Netrin-1-mediated axon outgrowth and cAMP production requires interaction with adenosine A2b receptor

The netrins, a family of laminin-related secreted proteins, are critical in controlling axon elongation and pathfinding. The DCC (for deleted in colorectal cancer) protein was proposed as a receptor for netrin-1 in the light of many observations including the inhibition of netrin-1-mediated axon outgrowth and attraction in the presence of an anti-DCC antiserum, the similitude of nervous system defects in DCC and netrin-1 knockout mice and the results of receptor swapping experiments. Previous studies have failed to show a direct interaction of DCC with netrin-1 (ref. 10), suggesting the possibility of an additional receptor or co-receptor. Here we show that DCC interacts with the membrane-associated adenosine A2b receptor, a G-protein-coupled receptor that induces cAMP accumulation on binding adenosine. We show that A2b is actually a netrin-1 receptor and induces cAMP accumulation on binding netrin-1. Finally, we show that netrin-1-dependent outgrowth of dorsal spinal cord axons directly involves A2b. Together our results indicate that the growth-promoting function of netrin-1 may require a receptor complex containing DCC and A2b.     

The neuronal repellent Slit inhibits leukocyte chemotaxis induced by chemotactic factors

Migration is a basic feature of many cell types in a wide range of species. Since the 1800s, cell migration has been proposed to occur in the nervous and immune systems, and distinct molecular cues for mammalian neurons and leukocytes have been identified. Here we report that Slit, a secreted protein previously known for its role of repulsion in axon guidance and neuronal migration, can also inhibit leukocyte chemotaxis induced by chemotactic factors. Slit inhibition of the chemokine-induced chemotaxis can be reconstituted by the co-expression of a chemokine receptor containing seven transmembrane domains and Roundabout (Robo), a Slit receptor containing a single transmembrane domain. Thus, there is a functional interaction between single and seven transmembrane receptors. Our results reveal the activity of a neuronal guidance cue in regulating leukocyte migration and indicate that there may be a general conservation of guidance mechanisms underlying metazoan cell migration. In addition, we have uncovered an inhibitor of leukocyte chemotaxis, and propose a new therapeutic approach to treat diseases involving leukocyte migration and chemotactic factors.     

Boc is a receptor for sonic hedgehog in the guidance of commissural axons

In the spinal cord, sonic hedgehog (Shh) is secreted by the floor plate to control the generation of distinct classes of ventral neurons along the dorsoventral axis. Genetic and in vitro studies have shown that Shh also later acts as a midline-derived chemoattractant for commissural axons. However, the receptor(s) responsible for Shh attraction remain unknown. Here we show that two Robo-related proteins, Boc and Cdon, bind specifically to Shh and are therefore candidate receptors for the action of Shh as an axon guidance ligand. Boc is expressed by commissural neurons, and targeted disruption of Boc in mouse results in the misguidance of commissural axons towards the floor plate. RNA-interference-mediated knockdown of Boc impairs the ability of rat commissural axons to turn towards an ectopic source of Shh in vitro. Taken together, these data suggest that Boc is essential as a receptor for Shh in commissural axon guidance.     

Wnt-mediated axon guidance via the Drosophila Derailed receptor

In nervous systems with bilateral symmetry, many neurons project axons across the midline to the opposite side. In each segment of the Drosophila embryonic nervous system, axons that display this projection pattern choose one of two distinct tracts: the anterior or posterior commissure. Commissure choice is controlled by Derailed, an atypical receptor tyrosine kinase expressed on axons projecting in the anterior commissure. Here we show that Derailed keeps these axons out of the posterior commissure by acting as a receptor for Wnt5, a member of the Wnt family of secreted signalling molecules. Our results reveal an unexpected role in axon guidance for a Wnt family member, and show that the Derailed receptor is an essential component of Wnt signalling in these guidance events.     

Semaphorin 7A promotes axon outgrowth through integrins and MAPKs

Striking parallels exist between immune and nervous system cellular signalling mechanisms. Molecules originally shown to be critical for immune responses also serve neuronal functions, and similarly neural guidance cues can modulate immune function. We show here that semaphorin 7A (Sema7A), a membrane-anchored member of the semaphorin family of guidance proteins previously known for its immunomodulatory effects, can also mediate neuronal functions. Unlike many other semaphorins, which act as repulsive guidance cues, Sema7A enhances central and peripheral axon growth and is required for proper axon tract formation during embryonic development. Unexpectedly, Sema7A enhancement of axon outgrowth requires integrin receptors and activation of MAPK signalling pathways. These findings define a previously unknown biological function for semaphorins, identify an unexpected role for integrins and integrin-dependent intracellular signalling in mediating semaphorin responses, and provide a framework for understanding and interfering with Sema7A function in both immune and nervous systems.     

Netrin-1 controls colorectal tumorigenesis by regulating apoptosis

The expression of the protein DCC (deleted in colorectal cancer) is lost or markedly reduced in numerous cancers and in the majority of colorectal cancers due to loss of heterozygosity in chromosome 18q, and has therefore been proposed to be a tumour suppressor. However, the rarity of mutations found in DCC, the lack of cancer predisposition of DCC mutant mice, and the presence of other tumour suppressor genes in 18q have raised doubts about the function of DCC as a tumour suppressor. Unlike classical tumour suppressors, DCC has been shown to induce apoptosis conditionally: by functioning as a dependence receptor, DCC induces apoptosis unless DCC is engaged by its ligand, netrin-1 (ref. 3). Here we show that inhibition of cell death by enforced expression of netrin-1 in mouse gastrointestinal tract leads to the spontaneous formation of hyperplastic and neoplastic lesions. Moreover, in the adenomatous polyposis coli mutant background associated with adenoma formation, enforced expression of netrin-1 engenders aggressive adenocarcinomatous malignancies. These data demonstrate that netrin-1 can promote intestinal tumour development, probably by regulating cell survival. Thus, a netrin-1 receptor or receptors function as conditional tumour suppressors.     

LysM蛋白在植物免疫中的作用

溶解素基序（LysM）是在多种蛋白质中普遍存在的结构域.植物LysM蛋白能够感知几丁质及其寡糖等分子配体，从而启动植物对病原菌的免疫反应.在水稻、拟南芥等植物免疫应答过程中，LysM蛋白作为一种重要的模式识别受体，通过不同形式的寡聚化，激活多种类受体胞质激酶及其下游的MAPK（mitogen activated protein kinase）级联反应传递信号.同时，蛋白质可逆磷酸化和蛋白质降解途径可以负调节LysM蛋白介导的防御信号转导.文章综述了植物免疫过程中LysM蛋白介导的信号转导分子机制.     

Netrin-1-mediated axon outgrowth requires deleted in colorectal cancer-dependent MAPK activation

Neuronal growth cones are guided to their targets by attractive and repulsive guidance cues. In mammals, netrin-1 is a bifunctional cue, attracting some axons and repelling others. Deleted in colorectal cancer (Dcc) is a receptor for netrin-1 that mediates its chemoattractive effect on commissural axons, but the signalling mechanisms that transduce this effect are poorly understood. Here we show that Dcc activates mitogen-activated protein kinase (MAPK) signalling, by means of extracellular signal-regulated kinase (ERK)-1 and -2, on netrin-1 binding in both transfected cells and commissural neurons. This activation is associated with recruitment of ERK-1/2 to a Dcc receptor complex. Inhibition of ERK-1/2 antagonizes netrin-dependent axon outgrowth and orientation. Thus, activation of MAPK signalling through Dcc contributes to netrin signalling in axon growth and guidance.     

Defining brain wiring patterns and mechanisms through gene trapping in mice

The search to understand the mechanisms regulating brain wiring has relied on biochemical purification approaches in vertebrates and genetic approaches in invertebrates to identify molecular cues and receptors for axon guidance. Here we describe a phenotype-based gene-trap screen in mice designed for the large-scale identification of genes controlling the formation of the trillions of connections in the mammalian brain. The method incorporates an axonal marker, which helps to identify cell-autonomous mechanisms in axon guidance, and has generated a resource of mouse lines with striking patterns of axonal labelling, which facilitates analysis of the normal wiring diagram of the brain. Studies of two of these mouse lines have identified an in vivo guidance function for a vertebrate transmembrane semaphorin, Sema6A, and have helped re-evaluate that of the Eph receptor EphA4.     

A kinase-regulated PDZ-domain interaction controls endocytic sorting of the beta2-adrenergic receptor.

A fundamental question in cell biology is how membrane proteins are sorted in the endocytic pathway. The sorting of internalized beta2-adrenergic receptors between recycling endosomes and lysosomes is responsible for opposite effects on signal transduction and is regulated by physiological stimuli. Here we describe a mechanism that controls this sorting operation, which is mediated by a family of conserved protein-interaction modules called PDZ domains. The phosphoprotein EBP50 (for ezrinradixin-moesin(ERM)-binding phosphoprotein-50) binds to the cytoplasmic tail of the beta2-adrenergic receptor through a PDZ domain and to the cortical actin cytoskeleton through an ERM-binding domain. Disrupting the interaction of EBP50 with either domain or depolymerization of the actin cytoskeleton itself causes missorting of endocytosed beta2-adrenergic receptors but does not affect the recycling of transferrin receptors. A serine residue at position 411 in the tail of the beta2-adrenergic receptor is a substrate for phosphorylation by GRK-5 (for G-protein-coupled-receptor kinase-5) and is required for interaction with EBP50 and for proper recycling of the receptor. Our results identify a new role for PDZ-domain-mediated protein interactions and for the actin cytoskeleton in endocytic sorting, and suggest a mechanism by which GRK-mediated phosphorylation could regulate membrane trafficking of G-protein-coupled receptors after endocytosis.     

UNC-6/netrin and its receptor UNC-5 locally exclude presynaptic components from dendrites

Polarity is an essential feature of many cell types, including neurons that receive information from local inputs within their dendrites and propagate nerve impulses to distant targets through a single axon. It is generally believed that intrinsic structural differences between axons and dendrites dictate the polarized localization of axonal and dendritic proteins. However, whether extracellular cues also instruct this process in vivo has not been explored. Here we show that the axon guidance cue UNC-6/netrin and its receptor UNC-5 act throughout development to exclude synaptic vesicle and active zone proteins from the dendrite of the Caenorhabditis elegans motor neuron DA9, which is proximal to a source of UNC-6/netrin. In unc-6/netrin and unc-5 loss-of-function mutants, presynaptic components mislocalize to the DA9 dendrite. In addition, ectopically expressed UNC-6/netrin, acting through UNC-5, is sufficient to exclude endogenous synapses from adjacent subcellular domains within the DA9 axon. Furthermore, this anti-synaptogenic activity is interchangeable with that of LIN-44/Wnt despite being transduced through different receptors, suggesting that extracellular cues such as netrin and Wnts not only guide axon navigation but also regulate the polarized accumulation of presynaptic components through local exclusion.     

SOL-1 is a CUB-domain protein required for GLR-1 glutamate receptor function in C. elegans

Ionotropic glutamate receptors (iGluRs) mediate most excitatory synaptic signalling between neurons. Binding of the neurotransmitter glutamate causes a conformational change in these receptors that gates open a transmembrane pore through which ions can pass. The gating of iGluRs is crucially dependent on a conserved amino acid that was first identified in the 'lurcher' ataxic mouse. Through a screen for modifiers of iGluR function in a transgenic strain of Caenorhabditis elegans expressing a GLR-1 subunit containing the lurcher mutation, we identify suppressor of lurcher (sol-1). This gene encodes a transmembrane protein that is predicted to contain four extracellular beta-barrel-forming domains known as CUB domains. SOL-1 and GLR-1 are colocalized at the cell surface and can be co-immunoprecipitated. By recording from neurons expressing GLR-1, we show that SOL-1 is an accessory protein that is selectively required for glutamate-gated currents. We propose that SOL-1 participates in the gating of non-NMDA (N-methyl-D-aspartate) iGluRs, thereby providing a previously unknown mechanism of regulation for this important class of neurotransmitter receptor.     

Co-localization of GABA receptors and benzodiazepine receptors in the brain shown by monoclonal antibodies

The most abundant inhibitory neurotransmitter in the central nervous system, gamma-aminobutyric acid (GABA), exerts its main effects via a GABAA receptor that gates a chloride channel in the subsynaptic membrane. These receptors can contain a modulatory unit, the benzodiazepine receptor, through which ligands of different chemical classes can increase or decrease GABAA receptor function. We have now visualized a GABAA receptor in mammalian brain using monoclonal antibodies. The protein complex recognized by the antibodies contained high- and low-affinity binding sites for GABA as well as binding sites for benzodiazepines, indicative of a GABAA receptor functionally associated with benzodiazepine receptors. As the pattern of brain immunoreactivity corresponds to the autoradiographical distribution of benzodiazepine binding sites, most benzodiazepine receptors seem to be part of GABAA receptors. Two constituent proteins were identified immunologically. Because the monoclonal antibodies cross-react with human brain, they provide a means for elucidating those CNS disorders which may be linked to a dysfunction of a GABAA receptor.     

Directional guidance of neuronal migration in the olfactory system by the protein Slit.

Although cell migration is crucial for neural development, molecular mechanisms guiding neuronal migration have remained unclear. Here we report that the secreted protein Slit repels neuronal precursors migrating from the anterior subventricular zone in the telencephalon to the olfactory bulb. Our results provide a direct demonstration of a molecular cue whose concentration gradient guides the direction of migrating neurons. They also support a common guidance mechanism for axon projection and neuronal migration and suggest that Slit may provide a molecular tool with potential therapeutic applications in controlling and directing cell migration.     

Cyclic AMP/GMP-dependent modulation of Ca2+ channels sets the polarity of nerve growth-cone turning

Signalling by intracellular second messengers such as cyclic nucleotides and Ca2+ is known to regulate attractive and repulsive guidance of axons by extracellular factors. However, the mechanism of interaction among these second messengers in determining the polarity of the guidance response is largely unknown. Here, we report that the ratio of cyclic AMP to cyclic GMP activities sets the polarity of netrin-1-induced axon guidance: high ratios favour attraction, whereas low ratios favour repulsion. Whole-cell recordings of Ca2+ currents at Xenopus spinal neuron growth cones indicate that cyclic nucleotide signalling directly modulates the activity of L-type Ca2+ channels (LCCs) in axonal growth cones. Furthermore, cGMP signalling activated by an arachidonate 12-lipoxygenase metabolite suppresses LCC activity triggered by netrin-1, and is required for growth-cone repulsion mediated by the DCC-UNC5 receptor complex. By linking cAMP and cGMP signalling and modulation of Ca2+ channel activity in growth cones, these findings delineate an early membrane-associated event responsible for signal transduction during bi-directional axon guidance.     

Multiple D2 dopamine receptors produced by alternative RNA splicing

Dopamine receptor belong to a large class of neurotransmitter and hormone receptors that are linked to their signal transduction pathways through guanine nucleotide binding regulatory proteins (G proteins). Pharmacological, biochemical and physiological criteria have been used to define two subcategories of dopamine receptors referred to as D1 and D2. D1 receptors activate adenylyl cyclase and are coupled with the Gs regulatory protein. By contrast, activation of D2 receptors results in various responses including inhibition of adenylyl cyclase, inhibition of phosphatidylinositol turnover, increase in K+ channel activity and inhibition of Ca2+ mobilization. The G protein(s) linking the D2 receptors to these responses have not been identified, although D2 receptors have been shown to both copurify and functionally reconstitute with both Gi and Go related proteins. The diversity of responses elicited by D2-receptor activation could reflect the existence of multiple D2 receptor subtypes, the identification of which is facilitated by the recent cloning of a complementary DNA encoding a rat D2 receptor. This receptor exhibits considerable amino-acid homology with other members of the G protein-coupled receptor superfamily. Here we report the identification and cloning of a cDNA encoding an RNA splice variant of the rat D2 receptor cDNA. This cDNA codes for a receptor isoform which is predominantly expressed in the brain and contains an additional 29 amino acids in the third cytoplasmic loop, a region believed to be involved in G protein coupling.     

Regulation of deactivation of photoreceptor G protein by its target enzyme and cGMP.

The photoreceptor G protein, transducin, is one of the class of heterotrimeric G proteins that mediates between membrane receptors and intracellular enzymes or ion channels. Light-activated rhodopsin catalyses the exchange of GDP for GTP on multiple transducin molecules. Activated transducin then stimulates cyclic GMP phosphodiesterase by releasing an inhibitory action of the phosphodiesterase gamma-subunits. This leads to a decrease in cGMP levels in the rod, and closure of plasma membrane cationic channels gated by cGMP. In this and other systems, turn-off of the response requires the GTP bound to G protein to be hydrolysed by an intrinsic GTPase activity. Here we report that the interaction of transducin with cGMP phosphodiesterase, specifically with its gamma-subunits, accelerates GTPase activity by several fold. Thus the gamma-subunits of the phosphodiesterase serve a function analogous to the GTPase-activating proteins that regulate the class of small GTP-binding proteins. The acceleration can be partially suppressed by cGMP, most probably through the non-catalytic cGMP-binding sites of phosphodiesterase alpha and beta-subunits. This cGMP regulation may function in light-adaptation of the photo-response as a negative feedback that decreases the lifetime of activated cGMP phosphodiesterase as light causes decreases in cytoplasmic cGMP.     

Requirement of TRPC channels in netrin-1-induced chemotropic turning of nerve growth cones

Ion channels formed by the TRP (transient receptor potential) superfamily of proteins act as sensors for temperature, osmolarity, mechanical stress and taste. The growth cones of developing axons are responsible for sensing extracellular guidance factors, many of which trigger Ca2+ influx at the growth cone; however, the identity of the ion channels involved remains to be clarified. Here, we report that TRP-like channel activity exists in the growth cones of cultured Xenopus neurons and can be modulated by exposure to netrin-1 and brain-derived neurotrophic factor, two chemoattractants for axon guidance. Whole-cell recording from growth cones showed that netrin-1 induced a membrane depolarization, part of which remained after all major voltage-dependent channels were blocked. Furthermore, the membrane depolarization was sensitive to blockers of TRP channels. Pharmacological blockade of putative TRP currents or downregulation of Xenopus TRP-1 (xTRPC1) expression with a specific morpholino oligonucleotide abolished the growth-cone turning and Ca2+ elevation induced by a netrin-1 gradient. Thus, TRPC currents reflect early events in the growth cone's detection of some extracellular guidance signals, resulting in membrane depolarization and cytoplasmic Ca2+ elevation that mediates the turning of growth cones.