Netrins are secreted proteins that play a crucial role in neuronal migration and in axon guidance during the development of the nervous system. To date, several Netrins have been described in mouse and humans: Netrin-1, -3/NTL2, -4/h and G-Netrins. Netrin-1 is the most studied member of the family and has been shown to play a crucial role in neuronal navigation during nervous system development mainly through its interaction with its receptors DCC and UNC5. Members of the Deleted in colorectal cancer (DCC) family- which includes DCC and Neogenin in vertebrates- mediate netrin-induced axon attraction, whereas the C. elegans UNC5 receptor and its four vertebrate homologs Unc5a-Unc5d mediate repulsion.
Liu G, Beggs H, Jürgensen C, Park HT, Tang H, Gorski J, Jones KR, Reichardt LF, Wu J, Rao Y.; ''Netrin requires focal adhesion kinase and Src family kinases for axon outgrowth and attraction.''; PubMedEurope PMCScholia
Barallobre MJ, Pascual M, Del Río JA, Soriano E.; ''The Netrin family of guidance factors: emphasis on Netrin-1 signalling.''; PubMedEurope PMCScholia
Stein E, Zou Y, Poo M, Tessier-Lavigne M.; ''Binding of DCC by netrin-1 to mediate axon guidance independent of adenosine A2B receptor activation.''; PubMedEurope PMCScholia
Millard TH, Sharp SJ, Machesky LM.; ''Signalling to actin assembly via the WASP (Wiskott-Aldrich syndrome protein)-family proteins and the Arp2/3 complex.''; PubMedEurope PMCScholia
Hu G, Fearon ER.; ''Siah-1 N-terminal RING domain is required for proteolysis function, and C-terminal sequences regulate oligomerization and binding to target proteins.''; PubMedEurope PMCScholia
Fuerst PG, Koizumi A, Masland RH, Burgess RW.; ''Neurite arborization and mosaic spacing in the mouse retina require DSCAM.''; PubMedEurope PMCScholia
Strübing C, Krapivinsky G, Krapivinsky L, Clapham DE.; ''Formation of novel TRPC channels by complex subunit interactions in embryonic brain.''; PubMedEurope PMCScholia
Li W, Lee J, Vikis HG, Lee SH, Liu G, Aurandt J, Shen TL, Fearon ER, Guan JL, Han M, Rao Y, Hong K, Guan KL.; ''Activation of FAK and Src are receptor-proximal events required for netrin signaling.''; PubMedEurope PMCScholia
Shekarabi M, Kennedy TE.; ''The netrin-1 receptor DCC promotes filopodia formation and cell spreading by activating Cdc42 and Rac1.''; PubMedEurope PMCScholia
Bretscher A, Edwards K, Fehon RG.; ''ERM proteins and merlin: integrators at the cell cortex.''; PubMedEurope PMCScholia
Moore SW, Tessier-Lavigne M, Kennedy TE.; ''Netrins and their receptors.''; PubMedEurope PMCScholia
Cooper HM, Gad JM, Keeling SL.; ''The Deleted in Colorectal Cancer netrin guidance system: a molecular strategy for neuronal navigation.''; PubMedEurope PMCScholia
Yamagata M, Sanes JR.; ''Dscam and Sidekick proteins direct lamina-specific synaptic connections in vertebrate retina.''; PubMedEurope PMCScholia
Li X, Gao X, Liu G, Xiong W, Wu J, Rao Y.; ''Netrin signal transduction and the guanine nucleotide exchange factor DOCK180 in attractive signaling.''; PubMedEurope PMCScholia
Li W, Guan KL.; ''The Down syndrome cell adhesion molecule (DSCAM) interacts with and activates Pak.''; PubMedEurope PMCScholia
Qin S, Yu L, Gao Y, Zhou R, Zhang C.; ''Characterization of the receptors for axon guidance factor netrin-4 and identification of the binding domains.''; PubMedEurope PMCScholia
Meyerhardt JA, Caca K, Eckstrand BC, Hu G, Lengauer C, Banavali S, Look AT, Fearon ER.; ''Netrin-1: interaction with deleted in colorectal cancer (DCC) and alterations in brain tumors and neuroblastomas.''; PubMedEurope PMCScholia
Briançon-Marjollet A, Ghogha A, Nawabi H, Triki I, Auziol C, Fromont S, Piché C, Enslen H, Chebli K, Cloutier JF, Castellani V, Debant A, Lamarche-Vane N.; ''Trio mediates netrin-1-induced Rac1 activation in axon outgrowth and guidance.''; PubMedEurope PMCScholia
Shekarabi M, Moore SW, Tritsch NX, Morris SJ, Bouchard JF, Kennedy TE.; ''Deleted in colorectal cancer binding netrin-1 mediates cell substrate adhesion and recruits Cdc42, Rac1, Pak1, and N-WASP into an intracellular signaling complex that promotes growth cone expansion.''; PubMedEurope PMCScholia
Martín M, Simon-Assmann P, Kedinger M, Martin M, Mangeat P, Real FX, Fabre M.; ''DCC regulates cell adhesion in human colon cancer derived HT-29 cells and associates with ezrin.''; PubMedEurope PMCScholia
Ren XR, Hong Y, Feng Z, Yang HM, Mei L, Xiong WC.; ''Tyrosine phosphorylation of netrin receptors in netrin-1 signaling.''; PubMedEurope PMCScholia
Agarwala KL, Ganesh S, Tsutsumi Y, Suzuki T, Amano K, Yamakawa K.; ''Cloning and functional characterization of DSCAML1, a novel DSCAM-like cell adhesion molecule that mediates homophilic intercellular adhesion.''; PubMedEurope PMCScholia
Li X, Meriane M, Triki I, Shekarabi M, Kennedy TE, Larose L, Lamarche-Vane N.; ''The adaptor protein Nck-1 couples the netrin-1 receptor DCC (deleted in colorectal cancer) to the activation of the small GTPase Rac1 through an atypical mechanism.''; PubMedEurope PMCScholia
Rouer E.; ''[Neuronal isoforms of Src, Fyn and Lck tyrosine kinases: A specific role for p56lckN in neuron protection].''; PubMedEurope PMCScholia
Rohatgi R, Ho HY, Kirschner MW.; ''Mechanism of N-WASP activation by CDC42 and phosphatidylinositol 4, 5-bisphosphate.''; PubMedEurope PMCScholia
Meriane M, Tcherkezian J, Webber CA, Danek EI, Triki I, McFarlane S, Bloch-Gallego E, Lamarche-Vane N.; ''Phosphorylation of DCC by Fyn mediates Netrin-1 signaling in growth cone guidance.''; PubMedEurope PMCScholia
Myosin-X, an unconventional myosin implicated in cell adhesion and filopodia elongation interacts with DCC and Neogenin and helps in their distribution in neurites. Myosin-X functions to transfer cargo proteins into filopodia and its hypothesized that Myosin-X may deliver DCC to filopodia on Netrin-1 stimulation.
Among netrin1 receptors neogenin is the only protein to interact with the repulsive guidance molecules (RGM). RGMs are membrane bound proteins involved in axon guidance in the visual system. Neogenin is the dependence receptor and cleaved by activated caspase-3 to trigger apoptotic cell death. RGM binding blocks the cleavage of neogenin so RGM functions as a cell survival factor.
Siah-1 binds DCC and promotes its proteolysis via the ubiquitin-proteasome pathway. Siah-1 contains an N-terminal RING domain that is involved in proteolysis function and a C-terminal sequence that is involved in its oligomerization and binding to target proteins, such as DCC.
Netrin-1 promotes attraction of the commissural neurons to midline cells. It is secreted in the ventral midline (also known as the floor plate). The transmembrane DCC receptor is a Netrin-1 receptor, involved in the attractive effects of Netrin-1. Contact-dependent mechanisms promote extension of growth cones across the floor plate to the contralateral side, whereupon growth cones acquire sensitivity to the midline repellent Slit and grow away from the midline. Netrin-1 binds directly to the fifth Fibronectin III motif of DCC, thereby inducing DCC clustering through the association between the DCC P3 domains, a process required for an attractive response.
FADK1 interacts with the C-terminal P3 domain of DCC complexed with Netrin-1, and undergoes tyrosine phosphorylation and activation. Netrin-1-DCC binding thus leads to the autophosphorylation of tyrosine 393 in FADK1.
UNC-5 uses DCC as a co-receptor and binds to the DCC P1 domain with its DB domain to repel axons at low netrin concentration. It is generally thought that UNC5 receptor alone transduces short range signals whereas DCC-Unc5 complex transduces long range signals important for neuron migration, neurite growth and axon repulsion.
Multiple sites on Unc5c are phosphorylated after netrin-1 stimulation. An activated Src tyrosine kinase induces phosphorylation of Unc5c at multiple cytoplasmic tyrosine residues including highly-conserved residues 449, 454, 568, 649 and 667. Phosphorylation of these residues creates potential binding sites for cytoplasmic signaling proteins.
Netrin-1, through its activation of DCC, triggers TRPC channel mediating the Ca+2 influx that is required for the growth cone turning. The effect of netrin-1 on TRP currents in the neurons is studied in Xenopus. In cultured Xenopus spinal neurons, Netrin-1 evoked Ca+2 influx and a depolarizing, TRPC-like current in both soma and growth cones. Inhibition of the Xenopus homologue of mammalian TRPC1 (XTRPC1) prevented Ca+2 influx, TRPC-like current activation and the chemotropic turning of the growth cone in response to a gradient of Netrin-1. Netrin-1 receptor signalling to TRPC channels is mediated via hydrolysis of PIP2 by PLCgamma which then activates TRPC channel activity through IP3 and DAG.
The adaptor protein Nck-1 binds to DCC and recruits Rac-1, Cdc42 and their effectors PAK-1 and N-WASP to the activated receptor. Both Cdc42 and Nck-1 are activators of N-WASP. Cdc42 in its active GTP bound form binds to the CRIB domain of N-WASP and this interaction along with PIP2 results in the activation of N-WASP. Nck-1 activate N-WASP via binding of its SH3 domain to the proline rich domain of N-WASP. Nck-1 also possess an SH2 domain that associates directly with activated tyrosine kinase receptors which can phosphorylate N-WASP.
Netrin-1 is not only involved as an axon guidance cue during the development of nervous system but is also involved in the morphogenesis of the mammary glands. Netrin-1 acts as a short-range attractant and has an adhesive, rather than a guidance, function during mammary gland morphogenesis. In the developing mammary gland, netrin-1 acts locally through neogenin to maintain close apposition of cap cells and prelumenal cells at the leading edge of the TEB (Terminal end bud).
Activated (phosphorylated) FADK1 acts as a scaffold and recruits src tyrosine kinases Src and Fyn to DCC. These tyrosine kinases phosphorylate DCC which is critical for Netrin-1 signaling.
Netrin-1 stimulates phosphorylation of DCC on serine, threonine, and tyrosine residues. The experimental data suggest that tyrosine phosphorylation of DCC is a prerequisite step for DCC phosphorylation on serine and threonine residues. Fyn initiates the phosphorylation of the tyrosine residue 1420 in the DCC cytoplasmic domain. This phosphorylation of DCC in turn facilitates the DCC-Fyn interaction, forming a positive reinforcement cycle.
Netrin-1-DCC mediated signaling rapidly phosphorylates PLCgamma. Netrin-1 mediated PLC activation depends on recruitment of PITPalpha to DCC. Stimulation of PLC signaling and hydrolysis of PIP2 by netrin-1 in neurons is time-dependent, with a maximal activity observed within 15 min of netrin-1 stimulation.
When Netrin-1 binds to the DCC-NCK1 complex, a conformational change in NCK1 promotes interaction between the SH2 domain proteins, leading to recruitment and activation of RhoGEFs DOCK180 and Trio. These GEFs mediate Netrin-1 signaling in axon outgrowth and guidance through their ability to activate Rac1 and Cdc42.
RhoGEF complexed with Netrin-1-DCC induces guanine nucleotide exchange by Cdc42, activating it. Activated Cdc42 activates N-WASP, which promotes the nucleation of F-actin via the Arp2/3 complex. Netrin-1, via DCC, influences cellular motility by regulating actin-based membrane extension through the activation of Cdc42.
DCC and UNC5A, are also receptors for Netrin-4. The LNT domain of Netrin-4 is the key domain for this specific binding. Netrin-4 might also mediate attractive action through DCC and repulsive action through UNC5A.
Ezrin is a member of the ezrin/radixin/moesin (ERM) family that acts as a linker between the plasma membrane and the actin cytoskeleton. Ezrin exists in a dormant, monomeric form in which its FERM/NERMAD and C-ERMAD domains are associated, masking membrane and F-actin binding regions. On production of PIP2, ezrin binds it, is recruited to the plasma membrane, and undergoes conformational changes unmasking the two binding sites.
DCC interacts with the SH3/SH2 adaptor NCK1 in commissural neurons. This interaction is direct and requires the SH3 but not SH2 domains of NCK1. NCK1 can recruit Rac, Cdc42 and their effectors Pak and N-WASP to the activated receptor, thereby providing a direct link between DCC, Rho GTPases and numerous downstream signaling components that regulate the actin cytoskeleton.
DCC and Robo1 heterodimerize via conserved sequence elements in their cytoplasmic domains, namely CC1 (conserved cytoplasmic region1) in Robo1 and P3 in DCC. The formation of this complex is dependent on the previous interaction between Robo and its ligand (Slit). This physical interaction between Robo Slit and DCC silences the attractive effect of Netrin DCC and regulates the midline crossing of axons. From the analysis of multiple double mutant combinations of the Robo Slit and Netrin DCC receptors it was deduced that Robo repulsion on its own is sufficient to prevent commissural axons from recrossing the midline, and that Netrin DCC is not the only source of attraction at the midline.
Phosphorylated Ezrin can link in microfilaments to the plasma membrane by direct association with transmembrane proteins such as the cytoplasmic domain of DCC.
PIP2 hydrolysis appers to be an important mechanism for netrin-1 mediated neurite elongation. Netrin-1 alone could not elicit hydrolysis of PIP2 but depends on the stimulation of DCC and PLCgamma.
Shp2 is recruited to the phosphorylated Unc5C receptor after netrin stimulation, in a fashion that requires binding of the Shp2 SH2 domains to the Tyr568 phosphorylated motif. The functional significance of the Unc5C-shp2 interaction has not been reported .Shp2 might negatively regulate tyrosine phosphorylation of Unc5C receptor, facilitating resensitization of the receptor to the netrin signal.
DCC interacts directly with PITPalpha and this interaction is enhanced in the presence of Netrin-1. The interaction of DCC with PITPalpha requires the carboxy-terminal dmain of both the proteins. PITPalpha signaling pathway is important for netrin-1 mediated axon outgrowth. Netrin-1 activates PITPalpha to regulate local phosphoinositide (PI) synthesis, which is important for PI3K dependent neurite elongation.
Unc-115/AbLIM is a key regulator of lamellipodia and filopodia in the growth cone during axon pathfinding and acts downstream of Rac GTPase signaling. It acts as a scaffold to recruit other actin-modulating complexes involved in lamellipodia and filopodia. Unc-115/AbLIM is locally recruited to the plasma membrane by activated Rac1 and subsequently dephosphorylated on serine 617. Dephosphorylated Unc-115/AbLIM then might interact with other molecules at the plasma membrane to induce formation of lamellipodia and filopodia by reorganization of the actin cytoskeleton.
PIKE-L a brain-specific GTPase selectively associates with UNC5B but not with other family members of UNC5. Netrin-1 enhances this interaction and this interaction triggers the activation of PI3K kinase signalling, prevents UNC5B's pro-apoptotic activity and enhances neuronal survival.
The carboxy (C) terminal domain of FADK1 interacts with the C-terminal P3 domain of DCC. This FADK1-DCC interaction is required for Netrin-1 to stimulate tyrosine phosphorylation and activation of FADK1.
Original Pathway at Reactome: http://www.reactome.org/PathwayBrowser/#DB=gk_current&FOCUS_SPECIES_ID=48887&FOCUS_PATHWAY_ID=373752
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DataNodes
Robo
SlitDCC oligomer
NetrinDCC Nck Cdc42-GTP Active N-WASP
PIP2DCC
PITPDCC oligomer pFAK Fyn Nck-1
Rho GEFs DOCK/TrioDCC
UNC5CDCC pFyn Nck Rac1-GTP
AblimDCC
pUNC5CpDCC dimer pFAK
Src/FynDCC oligomer pFADK1
Fyn/srcpUnc5C DCC
Shp2DCC oligomer
pFADK1pDCC oligomer pFAK Fyn
NCK1Annotated Interactions
DCC oligomer
NetrinDCC
PITPDCC
PITPDCC oligomer pFAK Fyn Nck-1
Rho GEFs DOCK/TrioDCC oligomer pFAK Fyn Nck-1
Rho GEFs DOCK/TrioDCC
UNC5CDCC
pUNC5CDCC
pUNC5CpDCC dimer pFAK
Src/FynpDCC dimer pFAK
Src/FynDCC oligomer pFADK1
Fyn/srcDCC oligomer
pFADK1DCC oligomer
pFADK1pDCC oligomer pFAK Fyn
NCK1Netrin-1 binds directly to the fifth Fibronectin III motif of DCC, thereby inducing DCC clustering through the association between the DCC P3 domains, a process required for an attractive response.
Netrin-1 receptor signalling to TRPC channels is mediated via hydrolysis of PIP2 by PLCgamma which then activates TRPC channel activity through IP3 and DAG.
From the analysis of multiple double mutant combinations of the Robo Slit and Netrin DCC receptors it was deduced that Robo repulsion on its own is sufficient to prevent commissural axons from recrossing the midline, and that Netrin DCC is not the only source of attraction at the midline.