Trio's Rho-specific GEF domain is the missing Gαq effector in C. elegans

The Gαq pathway is essential for animal life and is a central pathway for driving locomotion, egg laying, and growth in Caenorhabditis elegans, where it exerts its effects through EGL-8 (phospholipase Cβ [PLCβ]) and at least one other effector. To find the missing effector, forward genetic screens were performed to suppress the slow growth and hyperactive behaviors of mutants with an overactive Gαq pathway. Four suppressor mutations disrupted the Rho-specific guanine-nucleotide exchange factor (GEF) domain of UNC-73 (Trio). The mutations produce defects in neuronal function, but not neuronal development, that cause sluggish locomotion similar to animals lacking EGL-8 (PLCβ). Strains containing null mutations in both EGL-8 (PLCβ) and UNC-73 (Trio RhoGEF) have strong synthetic phenotypes that phenocopy the arrested growth and near-complete paralysis of Gαq-null mutants. Using cell-based and biochemical assays, it was shown that activated C. elegansq synergizes with Trio RhoGEF to activate RhoA. Activated Gαq and Trio RhoGEF appear to be part of a signaling complex, because they coimmunoprecipitate when expressed together in cells. These results show that Trio's Rho-specific GEF domain is a major Gαq effector that, together with PLCβ, mediates the Gαq signaling that drives the locomotion, egg laying, and growth of the animal (Williams, 2007).

The Gαq pathway is one of the major routes through which receptor-generated signals affect the state and function of neurons and other cells. Like other members of the family of heterotrimeric G proteins, Gαq becomes activated when a ligand, such as a neurotransmitter, binds to a Gαq-coupled receptor. This causes the receptor to act as a guanine-nucleotide exchange factor (GEF) that converts Gαq from its inactive GDP-bound state into its active GTP-bound state and facilitates its dissociation from the βγ subunits of the G protein (Williams, 2007).

In their activated state, the Gα and βγ subunits activate specific effector proteins that ultimately change the state of the cell. Thus, to understand how a G protein pathway affects a cell’s functional state, it is crucial to identify all of the effector proteins through which the G protein acts. Early pioneering studies of the Gαq pathway revealed that phospholipase Cβ (PLCβ) is an important Gαq effector protein. Gαq-activated PLCβ cleaves the lipid PIP2 into the small signaling molecules DAG and IP3. More recently, there has been an emerging body of evidence definitively linking Gαq-coupled receptors and activated Gαq to Rho activation, independent of effects on PLCβ. However, the biological significance of this newly discovered Gαq effector pathway has not been determined, thus leaving fundamental questions unanswered. For example, what is the relative importance of each effector pathway in the total scheme of Gαq signaling? Are both pathways active in the same cells at the same time, or are they specialized for different cell types or responses? Given the key role of Gαq signaling in many different cell types, these are important questions to answer (Williams, 2007).

Since the Gαq pathway is highly conserved in all animals (~82% identical between Caenorhabditis elegans and humans), genetic studies in model organisms provide a way to address the above questions. In C. elegans neurons, a synaptic signaling network of heterotrimeric G protein pathways, including a core Gαq pathway, controls synaptic activity to produce behaviors such as locomotion and egg laying. Under standard C. elegans culture conditions, animals lacking a Gαq pathway exhibit a larval growth arrest and strong paralysis that can be acutely rescued to wild-type levels of locomotion by applying phorbol esters, which are DAG analogs. Gαq reduction-of-function mutants survive to become sluggish or paralyzed egg-laying-defective adults with impaired neurotransmitter release (Williams, 2007).

Past genetic studies have shown that the C. elegansq ortholog, known as EGL-30 for its egg-laying-defective phenotype, exerts its effects in part through EGL-8, which is the only neuronal PLCβ ortholog in C. elegans. However, these studies all inferred at least one other Gαq effector pathway based on the finding that a Gαq-null mutant is much more impaired for locomotion, egg laying, and growth than a mutant lacking EGL-8 (PLCβ). In this study, two forward genetic screen strategies strategies were used to identify candidates for the missing Gαq effector. In so doing, mutations were recovered that disrupt the Rho-specific GEF domain of UNC-73 (Trio). Through complementary genetic, biochemical, and cell-based approaches, it was shown that Trio's Rho-specific GEF domain is a major Gαq effector that, together with PLCβ, mediates the Gαq signaling that drives the locomotion, egg laying, and growth of the animal. These results provide the first insights into the relative importance of the RhoGEF and PLCβ Gαq effector pathways in the context of a living animal (Williams, 2007).

C. elegans CARMIL negatively regulates UNC-73/Trio function during neuronal development

Whereas many molecules that promote cell and axonal growth cone migrations have been identified, few are known to inhibit these processes. In genetic screens designed to identify molecules that negatively regulate such migrations, CRML-1,the C. elegans homolog of CARMIL, also known as Lrrc16a, an actin-uncapping protein, was identified. Although mammalian CARMIL acts to promote the migration of glioblastoma cells, this study found that CRML-1 acts as a negative regulator of neuronal cell and axon growth cone migrations. Genetic evidence indicates that CRML-1 regulates these migrations by inhibiting the Rac GEF activity of UNC-73, a homolog of the Rac and Rho GEF Trio. The antagonistic effects of CRML-1 and UNC-73 can control the direction of growth cone migration by regulating the levels of the SAX-3 (a Robo homolog) guidance receptor. Consistent with the hypothesis that CRML-1 negatively regulates UNC-73 activity, these two proteins form a complex in vivo. Based on these observations, a role is proposed for CRML-1 as a novel regulator of cell and axon migrations that acts through inhibition of Rac signaling (Vanderzalm, 2009).

Cloning, structure and expression of Trio homologs

Although the integral membrane proteins that catalyze steps in the biosynthesis of neuroendocrine peptides are known to contain routing information in their cytosolic domains, the proteins recognizing this routing information are not known. Using the yeast two-hybrid system, P-CIP10 has been identified as a protein interacting with the cytosolic routing determinants of peptidylglycine alpha-amidating monooxygenase (PAM). P-CIP10 is a 217-kDa cytosolic protein with nine spectrin-like repeats and adjacent Dbl homology and pleckstrin homology domains typical of GDP/GTP exchange factors. In the adult rat, expression of P-CIP10 is most prevalent in the brain. Corticotrope tumor cells stably expressing P-CIP10 and PAM produce longer and more highly branched neuritic processes than nontransfected cells or cells expressing only PAM. The turnover of newly synthesized PAM is accelerated in cells co-expressing P-CIP10. P-CIP10 binds to selected members of the Rho subfamily of small GTP binding proteins (Rac1, but not RhoA or Cdc42). P-CIP10 (kalirin), a member of the Dbl family of proteins, may serve as part of a signal transduction system linking the catalytic domains of PAM in the lumen of the secretory pathway to cytosolic factors regulating the cytoskeleton and signal transduction pathways (Alam, 1997).

Huntington's disease (HD) occurs when the widely expressed protein huntingtin contains an expanded glutamine repeat. The selective degeneration and neuronal morphologic abnormalities of HD may involve interactions with proteins that bind to huntingtin, such as HAP1. The biological significance of this interaction is unclear because neither HAP1 nor huntingtin have significant homology to known proteins. Therefore, HAP1-binding proteins were sought. Using the yeast two-hybrid system, a rat cDNA encoding part of a protein that interacts with HAP1 was isolated, the specificity of this interaction was confirmed using an in vitro protein-binding assay. The protein is called Duo because it is closely related to the human protein Trio but is shorter. Northern blot analysis indicates brain-specific expression of Duo. Human Duo contains a guanine nucleotide exchange factor (GEF) domain that is likely to be rac1-specific, a pleckstrin homology (PH) domain and spectrin-like repeat units. These data support the hypothesis that huntingtin is involved in vesicle trafficking and cytoskeletal functions, and raise the possibility of a role for huntingtin in the regulation of a ras-related signaling pathway (Colomer, 1997).

unc-73 is required for cell migrations and axon guidance in C. elegans and encodes overlapping isoforms of 283 and 189 kDa that are closely related to the vertebrate Trio and Kalirin proteins, respectively. UNC-73A contains, in order, eight spectrin-like repeats, a Dbl/Pleckstrin homology (DH/PH) element, an SH3-like domain, a second DH/PH element, an immunoglobulin domain, and a fibronectin type III domain. UNC-73B terminates just downstream of the SH3-like domain. The first DH/PH element specifically activates the Rac GTPase in vitro and stimulates actin polymerization when expressed in Rat2 cells. Both functions are eliminated by introducing the S1216F mutation of unc-73(rh40) into this DH domain. These results suggest that UNC-73 acts cell autonomously in a protein complex to regulate actin dynamics during cell and growth cone migrations (Steven, 1998).

In both Caenorhabditis elegans and Drosophila, UNC-73/Trio functions in axon guidance by signaling through the Rac GTPase to regulate cytoskeletal rearrangements necessary for growth cone migrations. unc-73 encodes proteins with several domains, including two tandem RhoGEF and PH domain combinations, a Sec14p motif, eight spectrin-like repeats, a variant SH3 domain, an immunoglobulin domain (Ig), and a fibronectin type III (FnIII) domain. The N-terminal UNC-73 RhoGEF-1 domain specifically activates the Rac family GTPases CED-10 and MIG-2 in vitro, while the C-terminal RhoGEF-2 domain is specific to Rho. The complex C. elegans unc-73 gene encodes at least eight differentially expressed UNC-73 intracellular protein isoforms. Previously reported mutations affecting UNC-73 isoforms encoding the Rac-specific RhoGEF-1 domain cause uncoordinated movement, correlating with defects in axon guidance. Mutations in isoforms encoding the Rho-specific RhoGEF-2 domain, which are describe in this study, result in L1 stage larval lethality with no associated axon guidance defects. Isoform-specific rescue experiments reveal separate functions for the various RhoGEF-2-containing UNC-73 isoforms, which would not likely be discovered by conventional genetic screening. UNC-73 D1 and D2 appear to function redundantly in pharynx muscle to regulate the rate and strength of pharynx pumping, and in the HSN neurons and vulval muscles to control egg laying. Isoforms C1, C2, E, and F act redundantly within the nervous system to regulate the speed of locomotion. The multiple UNC-73 isoforms containing Rac- and Rho-specific RhoGEF domains therefore have distinct physiological functions. In addition to its previously identified role involving RhoGEF-1 in migrating cells and growth cones, these data indicate that UNC-73 signals through RhoGEF-2 to regulate pharynx and vulva musculature and to modulate synaptic neurotransmission (Steven, 2005).

Guanine nucleotide exchange factors for the Rho family of GTPases contain a Dbl homology (DH) domain responsible for catalysis and a pleckstrin homology (PH) domain whose function is unknown. A description is given of the solution structure of the N-terminal DH domain of Trio that catalyzes nucleotide exchange for Rac1. The all-alpha-helical protein has a very different structure compared to other exchange factors. Based on site-directed mutagenesis, functionally important residues of the DH domain were identified. They are all highly conserved and reside in close proximity on two alpha helices. In addition, a unique capability of the PH domain to enhance nucleotide exchange in DH domain-containing proteins has been discovered (Liu, 1998).

Mammalian Trio is a multifunctional, multidomain Rho guanine nucleotide exchange factor (GEF) closely related to Kalirin. Trio is important for proper axon guidance in Drosophila, and mice lacking Trio exhibit both skeletal muscle and neuronal disorders. Full length mammalian Trio and Kalirin both consist of a Sec14P-like domain, several spectrin-like domains, two Rho GEF domains each containing a Dbl-homology (DH) and a pleckstrin-homology (PH) domain, two src homology 3 domains (SH3), Ig/fibronectin-like domains (Ig/FN), and a kinase domain. Multiple isoforms of Kalirin derived through alternative splicing and multiple transcription start sites have been described, but multiple isoforms of Trio containing different functional domains have not yet been described. Using a new antibody directed against the spectrin-like region of rat Trio coupled with reverse transcription PCR and cDNA sequencing, four novel isoforms of Trio expressed in rat cortex and cerebellum have been identifed. Two isoforms, Trio 9S and Trio 9L, are derived through alternative splicing of Trio exon 48 and are abundantly expressed in rat brain. Trio 8 is expressed in postnatal day 30 and adult cerebellum, but not in cortex or skeletal muscle. Trio/duet is expressed in adult cortex and cerebellum. In the rat brain, each of these Trio isoforms is expressed at a higher level than full length Trio (McPherson, 2005).

Trio, a member of the Dbl family of guanine nucleotide exchange factors (GEFs), has a series of spectrin repeats, two GEF domains, protein interaction domains, and a putative kinase domain, potentially important in neuronal axon guidance and cell migration. Most knowledge about Trio is based on studies of Caenorhabditis elegans and Drosophila, while the function of Trio in vertebrates is unclear. The aim of these experiments was to establish the patterns of Trio expression in the postnatal rat brain. During postnatal development, high levels of Trio mRNA are found in the cerebral cortex, hippocampus, thalamus, caudate/putamen, and olfactory bulb, with lower levels in the septal nucleus, nucleus accumbens, amygdala, and hypothalamus. Except for the cerebellum, Trio mRNA in major brain areas is highest at P1, decreasing gradually during development, with low but detectable levels at P30. In P14 cerebral cortex, pyramidal neurons with strongly staining soma and dendrites are observed primarily in layer 5. In hippocampus, strong staining is observed in pyramidal neurons, granule cells, and isolated interneurons. Cerebellar Purkinje neurons exhibit intense staining in the soma and in extensive dendritic arbors at P7 and P14. Levels of Trio mRNA and the intensity of Trio immunostaining in cerebellar Purkinje cells increase from P1 to P30. Consistent with the in situ hybridization pattern, Western blot analyses show that Trio levels in the hippocampus and cortex are high at P1, decreasing until P30. The data suggest that Trio plays an important role during neuronal development (Ma, 2005).

Functional analysis of Trio homologs

The C. elegans genome contains three rac-like genes: ced-10, mig-2, and rac-2. ced-10, mig-2 and rac-2 act redundantly in axon pathfinding: inactivating one gene has little effect, but inactivating two or more genes perturbes both axon outgrowth and guidance. mig-2 and ced-10 also have redundant functions in some cell migrations. By contrast, ced-10 is uniquely required for cell-corpse phagocytosis, and mig-2 and rac-2 have only subtle roles in this process. Rac activators are also used differentially. The UNC-73 Trio Rac GTP exchange factor affects all Rac pathways in axon pathfinding and cell migration but does not affect cell-corpse phagocytosis. CED-5 DOCK180, which acts with CED-10 Rac in cell-corpse phagocytosis, acts with MIG-2 but not CED-10 in axon pathfinding. Thus, distinct regulatory proteins modulate Rac activation and function in different developmental processes (Lundquist, 2001).

Vulval development in the nematode C. elegans can be divided into a fate specification phase controlled in part by let-60 Ras, and a fate execution phase involving stereotypical patterns of cell division and migration controlled in part by lin-17 Frizzled. Since the small GTPase Rac has been implicated as a downstream target of both Ras and Frizzled and influences cytoskeletal dynamics, the role of Rac signaling during each phase of vulval development was investigated. The Rac gene ced-10 and the Rac-related gene mig-2 are redundantly required for the proper orientation of certain vulval cell divisions, suggesting a role in spindle positioning. ced-10 Rac and mig-2 are also redundantly required for vulval cell migrations and play a minor role in vulval fate specification. Constitutively active and dominant-negative mutant forms of mig-2 cause vulval defects that are very similar to those seen in ced-10;mig-2 double loss-of-function mutants, indicating that they interfere with the functions of both ced-10 Rac and mig-2. Mutations in unc-73 (a Trio-like guanine nucleotide exchange factor) cause similar vulval defects, suggesting that UNC-73 is an exchange factor for both CED-10 and MIG-2. The similarities and differences between the cellular defects seen in Rac mutants and let-60 Ras or lin-17 Frizzled mutants are discussed (Kishore, 2002).

Although the pathways through which Rac controls different cellular processes in vivo are still poorly defined, a large number of candidate Rac regulators and targets have been identified biochemically. These observations raise the question of whether Rac signals through multiple (perhaps redundant) pathways concomitantly, or whether Rac signals through different pathways in different cells to control different biological processes. A comparison of different Rac-dependent processes in C. elegans seems to support the latter model. For example, UNC-73 Trio is required for CED-10- and MIG-2-mediated vulva fate execution, but it is not required for CED-10-mediated cell corpse engulfment. Conversely, the adaptor proteins CED-2 CrkII and CED-5 Dock180 are required for cell corpse engulfment, but not for vulval fate execution since ced-2lf and ced-5lf mutants have wild-type vulval development. C. elegans vulval development will be a useful model system for elucidating specific Rac pathways involved in cell-fate specification, division axis orientation, and cell migration, and for testing the relationship between Rac and the Ras and Wnt signaling pathways (Kishore, 2002).

Rac GTPases act as molecular switch in various morphogenic events. However, the regulation of their activities during the development of multicellular organisms is not well understood. Caenorhabditis elegans rac genes ced-10 and mig-2 have been shown to act redundantly to control P cell migration and the axon outgrowth of D type motoneurons. ced-10 and mig-2 also control amphid axon outgrowth and amphid dendrite fasciculation in a redundant fashion. Biochemical and genetic data indicate that unc-73, which encodes a protein related to Trio-like guanine nucleotide exchange factor, acts as a direct activator of ced-10 and mig-2 during P cell migration and axon outgrowth of D type motoneurons and amphid sensory neurons. Furthermore, rac regulators ced-2/crkII and ced-5/dock180 function genetically upstream of ced-10 and mig-2 during axon outgrowth of D type motoneurons and act upstream of mig-2 but not ced-10 during P cell migration. However, neither ced-2/crkII nor ced-5/dock180 is involved in amphid axon outgrowth. Therefore, distinct rac regulators control ced-10 and mig-2 differentially in various cellular processes (Wu, 2002).

Biochemical data suggest that the GEF1 domain of UNC-73 has a guanine nucleotide exchange activity for both CED-10 and MIG-2 in vitro, consistent with the finding that the GEF1 domain of the Drosophila Trio acts on Drosophila Rac1 and Drosophila Mtl (MIG-2 like). RHO-1 has been shown to function in P cell migration, likely through activation by UNC-73 GEF2. Thus, UNC-73 likely functions as a common exchange factor for CED-10, MIG-2, and RHO-1 during P cell migration, with the GEF1 domain acting on CED-10 and MIG-2 and the GEF2 domain on RHO-1. UNC-73, CED-10, and MIG-2 also act in axon outgrowth of D-type motoneurons and amphid sensory neurons. Biochemical data and genetic analyses suggest that UNC-73 likely activates CED-10 and MIG-2 through the GEF1 domain to control the axon outgrowth of D-type motoneurons and amphid sensory neurons. Genetic analysis indicates that UNC-73 GEF2 likely functions in these axonal outgrowth processes as well. For example, axon outgrowth defects of unc-73(gm33) mutants are more severe than those of unc-73(rh40) mutants. The rh40 mutation abolishes UNC-73 GEF1 activities for RAC, and the gm33 mutation affects both GEF1 and GEF2 domains of UNC-73. UNC-73 GEF2 has a GEF activity for human RhoA but not RAC. Therefore, UNC-73 GEF2 probably act through rho-1 but not ced-10 or mig-2 to control axon outgrowth of DDs, VDs, and amphid neurons. An additional GEF besides UNC-73 may be important for the activation of CED-10 and MIG-2 during the axon outgrowth of DDs, VDs, and amphid neurons, since defects in these axon outgrowths in unc-73(rh40) mutants are not as severe as those in unc-73(rh40);mig-2(mu28), unc-73(rh40); ced-10 and ced-10;mig-2(mu28) double mutants. Therefore, multiple GEFs may regulate various GTPase activities during the development of DD, VD, and amphid axons, patterning axons by regulating their directional extension (Wu, 2002).

Trio contains two functional guanine nucleotide exchange factor (GEF) domains for the Rho-like GTPases and a serine/threonine kinase domain. In vitro, GEF domain 1(GEFD1) is specifically active on Rac1, while GEF domain 2 (GEFD2) targets RhoA. To determine whether Trio can activate Rac1 and RhoA in vivo, the effect of Trio on mitogen activated protein kinase (MAPK) pathways and cytoskeletal rearrangements events mediated by the two GTPases was measured. It is shown that: (1) the GEFD1 domain of Trio triggers the MAPK pathway leading to Jun kinase (JNK) activation and the production of membrane ruffles; (2) co-expression of the TrioGEFD1 domain with a dominant-negative form of Rac blocks JNK induction, whereas a dominant-negative form of Cdc42 does not; (3) a deletion mutant of TrioGEFD1 lacking a region important for exchange activity can not stimulate JNK activity; (4) in contrast, the TrioGEFD2 domain does not stimulate JNK activity and induces the formation of stress fibers, as does activated RhoA; (5) furthermore, co-expression of both GEF domains induces simultaneously the formation of ruffles and stress fibers. Therefore, Trio represents a unique member of the Rho-GEFs family possessing two functional domains of distinct specificities, that allow it to link Rho and Rac signaling pathway in vivo (Bellanger, 1998a).

Rho GTPases regulate the morphology of cells stimulated by extracellular ligands. Rho GTPase activation is controlled by guanine exchange factors (GEF) that catalyze their binding to GTP. The multidomain Trio protein represents an emerging class of Rho regulators that contain two GEF domains of distinct specificities. The characterization of Rho signaling pathways activated by the N-terminal GEF domain of Trio (TrioD1) is reported here. In fibroblasts, TrioD1 triggers the formation of particular cell structures, similar to those elicited by RhoG, a GTPase known to activate both Rac1 and Cdc42Hs. In addition, the activity of TrioD1 requires the microtubule network and relocalizes RhoG at the active sites of the plasma membrane. Using a classical in vitro exchange assay, TrioD1 displays a higher GEF activity on RhoG than on Rac1. In fibroblasts, expression of dominant negative RhoG mutants totally abolishes TrioD1 signaling, whereas dominant negative Rac1 and Cdc42Hs only lead to partial and complementary inhibitions. Finally, expression of a Rho Binding Domain that specifically binds RhoG(GTP) leads to the complete abolition of TrioD1 signaling; this strongly supports Rac1 not being activated by TrioD1 in vivo. These data demonstrate that Trio controls a signaling cascade that activates RhoG, which in turn activates Rac1 and Cdc42Hs (Blangy, 2000).

Rho family GTPases regulate diverse cellular processes, including extracellular signal-mediated actin cytoskeleton reorganization and cell growth. The functions of GTPases are positively regulated by guanine nucleotide exchange factors, which promote the exchange of GDP for GTP. Trio is a complex protein possessing two guanine nucleotide exchange factor domains, each with adjacent pleckstrin homology and SH3 domains, a protein serine/threonine kinase domain with an adjacent immunoglobulin-like domain and multiple spectrin-like domains. To assess the functional role of the two Trio guanine nucleotide exchange factor domains, NIH 3T3 cell lines stably expressing the individual guanine nucleotide exchange factor domains were established and characterized. Expression of the amino-terminal guanine nucleotide exchange factor domain results in prominent membrane ruffling, whereas cells expressing the carboxy-terminal guanine nucleotide exchange factor domain have lamellae that terminate in miniruffles. Moreover, cells expressing the amino-terminal guanine nucleotide exchange factor domain display more rapid cell spreading, haptotactic cell migration and anchorage-independent growth, suggesting that Trio regulates both cell motility and cell growth. Expression of full-length Trio in COS cells also alters actin cytoskeleton organization, as well as the distribution of focal contact sites. These findings support a role for Trio as a multifunctional protein that integrates and amplifies signals involved in coordinating actin remodeling, which is necessary for cell migration and growth (Seipel, 1999).

Rho-GTPases control a wide range of physiological processes by regulating actin cytoskeleton dynamics. Numerous studies on neuronal cell lines have established that Rac, Cdc42, and RhoG activate neurite extension, while RhoA mediates neurite retraction. Guanine nucleotide exchange factors (GEFs) activate Rho-GTPases by accelerating GDP/GTP exchange. Trio displays two Rho-GEF domains -- GEFD1, activating the Rac pathway via RhoG, and GEFD2, acting on RhoA -- and contains numerous signaling motifs whose contribution to Trio function has not yet been investigated. Genetic analyses in Drosophila and in Caenorhabditis elegans indicate that Trio is involved in axon guidance and cell motility via a GEFD1-dependent process, suggesting that the activity of its Rho-GEFs is strictly regulated. Human Trio induces neurite outgrowth in PC12 cells in a GEFD1-dependent manner. Interestingly, the spectrin repeats and the SH3-1 domain of Trio are essential for GEFD1-mediated neurite outgrowth, revealing an unexpected role for these motifs in Trio function. Moreover, Trio-induced neurite outgrowth is mediated by the GEFD1-dependent activation of RhoG, previously shown to be part of the NGF (nerve growth factor) pathway. The expression of different Trio mutants interferes with NGF-induced neurite outgrowth, suggesting that Trio may be an upstream regulator of RhoG in this pathway. In addition, Trio protein accumulates under NGF stimulation. Thus, Trio is the first identified Rho-GEF involved in the NGF-differentiation signaling (Estrach, 2002).

The morphogenesis of dendritic spines, the major sites of excitatory synaptic transmission in the brain, is important in synaptic development and plasticity. An ephrinB-EphB receptor trans-synaptic signaling pathway has been identified that regulates the morphogenesis and maturation of dendritic spines in hippocampal neurons. Activation of the EphB receptor induces translocation of the Rho-GEF kalirin (Drosophila ortholog: Trio) to synapses and activation of Rac1 and its effector PAK. Overexpression of dominant-negative EphB receptor, catalytically inactive kalirin or dominant-negative Rac1, or inhibition of PAK each eliminates ephrin-induced spine development. This novel signal transduction pathway may be critical for the regulation of the actin cytoskeleton controlling spine morphogenesis during development and plasticity (Penzes, 2003).

The role of the Rac1 effector p21-activated kinase PAK was examined. Several PAK proteins are expressed in the brain, and previous studies have shown that some of the effects of Rac1 on the cytoskeleton are mediated by PAK. In addition, genetic analysis in Drosophila has shown that PAK1 is genetically associated with Trio, the fly ortholog of kalirin, in the pathway through which Trio affects axon growth and guidance. Binding of activated Rac1 to PAK induces PAK autophosphorylation, which strongly correlates with its activation. To test whether ephrinB treatment induces activation of PAKs, an antibody detecting autophosphorylated PAK (P-PAK) was used. In addition, this experiment can be regarded as a way to visualize endogenous Rac1 activation. Treatment of hippocampal neurons with clustered ephrinB1 induce a dramatic increase in the number and size of clusters stained with the P-PAK antibody. This effect was confirmed by Western analysis with the P-PAK antibody of extracts of 4-week-old high-density cortical neurons treated with ephrinB1. Moreover, in hippocampal neurons, ephrinB1 treatment induces activation of PAK at synapses, as shown by P-PAK immunostaining coincident with synaptophysin (Penzes, 2003).

To test whether kalirin-7 was required for ephrinB1-induced PAK phosphorylation, the effect was examined of overexpressing the GEF inactive kal7-mut in hippocampal neurons on the ability of clustered ephrinB1 to induce phosphorylation of PAK. Therefore, DIV7 hippocampal neurons were transfected with myc-kal7-mut, and 2 days later the neurons were treated with clustered ephrinB1 for 2 hr, followed by fixation and immunostaining for myc and P-PAK. While ephrinB1 treatment induces an increased phosphorylation of PAK in nontransfected neurons, in neurons expressing kal7-mut, the level of P-PAK is visibly reduced compared to adjacent nontransfected neurons. Quantification of the ratios of P-PAK fluorescence intensities to total cell areas of nontransfected control neurons relative to the same ratios for neurons expressing myc-kal7-mut confirmed this observation (Penzes, 2003).

PAKs phosphorylate proteins involved in regulating the actin cytoskeleton and gene expression. To test whether PAK is an essential downstream component of ephrinB signaling in spine morphogenesis, GFP-transfected hippocampal neurons were treated with a fusion protein of the PAK1 inhibitory domain (PID) fused with the cell-penetrating peptide (TAT-PID) along with ephrinB1. These neurons exhibit a reduction in the number and size of spines, compared to the ephrinB1-treated neurons, while also showing a reduced phosphorylation level of PAK, confirming its inhibition by PID. Together, these data demonstrate that Rac1 and PAK are key downstream components of ephrinB regulation of spine morphogenesis (Penzes, 2003).

During development, it is necessary to coordinate accurately the formation and location of presynaptic active zones with those of the postsynaptic structures. This could be achieved by signaling from presynaptic ephrinB, clustered at active zones on axons, to activate postsynaptic EphB2, resulting in synaptogenesis on the apposing dendrites. Even in mature neurons, dendritic spines are very dynamic structures, and recent studies have demonstrated that LTP induces morphological changes in spines, which may contribute to plasticity in adult neurons. The rapid and dramatic effect of ephrinB on spine maturation suggests that ephrinB-EphB2 signaling may be a key component in the regulation of spine morphogenesis during plasticity. Other extracellular signals have been shown to regulate spine morphogenesis, such as K+ depolarization, glutamate action on NMDA receptors, and BDNF. It is possible that kalirin mediates the intracellular effects of these signals as well (Penzes, 2003).

The UNC-6/Netrin receptors UNC-40/DCC and UNC-5 inhibit growth cone filopodial protrusion via UNC-73/Trio, Rac-like GTPases and UNC-33/CRMP

UNC-6/Netrin is a conserved axon guidance cue that can mediate both attraction and repulsion. Previous studies have discovered that attractive UNC-40/DCC receptor signaling (see Drosophila Frazzled) stimulates growth cone filopodial protrusion and that repulsive UNC-40-UNC-5 heterodimers (see Drosophila Unc5) inhibit filopodial protrusion in C. elegans. This study identified cytoplasmic signaling molecules required for UNC-6-mediated inhibition of filopodial protrusion involved in axon repulsion. The Rac-like GTPases CED-10 and MIG-2, the Rac GTP exchange factor UNC-73/Trio, UNC-44/Ankyrin and UNC-33/CRMP act in inhibitory UNC-6 signaling. These molecules were required for the normal limitation of filopodial protrusion in developing growth cones and for inhibition of growth cone filopodial protrusion caused by activated MYR::UNC-40 and MYR::UNC-5 receptor signaling. Epistasis studies using activated CED-10 and MIG-2 indicated that UNC-44 and UNC-33 act downstream of the Rac-like GTPases in filopodial inhibition. UNC-73, UNC-33 and UNC-44 did not affect the accumulation of full-length UNC-5::GFP and UNC-40::GFP in growth cones, consistent with a model in which UNC-73, UNC-33 and UNC-44 influence cytoskeletal function during growth cone filopodial inhibition (Norris, 2014).

A Trio-Rac1-Pak1 signalling axis drives invadopodia disassembly

Rho family GTPases control cell migration and participate in the regulation of cancer metastasis. Invadopodia, associated with invasive tumour cells, are crucial for cellular invasion and metastasis. To study Rac1 GTPase in invadopodia dynamics, a genetically encoded, single-chain Rac1 fluorescence resonance energy (FRET) transfer biosensor was developed. The biosensor shows Rac1 activity exclusion from the core of invadopodia, and higher activity when invadopodia disappear, suggesting that reduced Rac1 activity is necessary for their stability, and Rac1 activation is involved in disassembly. Photoactivating Rac1 at invadopodia confirmed this previously unknown Rac1 function. This study describes an invadopodia disassembly model, where a signalling axis involving TrioGEF, Rac1, Pak1, and phosphorylation of cortactin, causes invadopodia dissolution. This mechanism is critical for the proper turnover of invasive structures during tumour cell invasion, where a balance of proteolytic activity and locomotory protrusions must be carefully coordinated to achieve a maximally invasive phenotype (Moshfegh, 2014).

Interaction of Trio homologs with surface receptors

rho-like GTP binding proteins play an essential role in regulating cell growth and actin polymerization. These molecular switches are positively regulated by guanine nucleotide exchange factors (GEFs) that promote the exchange of GDP for GTP. Using the interaction-trap assay to identify candidate proteins that bind the cytoplasmic region of the LAR transmembrane protein tyrosine phosphatase (PT-Pase), a cDNA was isolated encoding a 2861-amino acid protein termed Trio that contains three enzyme domains: two functional GEF domains and a protein serine/threonine kinase (PSK) domain. One of the Trio GEF domains (Trio GEF-D1) has rac-specific GEF activity, while the other Trio GEF domain (Trio GEF-D2) has rho-specific activity. The C-terminal PSK domain is adjacent to an Ig-like domain and is most similar to calcium/calmodulin-dependent kinases, such as smooth muscle myosin light chain kinase which similarly contains associated Ig-like domains. Near the N terminus, Trio has four spectrin-like repeats that may play a role in intracellular targeting. Northern blot analysis indicates that Trio has a broad tissue distribution. Trio appears to be phosphorylated only on serine residues, suggesting that Trio is not a LAR substrate, but rather that it forms a complex with LAR. Since the LAR PTPase localizes to the ends of focal adhesions, it is proposed that LAR and the Trio GEF/PSK may orchestrate cell-matrix and cytoskeletal rearrangements necessary for cell migration (Debant, 1996).

Par3 controls neural crest migration by promoting microtubule catastrophe during contact inhibition of locomotion

There is growing evidence that contact inhibition of locomotion (CIL) is essential for morphogenesis and its failure is thought to be responsible for cancer invasion; however, the molecular bases of this phenomenon are poorly understood. This study investigated the role of the polarity protein Par3 in CIL during migration of the neural crest, a highly migratory mesenchymal cell type. In epithelial cells, Par3 is localised to the cell-cell adhesion complex and is important in the definition of apicobasal polarity, but the localisation and function of Par3 in mesenchymal cells are not well characterised. In Xenopus and zebrafish it was shown that Par3 is localised to the cell-cell contact in neural crest cells and is essential for CIL. The dynamics of microtubules are different in different parts of the cell, with an increase in microtubule catastrophe at the collision site during CIL. Par3 loss-of-function affects neural crest migration by reducing microtubule catastrophe at the site of cell-cell contact and abrogating CIL. Furthermore, Par3 promotes microtubule catastrophe by inhibiting the Rac-GEF Trio, as double inhibition of Par3 and Trio restores microtubule catastrophe at the cell contact and rescues CIL and neural crest migration. These results demonstrate a novel role of Par3 during neural crest migration, which is likely to be conserved in other processes that involve CIL such as cancer invasion or cell dispersion (Moore, 2013).

Trio: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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