Structures of netrin-1 bound to two receptors provide insight into its axon guidance mechanism.

Science (Impact Factor: 31.48). 06/2014; 344:1275-1279. DOI: 10.1126/science.1255149

ABSTRACT Netrins are secreted proteins that regulate axon guidance and neuronal migration. Deleted in colorectal cancer (DCC) is a well-established netrin-1 receptor mediating attractive responses. We provide evidence that its close relative neogenin is also a functional netrin-1 receptor that acts with DCC to mediate guidance in vivo. We determined the structures of a functional netrin-1 region, alone and in complexes with neogenin or DCC. Netrin-1 has a rigid elongated structure containing two receptor-binding sites at opposite ends through which it brings together receptor molecules. The ligand/receptor complexes reveal two distinct architectures: a 2:2 heterotetramer and a continuous ligand/receptor assembly. The differences result from different lengths of the linker connecting receptor domains fibronectin type III domain 4 (FN4) and FN5, which differs among DCC and neogenin splice variants, providing a basis for diverse signaling outcomes.

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    ABSTRACT: Netrin-1, acting through its principal receptor DCC (deleted in colorectal cancer), serves as an axon guidance cue during neural development, and also contributes to vascular morphogenesis, epithelial migration, and the pathogenesis of some tumors. Several lines of evidence suggest that netrin-DCC signaling can regulate, and be regulated by, the cAMP-dependent protein kinase, PKA, although the molecular details of this relationship are poorly understood. Specificity in PKA signaling is often achieved through differential subcellular localization of the enzyme by interaction with A-kinase anchoring proteins (AKAPs). Here, we show that AKAP function is required for DCC-mediated activation of PKA and phosphorylation of cytoskeletal regulatory proteins of the Mena/VASP (vasodilator-stimulated phosphoprotein) family. Moreover, we show that DCC and PKA physically interact and that this association is mediated by the Ezrin-Radixin-Moesin (ERM) family of plasma membrane-actin cytoskeleton cross-linking proteins. Silencing of ERM protein expression inhibits DCC-PKA interaction, DCC-mediated PKA activation and phosphorylation of Mena/VASP proteins, as well as growth cone morphology and neurite outgrowth. Finally, while expression of wild-type radixin partially rescued growth cone morphology and tropism towards netrin in ERM-knockdown cells, expression of an AKAP-deficient mutant of radixin did not fully rescue growth cone morphology and switched netrin tropism from attraction to repulsion. These data support a model in which ERM-mediated anchoring of PKA activity to DCC is required for proper netrin/DCC-mediated signaling. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 01/2015; DOI:10.1074/jbc.M114.628644 · 4.60 Impact Factor
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    ABSTRACT: Development of neuronal circuits is controlled by evolutionarily conserved axon guidance molecules, including Slits, the repulsive ligands for roundabout (Robo) receptors, and Netrin-1, which mediates attraction through the DCC receptor. We discovered that the Robo3 receptor fundamentally changed its mechanism of action during mammalian evolution. Unlike other Robo receptors, mammalian Robo3 is not a high-affinity receptor for Slits because of specific substitutions in the first immunoglobulin domain. Instead, Netrin-1 selectively triggers phosphorylation of mammalian Robo3 via Src kinases. Robo3 does not bind Netrin-1 directly but interacts with DCC. Netrin-1 fails to attract pontine neurons lacking Robo3, and attraction can be restored in Robo3(-/-) mice by expression of mammalian, but not nonmammalian, Robo3. We propose that Robo3 evolution was key to sculpting the mammalian brain by converting a receptor for Slit repulsion into one that both silences Slit repulsion and potentiates Netrin attraction. Copyright © 2014 Elsevier Inc. All rights reserved.
    Neuron 11/2014; 84(6). DOI:10.1016/j.neuron.2014.11.004 · 15.98 Impact Factor
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    ABSTRACT: Angiogenesis, a process that newly-formed blood vessels sprout from pre-existing ones, is vital for vertebrate development and adult homeostasis. Previous studies have demonstrated that the neuronal guidance molecule netrin-1 participates in angiogenesis and morphogenesis of the vascular system. Netrin-1 exhibits dual activities in angiogenesis: either promoting or inhibiting angiogenesis. The anti-angiogenic activity of netrin-1 is mediated by UNC5B receptor. However, how netrin-1 promotes angiogenesis remained unclear. Here we report that CD146, an endothelial transmembrane protein of the immunoglobulin superfamily, is a receptor for netrin-1. Netrin-1 binds to CD146 with high affinity, inducing endothelial cell activation and downstream signaling in a CD146-dependent manner. Conditional knockout of the cd146 gene in the murine endothelium or disruption of netrin-CD146 interaction by a specific anti-CD146 antibody blocks or reduces netrin-1-induced angiogenesis. In zebrafish embryos, downregulating either netrin-1a or CD146 results in vascular defects with striking similarity. Moreover, knocking down CD146 blocks ectopic vascular sprouting induced by netrin-1 overexpression. Together, our data uncover CD146 as a previously unknown receptor for netrin-1 and also reveal a functional ligand for CD146 in angiogenesis, demonstrating the involvement of netrin-CD146 signaling in angiogenesis during vertebrate development.Cell Research advance online publication 6 February 2015; doi:10.1038/cr.2015.15.
    Cell Research 02/2015; 25(3). DOI:10.1038/cr.2015.15 · 11.98 Impact Factor


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Jul 8, 2014

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