BIOLOGICAL OVERVIEW

Rho family small GTPases, including Rho, Rac, and Cdc42, are strong candidates for transducing axon guidance information to the actin cytoskeleton. In nonneuronal cells, these molecular switches control distinct types of actin-based cell motility in response to extracellular cues. Work in nonneuronal systems indicates that small GTPase activation is initiated by the Dbl homology (DH) family of guanine nucleotide exchange factors (GEFs), which allow Rho family GTPases to release GDP and acquire the GTP necessary for the active state. It is thought that association of GEF proteins with complexes that bind the cytoplasmic domains of cell surface receptors provides the necessary link between extracellular cues and GTPase activation. Described here is the Drosophila homolog of Trio, a multidomain protein containing two DH domains whose vertebrate counterpart specifically activates Rac and Rho (Debant, 1996). A close relative of Trio has also been identified in C. elegans (unc-73; Steven, 1998), in which mutant analysis reveals a variety of axon guidance and cell migration phenotypes (Siddiqui, 1991; McIntire, 1992). Drosophila trio mediates the development of multiple embryonic axon pathways. trio shows dosage-sensitive, reciprocal genetic interactions with Abl tyrosine kinase, revealing Trio's role in axon pathfinding (Liebl, 2000). Thus genetic analysis in Drosophila reveals potent genetic interactions between trio and a number of signaling components thought to control actin dynamics, including Rac, Dlar, and components in the Abl tyrosine kinase pathway (Bateman, 2000). Trio is distributed along axons in the central nervous system (CNS) of embryos and is strongly expressed in subsets of brain regions, including the mushroom body (MB). Loss-of-function trio mutations result in the misdirection or stall of axons in embryos and also cause malformation of the MB. The MB phenotypes are attributed to alteration in the intrinsic nature of neurites, as revealed by clonal analyses. Thus, Trio is essential in order for neurites to faithfully extend on the correct pathways (Bateman, 2000; Liebl, 2000; Awasaki, 2000).

Cell transfection and in vitro nucleotide exchange assays with each DH domain of human Trio have suggested that GEF1 preferentially activates Rac, whereas GEF2 activates Rho (Debant, 1996; Bellanger, 1998a). To explore the relationship between trio and Rac in Drosophila, the compound eye was used as an established system to test for genetic interactions in GTPase signaling pathways. Rac overexpression under the control of the eye-specific promoter GMR creates a mispatterened 'rough' eye in which individual ommatidia are misshapen. However, removal of a single copy of trio causes a dramatic suppression of this Rac gain-of-function phenotype. This is true for additional trio alleles. In contrast, overexpression of Rho also generates a rough eye, but this phenotype is not significantly altered by reduction in trio activity. This suggests that in the Drosophila retina, trio functions to activate one or more of the Drosophila Rac-like genes but not Rho (Bateman, 2000).

In the embryo, previous studies have shown that the same nerve branches affected by trio mutations are also most sensitive to Rac perturbation. Although occasional ISNb stop short phenotypes are observed, the predominant ISNb and SNa bypass phenotypes induced by Drac1^N17 overexpression are distinct from phenotypes caused by loss of trio function. This difference likely reflects Drac1^N17 interference with multiple neural activators of Rac GTPases. However, it was reasoned that if trio is involved in Rac activation in the embryonic motor nervous system, the penetrance of the Drac1^N17 phenotype should be sensitive to changes in the genetic dose of trio. Consistent with this hypothesis, removal of a single copy of trio in embryos expressing Drac1^N17 causes a distinct increase in the penetrance of ISNb bypass. This was true for all alleles of trio tested. Moreover, coexpression of Drac1^N17 and a wild-type trio transgene results in a dramatic suppression of the ISNb bypass phenotype, consistent with the model that trio is an activator of Rac GTPases in the embryonic motor nervous system (Bateman, 2000).

Despite phenotypic differences between Drac1^N17 and trio in the motor nervous system, analysis of the CNS in embryos lacking Rac function reveals defects identical to those observed in trio mutants. Specifically, expression of the dominant-negative Drac1^N17, under the control of the neural-specific GAL4 driver C155, causes a failure of the lateralmost Fas II-positive longitudinal pathway to properly connect at stage 17 (13.7%). In contrast, neural expression of either Dcdc42^N17 or DRho1^N19 does not cause defects in longitudinal pathfinding, indicating that the CNS phenotype is specific to interference with Rac-like GTPase function. Thus, in the CNS, mutant phenotypes of Drac1^N17 and trio are consistent with disruption of a common pathway (Bateman, 2000).

The ISNb and longitudinal pathway defects observed in trio mutants are similar to those of phenotypes observed in embryos mutant for the Abl tyrosine kinase. Previous analysis has shown that a partial reduction in Abl function suppresses the bypass phenotype caused by mutations in the RPTP Dlar, implying an antagonistic relationship between kinase and phosphatase. To address trio function at this ISNb choice point, ISNb pathfinding was examined for dosage-sensitive interactions between Dlar and trio. In strong zygotic Dlar mutants, ISNb bypass was observed at a moderate frequency (18.4%, A2-A7 hemisegments). However, partial reduction of trio activity in this Dlar background enhances the ISNb bypass ~2-fold. Although this potentiation disagrees with a simple model in which trio and Abl function together to oppose phosphatase signaling, it is consistent with the observation that neural expression of Drac1^N17 enhances the frequency of bypass in Dlar mutants. Thus, although trio may collaborate with Abl at the CNS midline, it rather appears to cooperate with Dlar and Drac1 during ISNb ventral target entry. The absence of bypass phenotypes in trio single mutants is likely to reflect the existence of additional inputs to Rac family GTPases that would be susceptible to the Drac1^N17 dominant-negative effect (Bateman, 2000).

While the functional connection between Trio and Rac-like GTPases provides clear links to cytoskeletal events, recent results suggest that Trio may coordinate the activities of multiple signaling partners. In addition to genetic interactions between Drosophila trio, Abl, ena, and Dlar, vertebrate Trio has been shown to bind directly to Filamin (Bellanger, 1998b), a protein required for cell motility in a variety of cell types. In addition to forming direct orthogonal cross-links between microfilaments, characteristic of lamellipodia, Filamin also plays a role in RalA GTPase-mediated induction of filopodial structures and interacts with cell surface receptors. Recent data also reveal a role for Drosophila filamin in the construction of actin-based structures during oogenesis (Li, 1999; Sokol, 1999) and in the formation of embryonic axon pathways (N. Sheard, J. B., T. Hays, and D. V. V., unpublished data cited in Bateman, 2000). Thus, Filamin may provide another direct link between Trio and the cytoskeletal arrays that drive the motile leading edge (Bateman, 2000).

Additional partners for Trio may also come from genetic studies. For example, previous genetic screens reveal another gene with a trio-like ISNb phenotype called 'stop short' (or 'shot', also known as 'kakapo'); recent molecular cloning reveals that this gene encodes a protein with an actin-binding domain. Interestingly, kakapo is expressed in the same muscle attachment sites in which trio is expressed, suggesting a functional overlap in different cell types. The shared function of cytoskeletal proteins in many different morphogenetic events is a common theme in many organisms. While these important proteins and signaling pathways are used again and again, their roles in a particular context may be quite specific. The challenge for the future is to dissect the specific from the general in order to understand the information content of the many pathways that regulate cytoskeletal structure and dynamics (Bateman, 2000 and references therein).

Recent genetic and biochemical analyses have suggested functional interactions between tyrosine phosphorylation and microfilament dynamics in the growth cone. In Drosophila second site modifier screens have found that heterozygosity for enabled (ena) alleviates the Abl mutant phenotype. Enabled can bind to and be phosphorylated by Abl and can bind to and be dephosphorylated by Dlar; Enabled's phosphorylation state is believed to influence its protein-protein interactions. Enabled can bind Profilin and is colocalized with Abl and Dlar in axons. Tyrosine phosphorylation of Enabled may be one way to influence F-actin production in the growth cone and therefore may be an important influence on Drosophila axonal pathfinding. Work in mice suggests that similar molecular cascades function in mammals; Mena, which promotes actin polymerization and binds Profilin, is concentrated in filopodia tips of growth cones, and Mena null mice have axonal projection defects (Liebl, 2000 and references therein).

Genetic analysis in Drosophila has been used to identify a variety of other genes having dosage-sensitive interactions with Abl, since dosage-sensitive genetic interactions can occur between members of signal transduction networks. Reducing the gene dose by half of either disabled (dab), failed axon connections (fax), or Notch has been shown to worsen the Abl mutant phenotype, while reducing the dose of Abl by half has been shown to worsen the profilin (chic) mutant phenotype. Reducing the gene dose of Abl has also been shown to alleviate the Dlar mutant phenotype. It is likely that these genes' products are integrated into molecular networks regulating growth cone guidance, although the biochemical interaction between these molecules and Abl is not yet established in all cases (Liebl, 2000 and references therein).

A dosage-sensitive genetic interaction has been shown to occur between Abl and Trio. Not only do heterozygous mutations in trio dramatically enhance the Abl mutant phenotype, but reducing the gene dose of either Abl, fax, or dab enhances the trio mutant phenotype. Heterozygosity for ena partially alleviates the trio mutant phenotype. These dosage-sensitive genetic interactions between trio, Abl, and genes believed to be in Abl-mediated signal transduction networks, as well as the potential for Trio to regulate the actin cytoskeleton by influencing small GTPases, position the Trio protein as another candidate for the integration of tyrosine phosphorylation and microfilament dynamics in the growth cone (Liebl, 2000).

Control of dendritic morphogenesis by Trio in Drosophila melanogaster

Abl tyrosine kinase and its effectors among the Rho family of GTPases each act to control dendritic morphogenesis in Drosophila. It has not been established, however, which of the many GTPase regulators in the cell link these signaling molecules in the dendrite. In axons, the bifunctional guanine exchange factor, Trio, is an essential link between the Abl tyrosine kinase signaling pathway and Rho GTPases, particularly Rac, allowing these systems to act coordinately to control actin organization. In dendritic morphogenesis, however, Abl and Rac have contrary rather than reinforcing effects, raising the question of whether Trio is involved, and if so, whether it acts through Rac, Rho or both. This study shows that Trio is expressed in sensory neurons of the Drosophila embryo and regulates their dendritic arborization. trio mutants display a reduction in dendritic branching and increase in average branch length, whereas over-expression of trio has the opposite effect. It is further shown that it is the Rac GEF domain of Trio, and not its Rho GEF domain that is primarily responsible for the dendritic function of Trio. Thus, Trio shapes the complexity of dendritic arbors and does so in a way that mimics the effects of its target, Rac (Shivalkar, 2012).

Trio has been associated with both Rho family GTPases and the Abl tyrosine kinase. Both these pathways control dendritic arborization in Drosophila, but they do so in different ways, with Rac, for example, promoting dendritic branching and Abl limiting it. This made it important to determine whether Trio plays a role in dendrogenesis, and if so, whether it was functioning in association with Rac or with Rho, and how its effects compared with those of Abl. This study shows that Trio also shapes dendritic structure in the fly. In both simple Class I sensory neurons and complex Class IV sensory neurons, Trio promotes formation of dendritic branches: over-expression of trio produces more elaborately branched dendritic trees whereas loss of trio reduces the number of dendritic branches. In both cases, the effect of Trio is concentrated on higher-order branches, which others have shown to be actin-dominated and more dynamic, and not in the primary branches, which tend to be microtubule-dominated and more stable (Shivalkar, 2012).

Trio not only affects dendritic branching but also dendritic length. In most assays, Trio limits the average length of some or all orders of dendritic branches to a degree that roughly offsets the increase in branch number, leading to a modest net change or no change in total dendritic length. The compensation is not exact, however. For example, in trio mutants, while average dendritic length is unchanged in Class I neurons, an increase in average branch length is seen in Class IV neurons but it is not enough to counteract the decrease in branch number, leading to an overall decrease in total length. Conversely, in trio over-expression, both Class I neurons and Class IV neurons show no net change in total length in spite of an increase in the average length of dendrites. This variability may suggest that total dendritic length is not strictly invariant for a given sensory neuron, with a fixed length parceled among a variable number of branches, but rather that Trio may have separate, and opposite, effects on branch length and number. Further experiments will be necessary, however, to test this idea (Shivalkar, 2012).

Expression of constructs bearing mutations in each of its GEF domains suggests that Trio acts primarily through its Rac GEF domain, and not its Rho GEF domain, to affect dendritic morphogenesis of the PNS sensory neurons. Thus, a Trio derivative lacking Rac GEF activity does not alter dendritic structure whereas a derivative lacking Rho GEF activity produces effects that are indistinguishable from those of the wild type protein. This is consistent with the similarity between the phenotype observed for gain and loss of trio function and that reported for gain and loss of Rac, and also with data from axonal development, both in embryonic motor neurons and adult photoreceptors showing that the Rac-specific GEF1 domain is the key effector domain of Trio in axons. It is in contrast, however, to results from the adult Drosophila mushroom body, in which trio mutant clones showed overextension of neurites similar to that in RhoA mutant clones in the dendritic portion of the structure (the calyx). Perhaps Trio pairs with different GTPases in different developmental settings, as has been observed for C. elegans Trio. The results also indicate that the dendritic phenotypes seen upon over-expression of trio are not due to changes in expression of the important neuronal class specific transcription factors, Abrupt and Knot, thus arguing against the idea that changes of cell fate are responsible for changes in dendritic morphology in these experiments (Shivalkar, 2012).

In contrast to the concordance between the effects of Trio and Rac, the phenotypes produced by altering Trio activity are opposite to those from manipulation of the Abl tyrosine kinase pathway. This was surprising in light of prior work showing that the effects of Trio mimic those of Abl in axonal development, and that led to the suggestion that Trio is a core component of the Abl pathway. Two hypotheses could account for this discrepancy. First, it could be that the Trio-Rac module should be thought of as an adjunct to the Abl signaling network, with a variable and context-dependent relationship to Abl, rather than as itself being a core element of that pathway. Such a relationship would allow the Trio-Abl interaction to produce different morphological outcomes in different developmental settings. Alternatively, the possibility cannot be ruled out that the relationship of Trio to Abl at the molecular level is the same in dendrites as in axons, but it manifests in opposite morphological consequences due to the complexities of the relationship between signaling, cytoskeletal dynamics and morphology. Indeed, there are many examples of a cytoplasmic signaling protein producing seemingly opposite effects in different developmental contexts. In the current setting, however, this interpretation is not favored since such non-linear effects of signaling proteins in other systems typically lead to observation of contradictory phenotypes upon manipulating the activity of a gene across a wide dynamic range. In the case of Trio, in contrast, all of the gain- and loss-of function manipulations give a consistent set of effects on dendritic branching. Additional experiments will be required, however, to distinguish fully between these hypotheses (Shivalkar, 2012).

The data reported in this study show that Trio, like its effector Rac, regulates dendritic arborization in Drosophila sensory neurons. The data also suggest that the relationship of Trio to the Abl tyrosine kinase signaling network may be more nuanced than was previously appreciated. It seems likely that the interplay of these signaling modules channels the molecular machinery of morphogenesis in a variety of ways to help produce the vast range of neuronal shapes (Shivalkar, 2012).

The RhoGEF trio functions in sculpting class specific dendrite morphogenesis in Drosophila sensory neurons

As the primary sites of synaptic or sensory input in the nervous system, dendrites play an essential role in processing neuronal and sensory information. Moreover, the specification of class specific dendrite arborization is critically important in establishing neural connectivity and the formation of functional networks. Cytoskeletal modulation provides a key mechanism for establishing, as well as reorganizing, dendritic morphology among distinct neuronal subtypes. While previous studies have established differential roles for the small GTPases Rac and Rho in mediating dendrite morphogenesis, little is known regarding the direct regulators of these genes in mediating distinct dendritic architectures. This study demonstrates that the RhoGEF Trio is required for the specification of class specific dendritic morphology in dendritic arborization (da) sensory neurons of the Drosophila peripheral nervous system (PNS). Trio is expressed in all da neuron subclasses and loss-of-function analyses indicate that Trio functions cell-autonomously in promoting dendritic branching, field coverage, and refining dendritic outgrowth in various da neuron subtypes. Moreover, overexpression studies demonstrate that Trio acts to promote higher order dendritic branching, including the formation of dendritic filopodia, through Trio GEF1-dependent interactions with Rac1, whereas Trio GEF-2-dependent interactions with Rho1 serve to restrict dendritic extension and higher order branching in da neurons. Finally, it was shown that de novo dendritic branching, induced by the homeodomain transcription factor Cut, requires Trio activity suggesting these molecules may act in a pathway to mediate dendrite morphogenesis. Collectively, these analyses implicate Trio as an important regulator of class specific da neuron dendrite morphogenesis via interactions with Rac1 and Rho1 and indicate that Trio is required as downstream effector in Cut-mediated regulation of dendrite branching and filopodia formation (Iyer, 2012).

This analysis demonstrates that Trio functions in promoting and refining class specific dendritic arborization patterns via GEF1- and GEF2-dependent interactions with Rac1 and Rho1, respectively. It was also demonstrated that Trio is required in mediating Cut induced effects on dendritic branching and filopodia formation suggesting that these molecules may operate in a common pathway to direct dendritic morphogenesis. Giniger and colleagues (NINDS/NIH) have likewise been investigating Trio function in da neurons via a non-overlapping, complementary experimental approach, and that they arrived at conclusions regarding Trio function largely consistent with those reported in this study (Iyer, 2012).

Previous studies have demonstrated that Trio functions via its GEF1 domain in mediating the regulation of axon morphogenesis by modulating Rac1 activity, however much less is known regarding the potential in vivo functional role(s) of the Trio GEF2 domain. Intriguingly, a previous study demonstrated that trio mutant neuroblast clones display a neurite overextension phenotype from the dendritic calyx region of the mushroom body which strongly resembled the dendrite-specific overextension phenotype observed in RhoA mutant mushroom body clones suggesting that RhoA/Rho1 activation may be required for restricting dendritic extension. In Drosophila da neurons, trio loss-of-function analyses reveal a reduction in dendritic branching in three distinct da neuron subclasses (class I, III, and IV), indicating a functional role for Trio in promoting dendritic branching. However, class specific differences are observed with Trio gain-of-function studies in which Trio overexpression in class I neurons increases dendritic branching, whereas in class III neurons there is no change in overall dendritic branching, but rather a redistribution of branches, and in class IV there is a reduction in overall dendritic branching. The basis for these differences appear to lie in the observation that refinement of dendritic branching in da neurons is subject to the opposing roles of Rac1 and Rho1 activation via Trio-GEF1 and Trio-GEF2, respectively, where Trio-GEF1 activity promotes higher order dendritic branching, whereas Trio-GEF2 activity restricts higher order branching and also limits overall dendritic length/extension (Iyer, 2012).

One of the key distinctions between class I versus class III and IV neurons relates to inherent differences in normal dendritic branching complexity and the relative roles of dynamic actin cytoskeletal based processes in these neurons which are known to mediate higher order branching including the dendritic filopodia of class III neurons and fine terminal branching in class IV neurons, whereas the class I neurons do not normally exhibit this degree of higher order branching and are predominantly populated by stable, microtubule-based primary and secondary branches. As such, Trio overexpression in these distinct subclasses may yield different effects on overall dendritic branching morphology based upon the normal distribution of actin cytoskeleton within these subclasses leading to unique effects on class specific dendritic architecture. Both loss-of-function and gain-of-function results support this hypothesis as the predominant effects are restricted to actin-rich higher order branching, whereas the primary branches populated by microtubles are relatively unaffected. This is further supported by the demonstration that trio knockdown suppresses Cut induced formation of actin-rich dendritic filopodia. Moreover, phenotypic analyses revealed that co-expression of Cut and Trio-GEF1 synergistically enhance dendritic branching in class I neurons likely due to increased activation of Rac1, whereas co-expression of Cut and Trio-GEF2 lead primarily to increased dendritic extension likely due to increased activation of Rho1. Thus, Trio mediated regulation of Rac1 and/or Rho1 signaling has the potential for sculpting dendritic branching and outgrowth/extension depending upon the combinatorial and opposing effects of Rac1 and Rho1 (Iyer, 2012).

In contrast to Cut, which has been shown to be differentially expressed in da neuron subclasses and exert distinct effects on class specific dendritic arborization, this study has demonstrated that Trio is expressed in all da neuron subclasses and can exert distinct effects on class specific dendritic branching. For example, in all subclasses examined, loss-of-function analyses indicate Trio is required to promote dendritic branching and yet individual subclasses exhibit strikingly distinct dendritic morphologies. These results suggest that Trio is generally required in each of these subclasses to regulate branching, however alone is insufficient to drive these class specific morphologies solely via activation of Rac1 and/or Rho1 signaling. One logical hypothesis is that differential expression of RhoGAP family members in distinct da neuron subclasses may work in concert with Trio to refine class specific morphologies. The potential for combinatorial activity between Trio and various RhoGAPs is significant given that 20 RhoGAPs have been defined in the Drosophila genome. For example, given that class I da neurons exhibit a simple branching morphology which becomes more complex when Trio or Trio-GEF1 domains are overexpressed, perhaps there is higher expression of Rac-inactivating GAPs in class I neurons that function in limiting dendritic branching, whereas in the more complex class III or IV da neurons, there may be lower expression of RacGAPs. Since overexpression of Trio-GEF2 reduces dendritic branching complexity in all three da neuron subclasses analyzed, it might be predicted that Rho1 activation limits dendritic branching and that therefore the expression of RhoGAPs may be modulated to facilitate branching in class III and IV neurons relative to class I neurons. In concert, differential expression of RacGAPs and RhoGAPs together with the uniform expression of Trio in all da neuron subclasses could potentially account for differential levels of activation/inactivation of Rac1 and/or Rho1 in individual subclasses and thereby influence overall class specific dendritic architecture (Iyer, 2012).

In support of this hypothesis, class-specific microarray analyses conducted in class I, III, and IV da neurons indeed reveal differential gene expression levels for most of the 20 known RhoGAP family members at a class-specific level. These expression analyses reveal one trend whereby select RhoGAP encoding genes are upregulated in the more complex class III and IV da neurons relative to the simple class I da neurons, whereas select RacGAP encoding genes are downregulated in complex neurons relative to simple neurons. Moreover, it is known that individual RhoGAPs display differential specificities for Rac, Rho and Cdc42 in vivo, such that a given RhoGAP may function in activating one or more of these small G proteins thereby increasing the potential for fine-tuning activation levels of a particular G protein at a class specific level. Furthermore recent studies provide direct evidence of the importance of RhoGAP family members in regulating da neuron dendritic morphogenesis. Analyses of the tumbleweed (tum) gene, which encodes the GTPase activating protein RacGAP50C, demonstrate that tum mutants display excessive da neuron dendritic branching. The dendritic phenotype observed in tum mutant da neurons is strikingly similar to that observed with Trio-GEF1 overexpression which also leads to excessive dendritic branching. Together these data suggest that Trio-GEF1 functions in activating Rac1 to promote dendritic branching whereas Tum/RacGAP50C function in inactivating Rac1 via its GTPase activity and thereby limit dendritic branching. In contrast, mutant analyses of the RhoGAP encoding gene, crossveinless-c, whose target in da neurons is the Rho1 small G protein, reveal defects in directional growth of da neuron dendrites. These results indicate that Crossveinless-C is required to inactivate Rho1 in order to promote directional dendritic growth and further suggest that a failure to inactivate Rho1 leads to restricted dendritic growth consistent with the phenotypes observed with Trio-GEF2 overexpression in all da neuron subclasses examined. These results, together with those presented herein, suggest that potential combinatorial activity of Trio and RhoGAP family proteins may converge in shaping the class specific dendritic architecture. Ultimately, future functional studies will be required to validate this hypothesis (Iyer, 2012).

While previous studies have revealed Trio acts in concert with Abl and Ena in coordinately regulating axon guidance, the same regulatory relationship does not appear to operate in da neuron dendrites as Abl has been shown to function in limiting dendritic branching and the formation of dendritic filopoda, whereas both Ena functions in promoting dendritic branching. This study demonstrates that Trio functions in promoting dendritic branching, consistent with Ena activity, but in da neuron dendrites works in an opposite direction to Abl. These findings suggest that, at least in da neuron dendrites, Trio may operate in either an Abl-independent pathway or that Trio and Abl may exhibit a context dependent regulatory interaction that is distinctly different in dendrites versus axons (Iyer, 2012).

GENE STRUCTURE

cDNA clone length - 8033 bp

Bases in 5' UTR - 2099

Bases in 3' UTR - 347

PROTEIN STRUCTURE

Amino Acids - 2264

Structural Domains

trio was isolated by PCR based on its proposed similarity to mammalian Trio proteins (Bateman, 2000). Two other studies have identified trio on the basis of either a mutational phenotype (Liebl, 2000) or on the basis of sequence homology revealed in database searches (Awasaki, 2000). The predicted Trio peptide sequence was compared to the sequence of hTrio, its close relative, Kalirin (Alam, 1997), and UNC-73, as well as other less related proteins. Analysis of the Drosophila sequence using BLAST and PFAM algorithms predicts the presence of seven N-terminal spectrin-like domains (residues 313-1227) followed by a DH domain (DH1, residues 1287-1486), a pleckstrin homology domain (PH1, residues 1493-1581), a Src-homology 3 domain (SH3, residues 1645-1708), a second DH domain (DH2, residues 1945-2122), and a second PH domain (PH2, residues 2136-2245). Both DH domains contain all of the highly conserved residues in the GTPase-binding surface shown to be functionally important by site-directed mutagenesis (Liu, 1998) and are thus likely to be enzymatically functional (Bateman, 2000).

The relative amino acid identity for each domain and the organization of these domains reveal that the Drosophila sequence is not only conserved in overall structure with members of the Trio GEF family, but is in fact most closely related to hTrio. Despite the close relationship between these family members in fly, human, and worm, all proteins of this family are divergent in their extreme C-terminal domains. Although Drosophila Trio contains only 15 residues C-teminal to the PH2 domain, hTrio contains a serine/threonine kinase domain in this position (Debant, 1996), whereas UNC-73 lacks the kinase and instead contains a fibronectin-like domain (Steven, 1998). The functional significance of this diversity is currently unknown (Bateman, 2000). Although human Trio contains a C-terminal Ig-like domain, a second SH3-like domain, and a serine protein kinase domain (all of which are lacking in Drosophila Trio), the overall arrangement of the N-terminal domain, the spectrin repeats, the DH/PH domains, and the first SH3-like domain is highly similar in both Drosophila Trio and human Trio (Liebl, 2000).

date revised: 4 August 2000

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