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.

Download full-text


Available from: Nicolas Renier, Jul 08, 2014
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Netrin-1 is a guidance cue that can trigger either attraction or repulsion effects on migrating axons of neurons, depending on the repertoire of receptors available on the growth cone. How a single chemotropic molecule can act in such contradictory ways has long been a puzzle at the molecular level. Here we present the crystal structure of netrin-1 in complex with the Deleted in Colorectal Cancer (DCC) receptor. We show that one netrin-1 molecule can simultaneously bind to two DCC molecules through a DCC-specific site and through a unique generic receptor binding site, where sulfate ions staple together positively charged patches on both DCC and netrin-1. Furthermore, we demonstrate that UNC5A can replace DCC on the generic receptor binding site to switch the response from attraction to repulsion. We propose that the modularity of binding allows for the association of other netrin receptors at the generic binding site, eliciting alternative turning responses.
    Neuron 08/2014; 83(4). DOI:10.1016/j.neuron.2014.07.010 · 15.98 Impact Factor
  • [Show abstract] [Hide abstract]
    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
  • [Show abstract] [Hide abstract]
    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; 290(9). DOI:10.1074/jbc.M114.628644 · 4.60 Impact Factor