Human Trio was identified in the late 1990s in a two-hybrid screen using the cytoplasmic fragment of the transmembrane tyrosine phosphatase LAR as a bait (Debant et al. 1996). The name of Trio came from the observation that Trio comprises three enzymatic domains: two GEF domains predicted to activate Rho GTPases and one serine kinase domain. It was rapidly shown that both GEF domains are active, the GEFD1 activating Rac1/RhoG and the GEFD2 domain acting on RhoA. While the function of Trio was unclear at that time, the first indication of its function came from studies in C. elegans, when it was discovered that UNC-73, a major regulator of axon guidance and cell motility, was the ortholog of mammalian Trio (Steven et al. 1998). Two years later, the Drosophila trio gene was shown by different groups to be essential for axon guidance via the activation of Rac through its first GEF domain (Bateman and Van Vactor 2001). Since then, Trio has been established as a major regulator of neuronal development in mammals by activating the GTPase Rac1. Subsequent studies showed that Trio also plays a central role in cell adhesion, in the mitogenic response mediated by Gαq protein-coupled receptors, and in cell division, revealing that the function of Trio is not restricted to the nervous system. The prominent role of Trio in cellular physiology suggested that deregulation of its activity could participate to human pathology. Indeed, recent studies support a direct role of the deregulation of Trio’s function in cancer and in neurodevelopmental diseases.
The Trio RhoGEF Family
The small GTPases of the Rho family control a variety of cellular processes, including actin cytoskeleton remodeling, microtubule dynamics, gene transcription, and phospholipid metabolism. Thus, it is not surprising that Rho GTPases are involved in a wide range of major cellular functions such as cell polarity, adhesion and motility, proliferation, and differentiation. The GTPases oscillate between an inactive GDP-bound state and an active GTP-bound state. This oscillation is controlled by three types of regulators: (i) Rho guanine nucleotide exchange factors (GEF) activate Rho GTPases by accelerating the exchange of GDP for GTP; (ii) GTPase-activating proteins (GAP) inactivate the GTPases by promoting the hydrolysis of GTP; (iii) finally, guanine dissociation inhibitors (GDI) sequester the GTPases in the cytosol before they are targeted to the membrane. Once active, Rho GTPases interact and activate downstream effectors of actin cytoskeleton remodeling.
The RhoGEF Trio belongs to the Dbl family of GEFs, which all share a catalytic domain, called Dbl-homology (DH) domain in reference to Dbl, one of the first GEFs identified in mammalian cells, and a pleckstrin homology (PH) domain, which plays a role in GEF activation and localization. Trio contains two GEF domains of different specificity, the GEFD1 domain activating RhoG and Rac1 and the GEFD2 specifically acting on RhoA (Schmidt and Debant 2014). Trio also harbors a kinase domain, two SH3 motifs, a CRAL-Trio/Sec14 motif, an Ig-like motif, and several spectrin-like repeats. In addition to its complex modular structure, the trio gene encodes several isoforms containing one or the two GEF domains as a result of alternative splicing. The TrioA–E isoforms are specifically expressed in the nervous system, while Tgat, carrying only the RhoA-specific GEF domain, was isolated from ATL (adult T-cell leukemia) patients and triggers tumor formation of xenografts in nude mice (Schmidt and Debant 2014).
Functions of Trio
Trio and Neuronal Physiology
The functions of the Trio family proteins in neuronal physiology are conserved across evolution. Both Trio orthologs UNC-73 in C. elegans and Drosophila D-Trio play a major role in neuronal cell motility and axon guidance, and Kalirin is involved in synapse physiology and in the secretion of neuropeptides in mammals. The function of the trio gene as a major regulator of brain development in mammals has been established, thanks to several studies using gene targeting technologies in mice (Schmidt and Debant 2014). The total knockout of the trio gene in mice is embryonic lethal with embryos presenting defects in brain organization. Mice in which trio has been specifically deleted in the nervous system die perinatally, and the few mice that survive display defects in the migration of cerebellar granule cells and have severe ataxia. Finally, deletion of trio in the hippocampus and in the cortex during early embryogenesis results in aberrant organization of these structures, impairing the learning ability of these mice (Zong et al. 2015). All together, these in vivo data show that disruption of the functions of Trio results in multiple neurodevelopmental defects, which are not compensated by the other member of the family, Kalirin.
Mammalian Trio was first described to induce neurite outgrowth in response to nerve growth factor (NGF) in PC12 cells by activating RhoG through its GEFD1 domain (Estrach et al. 2002). In addition, it was reported that Trio binds the integral membrane protein KidIns220/ARMS, a downstream target of neurotrophins (Neubrand et al. 2010). This interaction has been proposed to target Trio to specific membrane sites and to regulate its activity, leading to Rac1 activation and neurite outgrowth.
The function of Trio in axon outgrowth and guidance is also regulated by the schizophrenia susceptibility gene disrupted-in-schizophrenia 1 (DISC1), which plays a major role in various aspects of neuronal development. DISC1 is involved in axon guidance in C. elegans through a Trio/Rac/PAK pathway. DISC1 binds to the spectrin repeats of Trio, which play a role in inhibiting Trio activity toward Rac1. It has been proposed that the binding of DISC1 to this region relieves an auto-inhibitory constraint, thereby facilitating Rac1 recruitment and activation by Trio (Schmidt and Debant 2014).
Until recently, most studies about the functions of Trio in neuronal physiology had focused on its role in axon outgrowth and guidance. Little was known about the function of Trio in synaptogenesis. In contrast, the role of the Kalirin-7 isoform in synaptic maintenance is well established, pointing to a role for Kalirin in the structural changes in dendritic spines accompanying a form of synaptic plasticity, called long-term potentiation (LTP). However, LTP is normal in the hippocampus of Kalirin k.o. mice, suggesting that another GEF is functionally redundant. Based on these observations, Herring and Nicoll investigated the potential functional redundancy of Trio and Kalirin during synaptogenesis. They have shown that both Kalirin and Trio positively regulate the function of excitatory synapses and play redundant roles during the LTP process (Herring and Nicoll 2016). However, the exact function of Trio in synapse physiology is still on debate, as a concomitant study proposed that Trio negatively regulates hippocampal synaptic strength by specifically affecting the endocytosis of the postsynaptic AMPA-type glutamate receptors (Ba et al. 2016). It will be important to clarify the exact role of Trio in synaptogenesis in light of the recent discovery that Trio could be a novel risk gene for neurological disorders (see Trio in pathologies section).
Trio and Cell Adhesion
Trio also participates to integrin-mediated cell adhesion processes such as leukocyte transendothelial migration (TEM). TEM is a process in which the binding of leukocytic ß2-integrin to the endothelial adhesion molecule ICAM-1 triggers endothelial signaling. It has been shown that ICAM-1 binds to and activates Trio, resulting in Rac1 and RhoG activation and the formation of endothelial docking structures (Van Rijssel et al. 2012). In addition, Trio activation upon ICAM-1 clustering requires the presence of another Trio partner, the actin cross-linker filamin A. From these data, the authors proposed that clustering of ICAM-1 induces the recruitment of filamin A, which in turn acts as a scaffold for subsequent activation of Trio, leading to actin cytoskeleton remodeling in endothelial cells.
Interestingly, Trio also contributes to the process of adhesion by controlling the expression of adhesive receptors at the transcriptional level through Rac1 activation. For example, in addition to being downstream of ICAM-1 signaling, Trio has been proposed to impinge on ICAM expression. The TNFα pathway induces the expression of adhesion molecules at the surface of the endothelium that promote leukocyte recruitment during inflammation. The TNFα pathway activates Trio, leading to Rac1-mediated activation of the transcription factor Ets2, which induces an increase in ICAM-1 expression (Van Rijssel et al. 2013). The second example refers to the effect of Trio on E-cadherin expression in epithelial cells. It has been shown that Trio-mediated Rac1 signaling induces the phosphorylation of the transcription repressor Tbx3, leading to a decrease in E-cadherin expression (Yano et al. 2011). The effect of Trio on E-cadherin expression is inhibited by the interaction of Trio with Trio-associated repeat on actin (Tara), a protein described as a F-actin-binding protein important for cell spreading. The molecular mechanisms by which Tara inhibits Trio-mediated Rac1 activation are still unknown (Fig. 4).
Trio and Cell Cycle Regulation and Mitogenic Signaling
During cytokinesis, cells undergo dramatic changes in shape and adhesion that depend on efficient actin cytoskeleton remodeling. Rac1 has to be inactivated by the GAP MgcRacGAP at the division plane for cytokinesis to occur. Until recently, the GEF controlling Rac1 in this process was not known. A recent study showed that Trio controls Rac1 activation in dividing cells and that Trio counteracts Rac1 inactivation by MgcRacGAP in cytokinesis, highlighting an unexpected role of Trio in the control of the cell cycle (Cannet et al. 2014).
Trio in Pathologies
Due to the central role of Trio in cell adhesion, motility, and cell division, a number of studies have initially investigated the impact of Trio on cancer progression. Indeed, many studies have shown that upregulation of Trio expression is associated with tumor progression and/or metastasis in different types of cancers and with poor patients’ survival (Schmidt and Debant 2014). Trio plays a role in the capacity of glioblastoma cells to invade in an ex vivo organotypic rat brain slice model and also participates to the invasion triggered by the TNF-like weak inducer of apoptosis (TWEAK) cytokine. The signaling pathways by which Trio triggers cancer progression often involve Rac1 activation and actin cytoskeleton remodeling. Tumor invasion requires the formation of actin-based protrusions called invadopodia in order to locally secrete metalloproteinases required for the degradation of the extracellular matrix. A recent study has shown that the Trio/Rac1/PAK pathway drives invadopodia disassembly, which has been proposed to be critical for a good balance between matrix degradation and cell motility (Moshfegh et al. 2015).
In addition to the upregulation of Trio expression, other mechanisms for the deregulation of Trio activity have been described, such as aberrant alternative splicing or translocation of the trio gene. For example, a screen designed to identify novel oncogenes that could participate to the malignant progression of adult T-cell leukemia (ATL) identified Tgat, an alternative splice variant of Trio harboring only the catalytic DH2 domain that activates RhoA (Schmidt and Debant 2014). Overexpression of Tgat induced cell transformation and tumor formation in nude mice. Tgat has been proposed to enhance tumor invasion by stimulating matrix metalloproteinases (MMPs) via the RECK protein and by activating the transcription factor NF-kappaB, which plays a crucial role in tumorigenesis, including ATL. However, the function of Tgat in the progression of this malignancy remains to be determined. Another example is the identification of the translocation of the trio gene in different sarcoma histiotypes. One translocation involved a chimeric transcript between the trio and the telomerase reverse transcriptase (tert) genes, leading to potential fusion proteins between Trio and TERT (Delespaul et al. 2017). TERT is an essential component of the complex controlling the length of the telomeres. The presence of these fusion transcripts does not trigger TERT reactivation in these tumors. Interestingly, specific knockdown of the Trio-TERT transcript significantly reduces cell proliferation. However, the functional consequences of the expression of these fusion proteins in tumor progression are not well understood yet.
Trio is an essential gene for the development of the mammalian nervous system, suggesting that deregulation of its activity could be involved in neurodevelopmental diseases, such as intellectual disability (ID) or autism spectrum disorders (ASD). These diseases are characterized by an extreme genetic heterogeneity. Thanks to advances in next-generation sequencing technologies, de novo mutations in coding sequences associated to ID and ASD have recently been discovered. Interestingly, whole-exome sequencing studies have identified several deleterious de novo mutations in the trio gene in ID and ASD patients (De Rubeis et al. 2014). It is noteworthy that the trio gene has been predicted to be intolerant to functional genetic variations. The trio gene has also been associated to developmental disorders together with 11 novel genes. Combined, these data suggest that trio is a novel risk gene for neurological disorders. Interestingly, some of these de novo mutations associated with ID and microcephaly affect Rac1 activation by Trio (Pengelly et al. 2016). It remains to be determined what the consequences of these mutations are on neuronal development and brain function.
The functions and the mechanisms of regulation of Trio have been extensively studied in the last two decades, but some important questions remain unanswered.
Trio is a major regulator of a variety of processes such as neuronal development, cell adhesion and motility, cell division, and proliferation. The involvement of Trio in these processes relies mainly on Rac1 activation, with the exception of the participation of Trio to the mitogenic response mediated by Gαq protein-coupled receptors, where both Rac1 and RhoA are required. It remains to be determined whether other processes involve the activation of RhoA by Trio. Interestingly, a recent study proposed that the Trio GEF domains might activate more GTPases than initially described (Peurois et al. 2017). The authors showed that the presence of lipidic membranes very efficiently stimulated the GEF activity of Trio in vitro and uncovered a previously unknown broader specificity toward Cdc42 that was undetectable in solution without the presence of membranes. This finding opens new perspectives for the study of Trio’s functions, but it remains to be determined whether Cdc42 is an in vivo target of Trio.
Until recently, Trio was best known for its role in actin cytoskeleton remodeling via the regulation of Rho GTPases. The recent discovery of an unexpected link between Trio and proteins related to microtubule dynamics represents a breakthrough in the understanding of Trio’s functions and opens up new perspectives in the field. The importance of this link has been demonstrated for the function of Trio in neurite outgrowth. It is very likely that the MT-binding ability of Trio will also turn out to be important in other physiological processes where the coordination between actin and MT cytoskeletons is essential, such as cell division, cell motility, or adhesion.
Finally, the contribution of Trio in pathologies is still an outstanding question. Given the pleiotropic effects of Trio, it is difficult to assess whether the global developmental delay presented by ID or ASD patients harboring mutations in the trio gene is due to a defect in neuronal migration, axon guidance, synaptogenesis, or even cell division. The generation of mutant trio knock-in models mimicking these neurodevelopmental diseases will help to address these questions.
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