 in mice resulted in drastic reduction of sensitivity to muscone, suggesting that MOR215-1 plays a critical role in muscone perception. Therefore, the current study reveals a clear link between the identified OR and muscone perception. Moreover, the strategy established for screening ligands for the muscone OR may facilitate the development of novel and commercially useful musk odors.

Abstract In this study, we examined the mode of metabolism of food odorant molecules in the human nasal/oral cavity in vitro and in vivo. We selected 4 odorants, 2-furfurylthiol (2-FT), hexanal, benzyl acetate, and methyl raspberry ketone, which are potentially important for designing food flavors. In vitro metabolic assays of odorants with saliva/nasal mucus analyzed by gas chromatography mass spectrometry revealed that human saliva and nasal mucus exhibit the following 3 enzymatic activities: (i) methylation of 2-FT into furfuryl methylsulfide (FMS); (ii) reduction of hexanal into hexanol; and (iii) hydrolysis of benzyl acetate into benzyl alcohol. However, (iv) demethylation of methyl raspberry ketone was not observed. Real-time in vivo analysis using proton transfer reaction-mass spectrometry demonstrated that the application of 2-FT and hexanal through 3 different pathways via the nostril or through the mouth generated the metabolites FMS and hexanol within a few seconds. The concentration of FMS and hexanol in the exhaled air was above the perception threshold. A cross-adaptation study based on the activation pattern of human odorant receptors suggested that this metabolism affects odor perception. These results suggest that some odorants in food are metabolized in the human nasal mucus/saliva, and the resulting metabolites are perceived as part of the odor quality of the substrates. Our results help improve the understanding of the mechanism of food odor perception and may enable improved design and development of foods in relation to odor.

Membrane ruffling induced by growth factor stimulation is caused by actin remodeling, which is mediated by various signaling molecules including Rac. We have shown that SWAP-70, which binds phosphatidylinositol trisphosphate, is one such molecule required for membrane ruffling in mouse kidney cells. Here, we show that SWAP-70 directly binds to F-actin. The bacterially expressed C-terminal region of SWAP-70 co-sedimented with non-muscle F-actin, suggesting direct binding of SWAP-70 to F-actin. The binding was much weaker in muscle F-actin. A truncated mutant of SWAP-70 containing only the C-terminal region readily colocalizes with F-actin, supporting this idea. Full-length SWAP-70 does not colocalize with F-actin unless cells are stimulated with growth factors, suggesting the presence of a stimuli-dependent regulatory mechanism for actin-binding activity in vivo. Overexpression of the mutant SWAP-70 lacking this binding domain inhibits the membrane ruffling induced by epidermal growth factor stimulation in COS7 cells. This dominant-negative effect is also observed in membrane ruffling induced by a dominant-active Rac, suggesting that SWAP-70 cooperates with Rac. These results suggest that the binding activity of SWAP-70 to non-muscle F-actin is required for membrane ruffling.