RhoGef2: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - RhoGEF2

Synonyms -

Cytological map position-53E4-53F1

Function - signaling

Keywords - gastrulation, rho pathway, cellular contraction

Symbol - RhoGEF2

FlyBase ID: FBgn0023172

Genetic map position - 2R

Classification - signaling

Cellular location - cytoplasmic

NCBI link: EntrezGene

RhoGEF2 orthologs: Biolitmine

Recent literature
Chang, Y. J., Zhou, L., Binari, R., Manoukian, A., Mak, T., McNeill, H. and Stambolic, V. (2016). The Rho guanine nucleotide exchange factor DRhoGEF2 is a genetic modifier of the PI3K pathway in Drosophila. PLoS One 11: e0152259. PubMed ID: 27015411
The insulin/IGF-1 signaling pathway mediates various physiological processes associated with human health. Components of this pathway are highly conserved throughout eukaryotic evolution. In Drosophila, the PTEN ortholog and its mammalian counterpart downregulate insulin/IGF signaling by antagonizing the PI3-kinase function. From a dominant loss-of-function genetic screen, this study discovered that mutations of a Dbl-family member, the guanine nucleotide exchange factor DRhoGEF2 (DRhoGEF22(l)04291), suppressed the PTEN-overexpression eye phenotype. dAkt/dPKB phosphorylation, a measure of PI3K signaling pathway activation, increased in the eye discs from the heterozygous DRhoGEF2 wandering third instar larvae. Overexpression of DRhoGEF2, and it's functional mammalian ortholog PDZ-RhoGEF (ArhGEF11), at various stages of eye development, resulted in both dPKB/Akt-dependent and -independent phenotypes, reflecting the complexity in the crosstalk between PI3K and Rho signaling in Drosophila.
Tamori, Y., Suzuki, E. and Deng, W. M. (2016). Epithelial tumors originate in tumor hotspots, a tissue-intrinsic microenvironment. PLoS Biol 14: e1002537. PubMed ID: 27584724
Malignant tumors are caused by uncontrolled proliferation of transformed mutant cells that have lost the ability to maintain tissue integrity. Although a number of causative genetic backgrounds for tumor development have been discovered, the initial steps mutant cells take to escape tissue integrity and trigger tumorigenesis remain elusive. This study shows through analysis of conserved neoplastic tumor-suppressor genes (nTSGs) in Drosophila wing imaginal disc epithelia that tumor initiation depends on tissue-intrinsic local cytoarchitectures, causing tumors to consistently originate in a specific region of the tissue. In this "tumor hotspot" where cells constitute a network of robust structures on their basal side, nTSG-deficient cells delaminate from the apical side of the epithelium and begin tumorigenic overgrowth by exploiting endogenous JAK/STAT signaling activity. Conversely, in other regions, the "tumor coldspot" nTSG-deficient cells are extruded toward the basal side and undergo apoptosis. When the direction of delamination is reversed through suppression of RhoGEF2, an activator of the Rho family small GTPases, and JAK/STAT is activated ectopically in these coldspot nTSG-deficient cells, tumorigenesis is induced. These data indicate that two independent processes, apical delamination and JAK/STAT activation, are concurrently required for the initiation of nTSG-deficient-induced tumorigenesis. Given the conservation of the epithelial cytoarchitecture, tumorigenesis may be generally initiated from tumor hotspots by a similar mechanism.
Sui, L. and Dahmann, C. (2020). Increased lateral tension is sufficient for epithelial folding in Drosophila. Development 147(23). PubMed ID: 33277300
The folding of epithelial sheets is important for tissues, organs and embryos to attain their proper shapes. Epithelial folding requires subcellular modulations of mechanical forces in cells. Fold formation has mainly been attributed to mechanical force generation at apical cell sides, but several studies indicate a role of mechanical tension at lateral cell sides in this process. However, whether lateral tension increase is sufficient to drive epithelial folding remains unclear. This study used optogenetics to locally increase mechanical force generation at apical, lateral or basal sides of epithelial Drosophila wing disc cells, an important model for studying morphogenesis. Optogenetic recruitment of RhoGEF2 to apical, lateral or basal cell sides leads to local accumulation of F-actin and increase in mechanical tension. Increased lateral tension, but not increased apical or basal tension, results in sizeable fold formation. These results stress the diversification of folding mechanisms between different tissues and highlight the importance of lateral tension increase for epithelial folding.
Lin, B., Luo, J. and Lehmann, R. (2022). An AMPK phosphoregulated RhoGEF feedback loop tunes cortical flow-driven amoeboid migration in vivo. Sci Adv 8(37): eabo0323. PubMed ID: 36103538
Development, morphogenesis, immune system function, and cancer metastasis rely on the ability of cells to move through diverse tissues. To dissect migratory cell behavior in vivo, this study developed cell type-specific imaging and perturbation techniques for Drosophila primordial germ cells (PGCs). PGCs were found to use global, retrograde cortical actin flows for orientation and propulsion during guided developmental homing. PGCs use RhoGEF2, a RhoA-specific RGS-RhoGEF, as a dose-dependent regulator of cortical flow through a feedback loop requiring its conserved PDZ and PH domains for membrane anchoring and local RhoA activation. This feedback loop is regulated for directional migration by RhoGEF2 availability and requires AMPK rather than canonical Gα(12/13) signaling. AMPK multisite phosphorylation of RhoGEF2 near a conserved EB1 microtubule-binding SxIP motif releases RhoGEF2 from microtubule-dependent inhibition. Thus, this study established the mechanism by which global cortical flow and polarized RhoA activation can be dynamically adapted during natural cell navigation in a changing environment.

Members of the Rho/Rac/Cdc42 superfamily of GTPases and their upstream activators, guanine nucleotide exchange factors (GEFs), have emerged as key regulators of actin and microtubule dynamics. In their GTP bound form, these proteins interact with downstream effector molecules that alter actin and microtubule behavior. During Drosophila embryogenesis, a Gα subunit (Concertina) and a Rho-type guanine nucleotide exchange factor (DRhoGEF2) have been implicated in the dramatic epithelial-cell shape changes that occur during gastrulation. Using Drosophila S2 cells as a model system, this study shows that DRhoGEF2 induces contractile cell shape changes by stimulating myosin II via the Rho1 pathway. Unexpectedly, it was found that DRhoGEF2 travels to the cell cortex on the tips of growing microtubules by interaction with the microtubule plus-end tracking protein EB1. The upstream activator Concertina, in its GTP but not GDP bound form, dissociates DRhoGEF2 from microtubule tips and also causes cellular contraction. It is proposed that DRhoGEF2 uses microtubule dynamics to search for cortical subdomains of receptor-mediated Gα activation, which in turn causes localized actomyosin contraction associated with morphogenetic movements during development (Rogers, 2004).

The cellular functions of the microtubule plus-end binding protein EB1 has been characterized in Drosophila S2 cells; this protein plays an important role in regulating microtubule dynamics and in the assembly and dynamics of the mitotic spindle (Rogers, 2002). In order to learn more about EB1's functions, attempts were made to identify EB1 binding partners with affinity purification. Recombinant Drosophila GST-EB1 bound to glutathione Sepharose beads was used as an affinity chromatography matrix to bind interacting partners from S2 cell extracts. Bound proteins were eluted from the beads and separated by SDS-PAGE, and excised bands were subjected to tryptic digestion and mass spectrometry fingerprinting (Rogers, 2004).

Twenty 'EB1-specific' proteins were identified over the course of five independent pull-down experiments. However, of these, only six candidates were identified in all five trials: CLIP190, the Drosophila ortholog of vertebrate CLIP-170, which localizes to the plus ends of microtubules, Orbit/MAST, a microtubule plus-end-associated protein that interacts with CLIP-170, nonmuscle myosin II heavy chain, the minus-end-directed kinesin, Ncd, Shortstop, a member of the spectraplakin family of actin/microtubule cross-linking proteins, and DRhoGEF2. This paper focuses on DRhoGEF2 for further study (Rogers, 2004).

The association of DRhoGEF2 with EB1 in vitro raised the possibility that this protein may localize to the tips of microtubules. To test this idea, polyclonal antibodies were generated against the C-terminal 720 amino acid residues of DRhoGEF2. These antibodies recognized a ~280 kDa polypeptide on immunoblots of S2 cell extracts; this polypeptide was eliminated after DRhoGEF2 RNAi treatment, indicating that the antibodies were reacting with the correct polypeptide (Rogers, 2004).

By immunofluorescence, anti-DRhoGEF2 antibodies recognized punctate structures distributed throughout the cell. Superimposed upon this punctate pattern, however, were short (~1 μm) linear tracks that colocalized with the tips of microtubules. Moreover, immunofluorescent staining of DRhoGEF2 in S2 cells expressing low amounts of EB1-EGFP indicated that these two proteins colocalize exactly at microtubule tips. In the perinuclear region of many cells, DRhoGEF2 antibodies also stained larger spots that costained with γ-tubulin, a centrosome marker. Depletion of DRhoGEF2 with RNAi eliminated antibody staining of all of these structures in S2 cells. Thus, these immunofluorescence experiments reveal that DRhoGEF2 exists in three pools within S2 cells: punctate throughout the cell, at microtubule tips, and on centrosomes (Rogers, 2004).

DRhoGEF2 tagged with green fluorescent protein (GFP) was tested to examine its dynamic behavior through time-lapse imaging with a spinning-disk microscope. As predicted from immunofluorescence data, 'comet-like' structures of DRhoGEF2-GFP moved from the cell center toward the periphery in a manner that was very similar to that observed for EB1-GFP. In many cells, an intense spot of DRhoGEF2-GFP was observed near the perinuclear region. This spot likely corresponded to the centrosome staining because the tips of nucleated microtubules emanated from this point in a radial pattern. Microtubule dynamics are essential for this movement because it could be eliminated with either 10 μM colchicine or 10 μM taxol. Thus, it is concluded that DRhoGEF2 associates with the tips of growing microtubules and exhibits plus-end tracking that is qualitatively similar to that described for EB1 (Rogers, 2004).

Because DRhoGEF2 was isolated based upon its association, direct or indirect, with EB1, EB1 was deleted from cells with RNAi and whether the association of DRhoGEF2 with microtubule tips was examined. In cultures treated with control dsRNA, scoring of fixed cells stained for DRhoGEF2 and microtubules revealed that 94% of the cells (n = 300) had DRhoGEF2 associated with the microtubule tip. In contrast, in S2 cells treated for 7 days with EB1 dsRNA, only 5% of the cells retained DRhoGEF2 at the plus ends. These results demonstrate that targeting of DRhoGEF2 to growing microtubule plus ends is an EB1-dependent process (Rogers, 2004).

To further understand DRhoGEF2 functions, how overexpression and depletion of the protein affects the morphology of S2 cells was examined. When S2 cells are plated on concanavalin A, they adopt a 'fried-egg' appearance with a dome-like central domain defined by the nucleus and perinuclear organelle-rich region and an extended, symmetrical lamella. In contrast, overexpressing DRhoGEF2 caused many cells to adopt a smaller, contracted footprint on the substrate and to become significantly taller than control cells. These overexpressing cells formed a skirt of abnormally large membrane ruffles that tapered to the base of a raised, organelle-rich compartment, and the overall morphology resembled a 'bonnet' shape. This result suggests that DRhoGEF2 can induce contractility, in agreement with the genetic phenotype of DRhoGEF2 mutations (Rogers, 2004).

Several genetic studies implicate DRhoGEF2 as a positive regulator of Rho1. To test whether Rho activation is involved in generating the unusual phenotype associated with DRhoGEF2 overexpression, cells were transfected with constitutively active Rho1V14 and transfected cells were identified with an antibody raised against Drosophila Rho1. As predicted, most of the Rho1V14-expressing cells duplicated the morphology produced by DRhoGEF2 overexpression. In order to next test if inhibition of Rho1 prevented DRhoGEF2-induced shape change, DRhoGEF2-EGFP was transfected into cells that had been treated with Rho RNAi. Depletion of Rho1 by RNAi produced large multinucleate cells that did not contract in response to DRhoGEF2 overexpression. In contrast, RNAi inhibition of the other six Rho family members did not block DRhoGEF2-induced contraction (Rogers, 2004).

Active Rho is known to stimulate nonmuscle myosin II, and a genetic interaction has been demonsrated between DRhoGEF2 and myosin II during Drosophila morphogenesis. One well-characterized mechanism by which Rho1 activates myosin II is Rho kinase (DROK in Drosophila) stimulation, which activates the motor by phosphorylating the myosin light chain and by inactivating myosin light chain phosphatase. In order to determine if DROK is indeed downstream of DRhoGEF2, DROK was depleted with RNAi or kinase activity was inhibited with Y-27632, a pharmacological inhibitor, and then cell morphology was examined after DRhoGEF2 overexpression. Both treatments significantly reduced the numbers of cells exhibiting the contracted morphology. From these data, it is concluded that DRhoGEF2 changes S2 cell morphology through Rho1 and its downstream effector, DROK (Rogers, 2004).

To confirm that myosin II is a downstream effector in the DRhoGEF2 pathway in this system, the behavior of GFP-tagged myosin II in control S2 cells on concanavalin A was compared with that of S2 cells overexpressing DRhoGEF2. To perform this analysis, a stable cell line was generated expressing the myosin II regulatory light chain (RLC), known by Drosophila nomenclature as spaghetti squash, under the control of the gene's endogenous promoter. Ectopic expression of RLC-GFP did not produce observable defects in actin organization or behavior; its distribution exactly coincided with the myosin II distribution determined by immunofluorescence staining of the same cells. RLC-GFP typically incorporated into punctae in the cell periphery and into higher-order structures in the central region of the cells. Time-lapse spinning-disk confocal microscopy revealed that punctae of RLC-GFP formed in the distal cell periphery and then translocated centripetally at a constant rate of 4.0 ± 0.3 μm/min toward the cell center. Such behavior of RLC-GFP is qualitatively very similar to the behavior of fluorescently labeled myosin II in cultured mammalian cells (Rogers, 2004).

Upon overexpression of DRhoGEF2, punctae of RLC-GFP were rarely observed. Instead, the majority of RLC-GFP signal was present in circular 'purse string' structures surrounding the organelle-dense region at the center of the cell. Time-lapse observation revealed that peripheral formation of RLC-GFP punctae and retrograde flow were infrequent in DRhoGEF2-overexpressing cells and that these RLC-GFP-containing purse strings were stable over a span of hours. The location and concentration of the myosin II suggests that actomyosin contraction is responsible for producing the bonnet-shaped appearance of these cells. From these observations, it is propose that DRhoGEF2 regulates myosin II dynamics and contractility in S2 cells (Rogers, 2004).

Genetic analyses of epithelial-sheet invagination in the early Drosophila embryo suggest that DRhoGEF2 may act downstream of the heterotrimeric Gα protein Concertina (Cta). To examine directly whether Concertina can activate DRhoGEF2, cells were transfected either with Myc-tagged wild-type Cta or Myc-tagged Cta bearing a constitutively activating point mutation (R277H) that inactivates GTPase activity, and the morphology of the transfected cells was examined. Cells expressing Myc-Cta were morphologically indistinguishable from untransfected cells, and only 3% of cells displayed a mildly contracted phenotype. In contrast, the majority of cells expressing Myc-CtaR277H exhibited the contracted morphology and myosin II purse string reminiscent of DRhoGEF2 overexpression. Similar results were obtained with three other constitutively activated Concertina constructs. However, the shape change was prevented in 88% of these cells (if they were pretreated for 7 days with dsRNA so that DRhoGEF2 was depleted. These results suggest that Concertina can act upstream of DRhoGEF2 to regulate S2 cell morphology (Rogers, 2004).

Next it was determined whether activation of DRhoGEF2 through Concertina affected its association with the microtubule cytoskeleton. Cells expressing Myc-Cta or Myc-CtaR277H were fixed and double stained for the Myc epitope tag and for DRhoGEF2. Overexpression of wild-type Concertina did not affect DRhoGEF2 association with microtubule plus ends or with the centrosome. However, constitutively activated Concertina resulted in DRhoGEF2 dissociation from microtubule tips; only 10% of the cells showed any colocalization of DRhoGEF2 with microtubule plus ends. Instead, DRhoGEF2 exhibited a diffuse staining pattern throughout the cell; this pattern likely represents association with the plasma membrane. Targeting of EB1 to the plus ends was not perturbed by CtaR277H, suggesting that Concertina signaling regulates the interactions between DRhoGEF2 and factors at microtubule tips (Rogers, 2004).

In an attempt to identify novel cellular factors that interact with EB1, this study unexpectedly discovered that DRhoGEF2, a key regulator of morphogenesis in Drosophila, associates with the tips of growing microtubules. This interesting type of intracellular motility required EB1 in a manner analogous to the EB1-dependent microtubule plus-end tracking of the vertebrate adenomatous polyposis coli (APC) tumor suppressor protein. This finding represents the first example of a regulator of the actin cytoskeleton that tracks along microtubule plus ends. Moreover, the dissociation of DRhoGEF2 from microtubule tips upon activation of Concertina also represents the first example of a regulated association of a protein with the microtubule plus end (Rogers, 2004).

The dissection of the DRhoGEF2 pathway at a cellular level is also consistent with genetic studies of Drosophila morphogenesis. These studies implicate Concertina in myosin II contractility through the Rho/Rho kinase pathway. The Rho1/Rho kinase/myosin II system is a widely employed module for bundling and contraction of actin filaments; it is involved in the formation of adhesion structures and stress fibers, retraction of the trailing edge in migrating cells, muscular contraction, morphogenetic cell shape changes, and construction of the cleavage furrow at the end of mitosis. Context- and location-specific activation of the Rho1/Rho kinase/myosin II module is likely to reside in the activation of specific RhoGEFs, over 20 of which reside within the Drosophila genome. This hypothesis is consistent with observations that inhibition of Rho1 or its downstream effectors causes a dramatic cytokinesis failure in S2 cells and embryos, but inhibition of DRhoGEF2 does not. Instead, DRhoGEF2 has been implicated in morphogenetic cell shape changes only in epithelial cells. Thus, it is believed that the signaling pathway that was engineered in S2 cells recapitulates events involved in the cellular shape changes preceding gastrulation in Drosophila blastula epithelia cells (Rogers, 2004).

However, in Drosophila development, this signaling pathway must be activated in a polarized manner by an unidentified receptor and its ligand so that myosin contraction occurs locally at the apical surface. In such a setting of asymmetric signaling, it is proposed that the intracellular transport of DRhoGEF2 on microtubule plus ends may play an important role in localized activation of the pathway. It is speculated that inactive DRhoGEF2 interacts with the tips of microtubules, whereupon these growing microtubules deliver 'packets' of DRhoGEF2 in the vicinity of the actin cortex. If DRhoGEF2 does not receive an activating input, it diffuses back into the cytoplasm to begin the transport cycle anew. However, if DRhoGEF2 is delivered to a subcortical region containing a high concentration of receptor-activated Concertina, DRhoGEF2 can locally activate the Rho1/Rho kinase/myosin II module. Moreover, because DRhoGEF2 possesses potential lipid (pleckstrin homology) and protein-protein (PDZ, RGS, and DH [Dbl homology]) interaction domains, microtubule-delivered DRhoGEF2 may be retained at the cortex if activated by Concertina. Although a microtubule-assisted activation of the Rho pathway during cellular shape changes during morphogenesis (such as in epithelial cells) is proposed, similar models that account for small GTPase activation during cellular motility have been suggested as well (Rogers, 2004).

In principle, interactions between DRhoGEF2 and its cortical activators could occur through diffusion within the cytoplasm. The evolution of this elaborate microtubule polymerization-based transport mechanism undoubtedly reflects some important property of the signaling pathway that is not yet understood. Perhaps the amount of DRhoGEF2 carried on the tip of a microtubule represents some quanta -- a critical concentration of the protein required either to respond to upstream inputs or to locally activate Rho1 in a cortical subdomain. This idea is supported by the observation that, at very low expression levels and without Concertina signaling, DRhoGEF2-GFP efficiently tracks microtubule ends without activating cellular contraction. Alternatively, it is possible that interaction with EB1 or some other protein at the microtubule plus end primes DRhoGEF2 for activation at the cortex. A third possibility is that microtubule dynamic instability is not uniform within a polarized cell but is locally modulated in order to deliver DRhoGEF2 to the cortex in a nonrandom manner. Testing between these hypotheses will require identification of the signaling components (i.e., the ligand-receptor pair) that act upstream of Concertina, reconstitution of the complete pathway in S2 cells, and the selective disruption of the association of DRhoGEF2 with microtubule tips in Drosophila embryos (Rogers, 2004).

Distinct RhoGEFs Activate Apical and Junctional Contractility under Control of G Proteins during Epithelial Morphogenesis

Small RhoGTPases direct cell shape changes and movements during tissue morphogenesis. Their activities are tightly regulated in space and time to specify the desired pattern of actomyosin contractility that supports tissue morphogenesis. This is expected to stem from polarized surface stimuli and from polarized signaling processing inside cells. This general problem was examined in the context of cell intercalation that drives extension of the Drosophila ectoderm. In the ectoderm, G protein-coupled receptors (GPCRs) and their downstream heterotrimeric G proteins (Galpha and Gbetagamma) activate Rho1 both medial-apically, where it exhibits pulsed dynamics, and at junctions, where its activity is planar polarized. However, the mechanisms responsible for polarizing Rho1 activity are unclear. This study reports that distinct guanine exchange factors (GEFs) activate Rho1 in these two cellular compartments. RhoGEF2 acts uniquely to activate medial-apical Rho1 but is recruited both medial-apically and at junctions by Galpha(12/13)-GTP, also called Concertina (Cta) in Drosophila. On the other hand, Dp114RhoGEF (Dp114), a newly characterized RhoGEF, is required for cell intercalation in the extending ectoderm, where it activates Rho1 specifically at junctions. Its localization is restricted to adherens junctions and is under Gbeta13F/Ggamma1 control. Furthermore, Gbeta13F/Ggamma1 activates junctional Rho1 and exerts quantitative control over planar polarization of Rho1. Finally, Dp114RhoGEF was absent in the mesoderm, arguing for a tissue-specific control over junctional Rho1 activity. These results clarify the mechanisms of polarization of Rho1 activity in different cellular compartments and reveal that distinct GEFs are sensitive tuning parameters of cell contractility in remodeling epithelia (Garcia De Las Bayonas, 2019).

Critical aspects of cell mechanics are governed by spatial-temporal control over Rho1 activity during Drosophila embryo morphogenesis. This work sheds new light on the mechanisms underlying polarized Rho1 activation during intercalation in the ectoderm. Rho1 activity was found to be driven by two complementary RhoGEFs under spatial control of distinct heterotrimeric G protein subunits. Notably, a regulatory module was uncovered specific for junctional Rho1 activation (Garcia De Las Bayonas, 2019).

Dp114RhoGEF was identified as a novel activator of junctional Rho1 in the extending ectoderm. Hence, two RhoGEFs, Dp114RhoGEF and RhoGEF2, coordinate independently the modular Rho signaling during tissue extension of the ectoderm. This has important implications, as it allows refinement of the nature of the interconnection between the two pools of Myo-II in this tissue. It has been shown previously that medial pulses of Myo-II flow toward and merge with the Myo-II pool at vertical junctions. However, to what extent these 'fusion' events contribute to junctional Myo-II was unclear. This study genetically uncoupled the regulation of both pools of Myo-II and showed that the loss of one pool does not compromise activation of Myo-II in the other. Indeed, junctional Myo-II levels and planar polarity are not affected in RhoGEF2 shRNA embryos or in RhoGEF2 germline clone where medial Myo-II is lost. This rules out the possibility of medial pulses being the main source of junctional Myo-II accumulation. Instead, it is concluded that actomyosin flow toward junctions contributes to junction shrinkage because it serves a distinct and direct mechanical function in junction remodeling rather than working by proxy by fueling junctional Myo-II (Garcia De Las Bayonas, 2019).

The division of labor in the molecular mechanisms of Rho1 activation in distinct cellular compartments lends itself to differential quantitative regulation. The activation kinetics of these different GEFs and nucleotide exchange catalytic efficiencies are likely to differentially impact Rho1 activity and therefore Myo-II activation at the junctional and medial-apical compartments. For example, RhoGEF2 mammalian orthologs, LARG and PDZ-RhoGEF, show a catalytic activity that is two orders of magnitude higher as compared with the Dp114RhoGEF orthologs subfamily. This may help to establish specific contractile regimes of actomyosin in given subcellular compartments. It is therefore important to tightly control RhoGEFs localization and activity to ensure a proper quantitative activation of the downstream GTPase (Garcia De Las Bayonas, 2019).

RhoGEF2 is a major regulator of medial-apical Rho1 activity during Drosophila gastrulation. Originally characterized in the invaginating mesoderm, it was found that RhoGEF2 also activates Rho1 medial-apical activity in the elongating ectoderm. There, RhoGEF2 localizes both medial-apically and at junctions where it is also planar polarized. Although RhoGEF2 and active Rho1 are both planar polarized at junctions, in RhoGEF2 mutants, junctional Rho1-GTP is not affected and ectopic recruitment of RhoGEF2 following expression of Gα12/13Q303L does not cause ectopic junctional Rho1-GTP accumulation. Thus, RhoGEF2 localization at the membrane is not strictly indicative of its activation status. Interestingly, Gα12/13/Cta is necessary for RhoGEF2 to translocate from microtubules plus ends to the plasma membrane where it signals. To date, experimental evidence favor a model whereby the binding of active Gα12/13/Cta to the RhoGEF in the vicinity of the cell membrane triggers its conformational change and stabilizes it in an open conformation able to bind to lipids via its PH domain and signal at the plasma membrane. There is no evidence that Gα12/13/Cta-GTP actively destabilizes RhoGEF2-EB1 interaction, but this is a formal possibility to be tested. Importantly, Gα12/13/Cta alone does not account for the restricted activation of Rho1 medial-apically (Garcia De Las Bayonas, 2019).

It is hypothesized that additional factors must regulate the spatial distribution of RhoGEF2 activity. In principle, RhoGEF2 signaling activity could either be specifically induced medial-apically independent of RhoGEF2 recruitment or RhoGEF2 could be inhibited at junctions and laterally. Sequestration of inactive RhoGEFs at cell junctions has been reported previously in mammalian cell cultures, suggesting that such mechanism could be evolutionary conserved. Phosphorylation can control the activity of the RH-RhoGEFs subfamily. Therefore, phosphorylation could promote activation or inhibition of RhoGEF2 activity in specific subcellular compartments in the ectoderm. RhoGEF2 is reported to be phosphorylated in the gastrulating embryo (Garcia De Las Bayonas, 2019).

Complementary to RhoGEF2, Dp114RhoGEF activates junctional Rho1 in the ectoderm. Dp114RhoGEF strictly localizes at junctions, providing a direct explanation for its junctional-specific effect. Gβ13F/G&gamma1 is also enriched at adherens junctions, where it controls Dp114RhoGEF junctional recruitment together with additional upstream regulators. Therefore, it is suggested that Gβ13F/Gγ1-dependent tuning of junctional Rho1 activation could be achieved through its ability to concentrate the GEF at junctions. Gβ/Gγ-dependent regulation of RhoGEFs has been described in mammals. One study proposes that mammalian p114RhoGEF may bind and be activated by Gβ1/Gγ2. Interestingly, recent work demonstrates that Gα12 can also recruit p114RhoGEF at cell junctions under mechanical stress in mammalian cell cultures where it promotes RhoA signaling. However, the region of mammalian p114RhoGEF that binds to Gα12 is absent in invertebrate RhoGEFs. How Gβ13F/Gγ1 controls Dp114RhoGEF at junctions in the Drosophila embryo remains an open question. A recent study reports that Dp114RhoGEF localizes at adherens junctions in the Drosophila ectoderm through multiple mechanisms, including interactions with Baz/Par3 and the Crumbs complex. Therefore, investigating a possible connection between Gβ13F/Gγ1 signaling and Baz/Crumbs should help decipher the mechanisms of Dp114RhoGEF localization (Garcia De Las Bayonas, 2019).

Importantly, neither Gβ13F/Gγ1 nor Dp114RhoGEF are themselves planar polarized at junctions. Hence, their distribution alone cannot explain polarized Rho1 activity at junctions. Strikingly, an increase in Gβ13F/Gγ1 dimers was found to hyperpolarize Rho1 activity and Myo-II at vertical junctions. Gβ13F/Gγ1 overexpression also leads to an overall increase in Dp114RhoGEF levels at junctions, although Dp114RhoGEF is not planar polarized in this condition. This indicates that recruitment at the plasma membrane and activation of Dp114RhoGEF are independently regulated, similar to RhoGEF2. In contrast, Dp114RhoGEF overexpression increases Myo-II at both transverse and vertical junctions, although a slightly stronger accumulation is observed at vertical junctions. Therefore, although Dp114RhoGEF junctional levels are increased in both experiments, only Gβ13F/Gγ1 overexpression leads to an increased planar polarization of Rho1-GTP and Myo-II at vertical junctions. This points to a key role for Gβ13F/Gγ1 subunits in the planar-polarization process associated with but independent from the sole recruitment of Dp114RhoGEF at junctions. In principle, Gβ13F/Gγ1 could bias junctional Rho1 signaling either by promoting its activation at vertical junctions or by inhibiting it at transverse junctions (e.g., RhoGAP polarized activation). Gβ13F/Gγ1 could also control active Rho1 distribution independent of its activation. For instance, a scaffolding protein binding to Rho1-GTP at junctions could be polarized by Gβ13F/Gγ1 to bias Rho1-GTP distribution downstream of its activation. Anillin, a Rho1-GTP anchor known to stabilize Rho1 signaling at cell junctions is a potential candidate in the ectoderm. Last, Toll receptors control Myo-II planar polarity in the ectoderm. Whether Gβ13F/Gγ1 and Tolls are part of the same signaling pathway is an important point yet to address in the future (Garcia De Las Bayonas, 2019).

Finally, this study sheds light on new regulatory differences underlying tissue invagination and tissue extension. This study found that Dp114RhoGEF localizes at junctions in the ectoderm, where it activates Rho1 and Myo-II. In contrast, maternally and zygotically supplied Dp114RhoGEF::GFP is not detected at junctions in the mesoderm. Little if any cytoplasmic signal is seen in this condition, suggesting that Dp114RhoGEF::GFP could be degraded in these cells. Thus, repression of Dp114RhoGEF protein in the mesoderm could be an important mechanism for cell apical constriction and proper tissue invagination. Of interest, Rho1 signaling is absent at junctions in the mesoderm. Therefore, it is tempting to suggest that the absence of Dp114RhoGEF at junction in the mesoderm accounts for cells' inability to activate Rho1 in this compartment. Importantly, the GPCR Smog and Gβ13F/Gγ1 subunits, found to control junctional Rho1 in the ectoderm, are common to both tissues. Dp114RhoGEF differential expression and/or subcellular localization could be a key element to bias signaling toward junctional compartment in the ectoderm (Garcia De Las Bayonas, 2019).

Cell contractility necessitates activation of the Rho1-Rock-MyoII core pathway. During epithelial morphogenesis, tissue- and cell-specific regulation of Rho1 signaling requires the diversification of Rho1 regulators, in particular RhoGEFs, as shown in this study, and RhoGAPs. Some of them are tissue specific with given subcellular localizations and activation mechanisms. The identification of signaling modules, namely Gα12/13-RhoGEF2 and Gβ13F/Gγ1-Dp114RhoGEF, provides a simple mechanistic framework for explaining how tissue-specific modulators control Rho1 activity in a given subcellular compartment in a given cell type. Therefore, it is suggested that the variation of (1) ligands, GPCRs, and associated heterotrimeric G proteins and (2) types of RhoGEFs and RhoGAPs as well as their combination, activation, and localization by respective co-factors underlies the context-specific control of Rho1 signaling during tissue morphogenesis. How developmental patterning signals ultimately control Rho regulators is an exciting area for future investigations (Garcia De Las Bayonas, 2019).


cDNA clone length - 7380 (transcript variant E)

Bases in 5' UTR - 422

Exons - 8

Bases in 3' UTR - 340


Amino Acids - 2559

Structural Domains

DRhoGEF2 was identified in a genetic screen for Rho signaling pathway components in Drosophila. DRhoGEF2 was found to encode a Rho-specific guanine nucleotide exchange factor (Barrett, 1997). The same gene, DRhoGEF2 was similarly identified in an independent screen carried out by Hacker (1998), a screen designed to characterize the maternal effects of zygotic lethal mutations. The gene was independently cloned by Werner (1997). DRhoGEF2 encodes a protein that contains a PDZ domain near the amino terminus, and a cystine-rich butterfly motif in the central region that is present in isoforms of protein kinase C and the mouse Dbl family oncoprotein Lfc. The C-terminal region of DRhoGEF2 contains an extensive region of homology with two separate protein motifs characteristic of the Dbl family of oncoproteins. The first motif, termed the Dbl homology domain promotes the exchange of guanine nucleotides within Rho family GTPases. The second domain, juxtaposed to the Dbl homology domain and C-terminal to it, is a Pleckstrin homology domain (Hacker, 1998).

The Guanine nucleotide exchange factor RhoGEF2 possesses potential lipid (pleckstrin homology) and protein-protein (PDZ, RGS, and DH [Dbl homology]) interaction domains (Rogers, 2004)

RhoGef2: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 2 January 2023

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