The chemotropic guidance cue netrin-1 promotes neurite outgrowth through its receptor Deleted in Colorectal Cancer (DCC) via activation of Rac1. The guanine nucleotide exchange factor (GEF) linking netrin-1/ DCC to Rac1 activation has not yet been identified. Here, we show that the RhoGEF Trio mediates Rac1 activation in netrin-1 signaling. We found that Trio interacts with the netrin-1 receptor DCC in mouse embryonic brains and that netrin-1-induced Rac1 activation in brain is impaired in the absence of Trio. Trio ؊/؊ cortical neurons fail to extend neurites in response to netrin-1, while they are able to respond to glutamate. Accordingly, netrin-1-induced commissural axon outgrowth is reduced in Trio ؊/؊ spinal cord explants, and the guidance of commissural axons toward the floor plate is affected by the absence of Trio. The anterior commissure is absent in Trio-null embryos, and netrin-1/DCC-dependent axonal projections that form the internal capsule and the corpus callosum are defective in the mutants. Taken together, these findings establish Trio as a GEF that mediates netrin-1 signaling in axon outgrowth and guidance through its ability to activate Rac1.During the development of the central nervous system, axons are guided to their targets in response to molecular cues that can be either membrane-bound factors or secreted molecules, acting over short or long distances. The neuronal growth cone is a specialized structure found at the tip of the axon that integrates attractive and repulsive signals elicited by these extracellular cues and responds to them by triggering signaling pathways that regulate growth cone motility (16,18). Netrins are a family of secreted proteins that control axon outgrowth and guidance in multiple vertebrate and invertebrate species (3). Netrin-1 is a bifunctional molecule that attracts and repels different classes of axons. In vertebrates, netrin-1 was first shown to attract commissural axons of the developing spinal cord toward the ventral midline (21, 44). Since then, netrin-1 has been shown to promote outgrowth of a wide variety of axons, including growing cortical axons (30, 41). Two families of netrin-1 receptors in mammals have been identified: the Deleted in Colorectal Cancer (DCC) family, comprising DCC and neogenin, and the UNC-5 family of proteins (1,20,25). DCC mediates growth cone attraction induced by netrin-1 (1, 20, 25, 43) whereas the repulsive effect of netrin-1 is mediated by the UNC-5 family of netrin receptors, alone or in combination with DCC (17,22,35).DCC is a transmembrane protein without any obvious catalytic activity in its intracellular domain, and for this reason, it was unclear until recently how the intracellular signaling machinery leading to axon outgrowth was initiated. This process has begun to be elucidated with the identification in cortical and commissural neurons of different DCC-binding proteins, including the protein tyrosine kinases focal adhesion kinase, Src, and Fyn; the Nck adaptor protein; and phosphatidylinositol transfer protein ␣ (26...
Background information. The large family of GEFs (guanine nucleotide-exchange factors) for Rho GTPases activate the GTPases by accelerating their GDP/GTP exchange. The multidomain protein Trio is the founding member of an intriguing subfamily of Rho-GEFs exhibiting two Rho-GEF and numerous additional domains. The members of the Trio family play an important role in neuronal physiology, and their structural organization is very well conserved through evolution. It has previously been shown that all the members, except mammalian Trio, display several isoforms, the functions of which have been well established.Results. In this study, we have identified, by a combination of different approaches, novel Trio isoforms that have been generated by alternative splicing, giving rise to proteins that exhibit one or two Rho-GEF domains (GEFDs). These isoforms are specifically expressed in the nervous system, at a higher level than the full-length Trio, which is ubiquitously expressed. In addition, we show that all the GEFD1-containing isoforms induce neurite outgrowth in neuroblastoma cells. Conclusions.We have identified neuronal specific isoforms of Trio which could be essential for Trio function in neuronal morphology. IntroductionRho GTPases are involved in numerous processes, such as proliferation, transformation, cell migration and morphogenesis, by their ability to direct the remodelling of the actin cytoskeleton in different cellular systems (Etienne-Manneville and Hall, 2002). GTPases are molecular switches that alternate between an inactive GDP-bound state and an active GTP-bound state. The large family of GEFs (guanine nucleotide-exchange factors) for Rho-GTPases activate the GTPases by accelerating the GDP/GTP exchange. Nearly all proteins with Rho-GEF activity contain a conserved catalytic region called the DH (Dbl homology) domain, adjacent to a PH (pleckstrin homology) domain (Schmidt and Hall, 2002).
Guanine nucleotide exchange factors for Rho-GTPases (Rho-GEFs) invariably share a catalytic Dbl-Homology (DH) domain associated with a Pleckstrin Homology (PH) domain, whose function in Rho-GEF activation is not well understood. Trio is the first member of an emerging family of Dbl proteins containing two Rho-GEF domains (GEFD1 and GEFD2). TrioGEFD1 activates the GTPases RhoG and Rac1, while TrioGEFD2 acts on RhoA. In this study, we have investigated the roles of the two PH domains of Trio in Rho-GEF activity. We show that TrioPH1 is required for GEFD1-mediated induction of actin cytoskeleton remodeling and JNK activation. TrioPH1 is involved both in the catalytic activity and in the subcellular localization of its associated DH domain, by acting as a cytoskeletal targeting signal. Moreover, TrioPH1 in association with DH2 activates the JNK pathway, by an unknown mechanism independent of DH2 catalytic activity. TrioPH2 does not behave as a targeting module in intact cells. TrioPH2 inhibits DH2-dependent stress fiber formation, which correlates with the TrioPH2-mediated inhibition of DH2 GEF activity. In addition, expression in the neuron-like PC12 cell line of the intact Trio protein deleted of each PH domain shows that only TrioPH1 is required for Trio-induced neurite outgrowth. Taken together, these data demonstrate that the two PH domains play a different role in the control of Trio Rho-GEF function.
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