Table of contents

Rho family members and Drosophila small GTPases

Eight Drosophila small GTPases have been isolated. They can be classified into three rab family genes (Drab2, Drab5, Drab11) and five rho family genes (Drac1a, Drac1b, Drac3, Dcdc42, DrhoA). While Drac3 is a novel type of rac gene, others are homologs of known mammalian genes for small GTPases. Northern blot analyses show that all the genes are expressed throughout all developmental stages from embryo to adult. In situ hybridization to embryos reveals that Drab2, Drac1b, and Drac3 are highly expressed in the nervous system, in the trunk mesoderm, and in the cephalic mesoderm, respectively. Since hemocytes are derived from the cephalic mesoderm, double stainings were carried out using a hemocyte marker anti-peroxidasin antibody and Drac3 in situ hybridization. Drac3 is expressed in hemocyte precursor cells. In the Drac3 deficiency embryos, the hemocyte precursor cells start to differentiate normally, but never develop into mature hemocytes, indicating that Drac3 is essential for their maturation. The DrhoA and Dcdc42 genes complemented S. cerevisiae rho1 and cdc42 mutations in the same manner as human rhoA and CDC42, respectively. These results suggest functional similarity between Drosophila and mammalian small GTPase genes (Sasamura, 1997).

A new member of the Rho family from Drosophila has been isolated, named RhoL, which is equally similar to Rac, Rho, and Cdc42. Mutant forms of Cdc42 mimicked this effect. All three activities are necessary for normal transfer of nurse cell cytoplasm to the oocyte. Rac is involved specifically in border cell migration. Analysis of heat shock regulated Rac1 indicates a requirement both for the initiation and continuation of migration. For more information about border cell migration see Slow border cells (Slbo). Expression of constitutively active Rac is unable to rescue the border cell migration defect in mutant slbo, suggesting that the two function in different pathways. Rac, Rho and Cdc42 are also required in the germ line for proper nurse cell cytoplamic transport. At stage 11 of oogenesis, a network of actin filaments polymerizes to form a cage around nurse cell nuclei, apparently preventing their movement during the transfer of nurse cell cytoplasm to the oocyte. This transfer process, known as "dumping," appears to result from the myosin-based contraction of subcortical actin. Limiting the amounts of each of the Rho family members results in the absence of the actin cage that normally surrounds stage 11 nurse cell nuclei. This results in the obstruction of ring canals and the failure of complete transfer, results similar to those occurring in other mutants that affect the cytoskeleton, such as chickadee, singed and quail. Expression of constitutively active RhoL leads to nurse cell subcortical actin breakdown and disruption of nurse cell-follicle cell contacts, followed by germ cell apoptosis (Murphy, 1996 and references).

Rho small GTPase regulates cell morphology, adhesion and cytokinesis through the actin cytoskeleton. A protein, p140mDia, has been identified as a downstream effector of Rho. It is a mammalian homolog of Drosophila Diaphanous, a protein required for cytokinesis, and belongs to a family of formin-related proteins containing repetitive polyproline stretches. p140mDia binds selectively to the GTP-bound form of Rho and also binds to profilin. p140mDia, profilin and RhoA are co-localized in the spreading lamellae of cultured fibroblasts. They are also co-localized in membrane ruffles of phorbol ester-stimulated sMDCK2 cells, which extend these structures in a Rho-dependent manner. The three proteins are recruited around phagocytic cups induced by fibronectin-coated beads. Their recruitment is not induced after Rho is inactivated by microinjection of botulinum C3 exoenzyme. Overexpression of p140mDia in COS-7 cells induces homogeneous actin filament formation. These results suggest that Rho regulates actin polymerization by targeting profilin beneath the specific plasma membranes via p140mDia (N. Watanabe, 1997).

Members of the Rho GTPase family regulate the organization of the actin cytoskeleton in response to extracellular growth factors. Three proteins have been identified that form a distinct branch of the Rho family: Rnd1, expressed mostly in brain and liver; Rnd2, highly expressed in testis; and Rnd3/RhoE, showing a ubiquitous low expression. Rnd proteins share 54-63% identity pairwise, ~45-49% identity with Rho, and slightly less identity with other Rho family members, Rac or Cdc42. It is concluded that the three Rnd proteins form a new branch of the Rho family. Rnd proteins display striking differences from other members of the Rho family in their size, their charge, and biochemical properties. Their expected molecular weights are higher due to NH2-terminal extensions for Rnd1 and Rnd3/RhoE and COOH-terminal extensions of ~30 amino acids for all three. Their apparent molecular mass on SDS-PAGE is ~32 kD, whereas Rho migrates at ~24 kD. Rnd proteins end with a "CAAX box" motif for prenylation, but unlike Rho, Rac, and Cdc42, which have a COOH-terminal leucine and are predominantly geranyl-geranylated, Rnd proteins end with a methionine residue; this suggests that the proteins are likely to be farnesylated. The three guanine-binding motifs are conserved in Rnd proteins; the two loops and a conserved threonine involved in phosphate binding can also be recognized, and the three major residues that coordinate magnesium in the GTP-bound form of Ras (T 17, T 35, and D 57) are strictly conserved. However, three residues of the phosphate-binding site that are important for the intrinsic GTPase activity of Ras proteins differ in Rnd. The equivalent of Ras glycine 12 is replaced by valine, alanine, or serine in Rnd proteins; Ras glycine 13 is replaced by glutamine or glutamic acid, and Ras alanine 59 and glutamine 61 are both replaced by serine. Any one of these substitutions in Ras decreases its intrinsic GTPase rate and prevents GAP-mediated GTPase stimulation, leading to constitutive activation of the protein. In viral Ras, the presence of two substitutions at positions 12 and 59 (replaced by threonine) decreases GTPase activity more than individual substitutions. Based on the effects of these substitutions on Ras, the prediction is that Rnd proteins will have little or no intrinsic GTPase activity (Nobes, 1998).

At the subcellular level, Rnd1 is concentrated at adherens junctions both in confluent fibroblasts and in epithelial cells. Rnd1 has a low affinity for GDP and spontaneously exchanges nucleotide rapidly in a physiological buffer. Rnd1 lacks intrinsic GTPase activity, suggesting that in vivo, it might be constitutively in a GTP-bound form. Expression of Rnd1 or Rnd3/RhoE in fibroblasts inhibits the formation of actin stress fibers, membrane ruffles, and integrin-based focal adhesions and induces loss of cell-substrate adhesion leading to cell rounding (hence Rnd for "round"). It is suggested that these proteins control rearrangements of the actin cytoskeleton and changes in cell adhesion (Nobes, 1998).

Signaling proteins from the same family can have markedly different roles in a given cellular context. Expression of one hundred constitutively active human small GTPases is found to induce cell morphologies that fall into nine distinct classes. An algorithm is developed for pairs of classes that predicted amino acid positions that can be exchanged to create mutants with switched functionality. The algorithm was validated by creating switch-of-function mutants for Rac1, CDC42, H-Ras, RalA, Rap2B, and R-Ras3. Contrary to expectations, the relevant residues are mostly outside known interaction surfaces and are structurally far apart from one another. This study shows that specificity in protein families can be explored by combining genome-wide experimental functional classification with the creation of switch-of-function mutants (Heo, 2003).

Fifty six of the expressed small GTPase constructs triggered no significant morphology changes, while 44 others induced marked morphology changes. The induced morphologies were clearly distinguishable from one another and fell into only nine distinct classes. The Rho family members Rho6, Rho7, RhoE, and ARHE induced a marked cell rounding. Cells transfected with CDC42, CDC42h, TC10, and TCL constructs showed extensions of thin processes that have been termed filopodia, while cells transfected with Rac1, Rac2, Rac3, and RhoG constructs extended lamellipodia that consisted of mostly circular membrane sheets. Transfection of RhoA, RhoB, and RhoC constructs induced polymerized actin bundles or stress fibers that reached across the cell. Only RhoD and RhoH did not show a significant morphology change (Heo, 2003).

Arf family small GTPases induced two types of morphologies. Several members of the Arl family induced a shrunken morphology, while Arf6 had one of the most distinct morphologies with multiple characteristics that include broader cell arms, local membrane spreading, filopodia extensions as well as actin polymerization throughout the cell body and along the cell periphery. Within the shrunken morphology class, Arl 1, Arl 2, and Arl 3 could be considered as a subclass with less pronounced shrinkage and occasional induction of short filopodia type processes that have been termed microspikes in other studies (Heo, 2003).

Cells transfected with Ras family small GTPases also show two distinguishable morphology classes. The oncogenic H-, K-, and N-Ras induce a marked polarized morphology with membrane ruffles and strong actin staining at a polar end of the cells, while cells transfected with most of the remaining members show cell spreading combined with hairlike filopodia formation with pronounced polymerized actin boutons at their ends. The spreading of these cells has a resemblance to eyelashes and looks markedly different from the morphology of lamellipodia induced by Rac or RhoG or the polarized morphology induced by Ras (Heo, 2003).

Finally, several of the Rab family members also have a strong effect on cell morphology. Rab4B, Rab13, Rab22A, Rab23, and Rab35 induce a local spread morphology characterized by local lamellipodia extensions and occasional filopodia induction. Rab8 and Rab8B have the most dramatic effect on cell morphology of all constructs tested and, like Arf6, fall into the multiple morphology class characterized by large branched structures with local lamellipodia and filopodia (Heo, 2003).

In conclusion, this study shows that the structural fold of Ras superfamily small GTPases can induce nine different morphology classes. Furthermore, the residues have been discovered that define the filopodia, lamellipodia, polar, and eyelash morphologies and it was unexpectedly found that the locations of the switch-of-function sites are mostly outside the known effector interaction surfaces and are far apart from each other. These engineered small GTPases with a changed functional selectivity will be useful as tools in pull-down assays to identify the function-specific binding partners as perturbation constructs to investigate crosstalk between signaling processes and for testing whether particular cell functions are physiologically relevant by creating mutant model organisms. Finally, this study introduced an algorithm and a genome-based experimental classification strategy that can be employed to classify the functional space of protein families and to understand the structural basis of functional specificity (Heo, 2003).

Rho GEFs (Rho activating proteins)

Signaling pathways that link extracellular factors to activation of the monomeric guanosine triphosphatase (GTPase) Rho protein control cytoskeletal rearrangements and cell growth. Heterotrimeric guanine nucleotide-binding proteins (G proteins) participate in several of these pathways, although their mechanisms are unclear. The GTPase activities of two G protein alpha subunits, Galpha12 and Galpha13, are stimulated by the Rho guanine nucleotide exchange factor p115 RhoGEF. Activated Galpha13 binds tightly to p115 RhoGEF and stimulates its capacity to catalyze nucleotide exchange on Rho. In contrast, activated Galpha12 inhibits stimulation by Galpha13. Thus, p115 RhoGEF can directly link heterotrimeric G protein alpha subunits to regulation of Rho (Hart, 1998).

Members of the regulators of G protein signaling (RGS) family stimulate the intrinsic guanosine triphosphatase (GTPase) activity of the alpha subunits of certain heterotrimeric guanine nucleotide-binding proteins (G proteins). The guanine nucleotide exchange factor (GEF) for Rho, p115 RhoGEF, has an amino-terminal region with similarity to RGS proteins. Recombinant p115 RhoGEF and a fusion protein containing the amino terminus of p115 have specific activity as GTPase activating proteins toward the alpha subunits of the G proteins G12 and G13, but not toward members of the Gs, Gi, or Gq subfamilies of Galpha proteins. This GEF may act as an intermediary in the regulation of Rho proteins by G13 and G12. A family of mammalian RGS-containing proteins has been identified, and most members stimulate the GTPase activity of mammalian G proteins. The RGS sequence therefore defines a family of GTPase-activating proteins (GAPs) capable of downregulating heterotrimeric G proteins. RhoGEF is the first identified target for the G12/13 family of G proteins and serves as a biochemical link between G-protein coupled receptors and activation of Rho (Kozasa, 1998).

Signal transduction pathways that mediate activation of serum response factor (SRF) by heterotrimeric G protein alpha subunits were characterized in transfection systems. Galphaq, Galpha12, and Galpha13 (but not Galphai) activate SRF through RhoA. When Galphaq, alpha12, or alpha13 are coexpressed with a Rho-specific guanine nucleotide exchange factor GEF115, Galpha13 (but not Galphaq or Galpha12) shows synergistic activation of SRF with GEF115. The synergy between Galpha13 and GEF115 depends on the N-terminal part of GEF115, and there is no synergistic effect between Galpha13 and another Rho-specific exchange factor: Lbc. In addition, the Dbl-homology (DH)-domain-deletion mutant of GEF115 inhibits Galpha13- and Galpha12-induced SRF activation, but not GEF115- or Galphaq-induced SRF activation. The DH-domain-deletion mutant also suppresses thrombin- and lysophosphatidic acid-induced SRF activation in NIH 3T3 cells, probably by inhibition of Galpha12/13. The N-terminal part of GEF115 contains a sequence motif that is homologous to the regulator of G protein signaling (RGS) domain of RGS12. RGS12 can inhibit both Galpha12 and Galpha13. Thus, the inhibition of Galpha12/13 by the DH-deletion mutant may be due to the RGS activity of the mutant. The synergism between Galpha13 and GEF115 indicates that GEF115 mediates Galpha13-induced activation of Rho and SRF (Mao, 1998b).

A cDNA for a novel human protein named CDEP was cloned using the subtractive hybridization method between dedifferentiated cartilage cells and overtly differentiated cartilage cells. CDEP cDNA contains an open reading frame encoding 1,045 amino acids in a total length of 3.4 kb. The deduced amino acid sequence reveals that a single polypeptide contains the ezrin-like domain, which is found in cytoskeleton-associated proteins of the band 4.1 superfamily, and the Dbl homology (DH) and pleckstrin homology (PH) domains, which are conserved in the Rho GEF (guanine nucleotide exchange factor) family. Northern blot analysis demonstrates that CDEP mRNA is expressed not only in the differentiated chondrocytes but also in various fetal and adult tissues. Since members of the band 4.1 superfamily and the Rho GEF family are crucial for microfilament organization, the novel protein CDEP may be involved in the adhesion, proliferation, and differentiation of some cell types, including chondrocytes via changes in the cytoskeleton (Koyano, 1997).

Rom2p is a GDP/GTP exchange factor for Rho1p and Rho2p GTPases. Rho proteins have been implicated in control of actin cytoskeletal rearrangements. ROM2 and RHO2 have been identified in a screen for high-copy number suppressors of cik1 delta, a mutant defective in microtubule-based processes in Saccharomyces cerevisiae. A Rom2p::3XHA fusion protein localizes to sites of polarized cell growth, including incipient bud sites, tips of small buds, and tips of mating projections. Disruption of ROM2 results in temperature-sensitive growth defects at 11 degrees C, and at 37 degrees C. rom2 delta cells exhibit morphological defects. At permissive temperatures, rom2 delta cells often form elongated buds and fail to form normal mating projections after exposure to pheromone; at the restrictive temperature, small budded cells accumulate. High-copy number plasmids containing either ROM2 or RHO2 suppress the temperature-sensitive growth defects of cik1 delta and kar3 delta strains. KAR3 encodes a kinesin-related protein that interacts with Cik1p. rom2 delta strains exhibit increased sensitivity to the microtubule depolymerizing drug, benomyl. These results suggest a role for Rom2p in both polarized morphogenesis and the functions of the microtubule cytoskeleton (Manning, 1997).

The pleckstrin homology (PH) domain is an approximately 100 amino acid structural motif found in many cellular signaling molecules, including the Dbl oncoprotein and related, putative guanine nucleotide exchange factors (GEFs). The role of the Dbl PH (dPH) domain has been examined in the activities of oncogenic Dbl. The dPH domain is not involved in the interaction of Dbl with small GTP-binding proteins and is incapable of transforming NIH 3T3 fibroblasts. In contrast, co-expression of the dPH domain with oncogenic Dbl inhibits Dbl-induced transformation. A deletion mutant of Dbl, which lacks a significant portion of the PH domain, retains full GEF activity, but is completely inactive in transformation assays. Replacement of the PH domain by the membrane-targeting sequence of Ras is not sufficient for the recovery of transforming activity. However, subcellular fractionations of Dbl and Dbl mutants reveal that the PH domain is necessary and sufficient for the association of Dbl with the Triton X-100-insoluble cytoskeletal components. Thus, these results suggest that the dPH domain mediates cellular transformation by targeting the Dbl protein to specific cytoskeletal locations to activate Rho-type small GTP-binding proteins (Zheng, 1996).

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 Rho bound 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) (see Drosophila Dlar), a cDNA was isolated encoding a 2861-amino acid protein termed Trio, containing 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. As 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).

The DH domain protein mNET1, a Rho-family guanine nucleotide exchange factor (GEF) has been characterized. N-terminal truncation of mNET1 generates an activated transforming form of the protein, mNET1DeltaN, which acts as a GEF for RhoA but not Cdc42 or Rac1. In NIH 3T3 cells, activated mNET1 induces formation of actin stress fibers and potentiates activity of the transcription factor serum response factor. Inhibitor studies show that these processes are dependent on RhoA and independent of Cdc42 or Rac1. However, in contrast to the GTPase-deficient RhoA.V14 mutant, expression of activated mNET1 also activates the SAPK/JNK pathway. This requires mNET1 GEF activity, since activation is blocked by point mutations in mNET1's DH domain and its C-terminal pleckstrin homology (PH) domain, and by the dominant-interfering RhoA mutant RhoA.N19. Although mNET1DeltaN-induced SAPK/JNK activation requires a C3 transferase-sensitive GTPase, activation occurs independent of the generation of titratable GTP-bound RhoA. Thus, mNET1 can activate signaling pathways in addition to those directly controlled by activated RhoA (Alberts, 1998b).

Two different families of Rho GEFs have been identified that differ in the structure of their catalytic domains. The first group is composed of Rho GDP dissociation stimulators (GDS), a family of proteins distantly related to the Cdc25 homology regions present in Ras GEFs. GDSs work at stoichiometric concentrations and have a rather broad catalytic specificity, being active on prenylated K-Ras, Rho and Rap proteins. The second subset of Rho activators comprises an extensive number of enzymes containing Dbl-homology (DH) domains with catalytic activity exclusively directed towards Rho/Rac GTPases. The majority of these GEFs are highly transforming when overexpressed either as wild-type or truncated proteins, a property that highlights their importance as regulators of mitogenic processes. Although Rho GEFs have been characterized extensively both biochemically and oncogenically, little information is available regarding the mechanism by which they become activated during signal transduction. To date, the best example for the participation of a DH-containing protein in receptor-mediated cell signaling is the product of the vav proto-oncogene, a protein preferentially expressed in the hematopoietic system. Vav-2, a member of the Vav family of oncoproteins, acts as a guanosine nucleotide exchange factor (GEF) for RhoG and RhoA-like GTPases in a phosphotyrosine-dependent manner. Vav-2 oncogenic activation correlates with the acquisition of phosphorylation-independent exchange activity. In vivo, wild-type Vav-2 is activated oncogenically by tyrosine kinases, an effect enhanced further by co-expression of RhoA. Likewise, the Vav-2 oncoprotein synergizes with RhoA and RhoB proteins in cellular transformation. Transient transfection assays in NIH-3T3 cells show that phosphorylated wild-type Vav-2 and the Vav-2 oncoprotein induce cytoskeletal changes resembling those observed by the activation of the RhoG pathway. In contrast, the constitutive expression of the Vav-2 oncoprotein in rodent fibroblasts leads to major alterations in cell morphology and to highly enlarged cells in which karyokinesis and cytokinesis frequently are uncoupled. These results identify a regulated GEF for the RhoA subfamily, provide a biochemical explanation for vav family oncogenicity, and establish a new signaling model in which specific Vav-like proteins couple tyrosine kinase signals with the activation of distinct subsets of the Rho/Rac family of GTPases (Schuebel, 1998).

Small GTP-binding proteins of the Rho family play a critical role in signal transduction. However, there is still very limited information on how they are activated by cell surface receptors. A consensus sequence for Dbl domains of Rho guanine nucleotide exchange factors (GEFs) was used to search DNA data bases, and a novel human GEF for Rho-related GTPases was identified harboring structural features indicative of its possible regulatory mechanism(s). This protein contains a tandem DH/PH domain closely related to those of Rho-specific GEFs, a PDZ domain, a proline-rich domain, and an area of homology to Lsc, p115-RhoGEF, and a Drosophila RhoGEF. The repeat domain has been termed the Lsc-homology (LH) domain. This novel molecule, designated PDZ-RhoGEF, activates biological and biochemical pathways specific for Rho, and activation of these pathways requires an intact DH and PH domain. However, the PDZ domain is dispensable for these functions, and mutants lacking the LH domain are more active, suggesting a negative regulatory role for the LH domain. A search for additional molecules exhibiting an LH domain reveals a limited homology with the catalytic region of RGS14, a newly identified GTPase-activating protein for heterotrimeric G proteins. This prompted an investigation to see whether PDZ-RhoGEF could interact with representative members of each G protein family. PDZ-RhoGEF is able to form, in vivo, stable complexes with two members of the Galpha12 family (Galpha12 and Galpha13), and this interaction is mediated by the LH domain. Evidence suggests that PDZ-RhoGEF mediates the activation of Rho by Galpha12 and Galpha13. Together, these findings suggest the existence of a novel mechanism whereby the large family of cell surface receptors that transmit signals through heterotrimeric G proteins activate Rho-dependent pathways: they appear to do this by stimulating the activity of members of the Galpha12 family, which, in turn, activate an exchange factor acting on Rho (Fukuhara, 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).

Production of the essential phospholipid PI4P at the Golgi by the Pik1 kinase is required for protein secretion, while a distinct pool of PI4P generated by the Stt4 kinase is critical for normal actin cytoskeleton organization. A transmembrane protein has been identified in yeast that stabilizes Stt4 at the plasma membrane where it directs synthesis of PI4P, which is required for activation of the Rho1/Pkc1-mediated MAP kinase cascade. Inactivation of Stt4 or the PI4P 5-kinase Mss4 results in mislocalization of the Rho-GTPase GEF Rom2. Rom2 binds PI4,5P2 through its PH domain and represents the first identified effector in the Stt4-Mss4 pathway. Based on these results, it is proposed that Stt4-Mss4 generates PI4,5P2 at the plasma membrane, required to recruit/activate effector proteins such as Rom2 (Audhya, 2002).

Plexins represent a novel family of transmembrane receptors that transduce attractive and repulsive signals mediated by the axon-guiding molecules semaphorins. Emerging evidence implicates Rho GTPases in these biological events. However, Plexins lack any known catalytic activity in their conserved cytoplasmic tails, and how they transduce signals from semaphorins to Rho is still unknown. This study shows that Plexin B2 associates directly with two members of a recently identified family of Dbl homology/pleckstrin homology containing guanine nucleotide exchange factors for Rho, PDZ-RhoGEF, and Leukemia-associated Rho GEF (LARG). This physical interaction is mediated by their PDZ domains and a PDZ-binding motif found only in Plexins of the B family. In addition, ligand-induced dimerization of Plexin B is sufficient to stimulate endogenous RhoA potently and to induce the reorganization of the cytoskeleton. Moreover, overexpression of the PDZ domain of PDZ-RhoGEF but not its regulator of G protein signaling domain prevents cell rounding and neurite retraction of differentiated PC12 cells induced by activation of endogenous Plexin B1 by semaphorin 4D. The association of Plexins with LARG and PDZ-RhoGEF thus provides a direct molecular mechanism by which semaphorins acting on Plexin B can control Rho, thereby regulating the actin-cytoskeleton during axonal guidance and cell migration (Perrot, 2002).

XGef was isolated in a screen for proteins interacting with CPEB, a regulator of mRNA translation in early Xenopus development. XGef is a Rho-family guanine nucleotide exchange factor and activates Cdc42 in mammalian cells. Endogenous XGef (58 kDa) interacts with recombinant CPEB, and recombinant XGef interacts with endogenous CPEB in Xenopus oocytes. Injection of XGef antibodies into stage VI Xenopus oocytes blocks progesterone-induced oocyte maturation and prevents the polyadenylation and translation of c-mos mRNA; injection of XGef rescues these events. Overexpression of XGef in oocytes accelerates progesterone-induced oocyte maturation and the polyadenylation and translation of c-mos mRNA. Overexpression of a nucleotide exchange deficient version of XGef, which retains the ability to interact with CPEB, no longer accelerates oocyte maturation or Mos synthesis, suggesting that XGef exchange factor activity is required for the influence of overexpressed XGef on oocyte maturation. XGef overexpression continues to accelerate c-mos polyadenylation in the absence of Mos protein, but does not stimulate MAPK phosphorylation, MPF activation, or oocyte maturation, indicating that XGef may function through the Mos pathway to influence oocyte maturation. These results suggest that XGef may be an early acting component of the progesterone-induced oocyte maturation pathway (Reverte, 2003).

Focal adhesion kinase (FAK) is a protein-tyrosine kinase that associates with multiple cell surface receptors and signaling proteins through which it can modulate the activity of several intracellular signaling pathways. FAK activity can influence the formation of distinct actin cytoskeletal structures such as lamellipodia and stress fibers in part through effects on small Rho GTPases, although the molecular interconnections of these events are not well defined. This study reports that FAK interacts with p190RhoGEF, a RhoA-specific GDP/GTP exchange factor, in neuronal cells and in brain tissue extracts by co-immunoprecipitation and co-localization analyses. Using a two-hybrid assay and deletion mutagenesis, the binding site of the FAK C-terminal focal adhesion targeting (FAT) domain was identified within the C-terminal coiled-coil domain of p190RhoGEF. Binding was independent of a LD-like binding motif within p190RhoGEF, yet FAK association was disrupted by a mutation (Leu-1034 to Ser) that weakens the helical bundle structure of the FAK FAT domain. Neuro-2a cell binding to laminin increased endogenous FAK and p190RhoGEF tyrosine phosphorylation, and co-transfection of a dominant-negative inhibitor of FAK activity, termed FRNK, inhibited lamininstimulated p190RhoGEF tyrosine phosphorylation and p21 RhoA GTP binding. Overexpression of FAK in Neuro-2a cells increased both endogenous p190RhoGEF tyrosine phosphorylation and RhoA activity, whereas these events were inhibited by FRNK co-expression. Because insulin-like growth factor 1 treatment of Neuro-2a cells increased FAK tyrosine phosphorylation and enhanced p190RhoGEF-mediated activation of RhoA, these results support the conclusion that FAK association with p190RhoGEF functions as a signaling pathway downstream of integrins and growth factor receptors to stimulate Rho activity (Zhai, 2003).

Small GTPases of the Rho family are crucial regulators of actin cytoskeleton rearrangements. Rho is activated by members of the Rho guanine-nucleotide exchange factor (GEF) family; however, mechanisms that regulate RhoGEFs are not well understood. This report demonstrates that PDZ-RhoGEF, a member of a subfamily of RhoGEFs that contain regulator of G protein signaling domains, is partially localized at or near the plasma membranes in 293T, COS-7, and Neuro2a cells, and this localization is coincident with cortical actin. Disruption of the cortical actin cytoskeleton in cells by using latrunculin B prevents the peri-plasma membrane localization of PDZ-RhoGEF. Coimmunoprecipitation and F-actin cosedimentation assays demonstrate that PDZ-RhoGEF binds to actin. Extensive deletion mutagenesis revealed the presence of a novel 25-amino acid sequence in PDZ-RhoGEF, located at amino acids 561-585, that is necessary and sufficient for localization to the actin cytoskeleton and interaction with actin. Last, PDZ-RhoGEF mutants that fail to interact with the actin cytoskeleton display enhanced Rho-dependent signaling compared with wild-type PDZ-RhoGEF. These results identify interaction with the actin cytoskeleton as a novel function for PDZ-RhoGEF, thus implicating actin interaction in organizing PDZ-RhoGEF signaling (Banerjee, 2004).

Calcium sensitization in smooth muscle is mediated by the RhoA GTPase, activated by hitherto unspecified nucleotide exchange factors (GEFs) acting downstream of Galphaq/Galpha(12/13) trimeric G proteins. At least one potential GEF, the PDZRhoGEF, is present in smooth muscle, and its isolated DH/PH fragment induces calcium sensitization in the absence of agonist-mediated signaling. In vitro, the fragment shows high selectivity for the RhoA GTPase. Full-length fragment is required for the nucleotide exchange, as the isolated DH domain enhances it only marginally. The DH/PH fragment of PDZRhoGEF was crystallized in complex with nonprenylated human RhoA and the structure was determined at 2.5 Å resolution. The refined molecular model reveals that the mutual disposition of the DH and PH domains is significantly different from other previously described complexes involving DH/PH tandems, and that the PH domain interacts with RhoA in a unique mode. The DH domain makes several specific interactions with RhoA residues not conserved among other Rho family members, suggesting the molecular basis for the observed specificity (Derewenda, 2004).

The activity of Rho GTPases is carefully timed to control epithelial proliferation and differentiation. RhoA is downregulated when epithelial cells reach confluence, resulting in inhibition of signaling pathways that stimulate proliferation. GEF-H1/Lfc, a guanine nucleotide exchange factor for RhoA, directly interacts with cingulin, an adaptor protein that is a component of vertebrate tight junctions. Cingulin binding inhibits RhoA activation and signaling, suggesting that the increase in cingulin expression in confluent cells causes downregulation of RhoA by inhibiting GEF-H1/Lfc. In agreement, RNA interference of GEF-H1 or transfection of GEF-H1 binding cingulin mutants inhibit G1/S phase transition of MDCK cells, and depletion of cingulin by regulated RNA interference results in irregular monolayers and RhoA activation. These results indicate that forming epithelial tight junctions contribute to the downregulation of RhoA in epithelia by inactivating GEF-H1 in a cingulin-dependent manner, providing a molecular mechanism whereby tight junction formation is linked to inhibition of RhoA signaling (Aijaz, 2005).

During Xenopus development, convergent extension movements mediated by cell intercalation drive axial elongation. While many genes required for convergent extension have been identified, little is known of regulation of the cytoskeleton during these cell movements. Although microtubules are required for convergent extension, this applies only to initial stages of gastrulation, between stages 10 and 10.5. To examine the cytoskeleton more directly during convergent extension, actin and microtubules were visualized simultaneously in live explants using spinning disk confocal fluorescence microscopy. Microtubule depolymerization by nocodazole inhibits lamellipodial protrusions and cell-cell contact, thereby inhibiting convergent extension. However, neither taxol nor vinblastine, both of which block microtubule dynamics while stabilizing a polymer form of tubulin, inhibits lamellipodia or convergent extension. This suggests an unusual explanation: the mass of polymerized tubulin, not dynamics of the microtubule cytoskeleton, is crucial for convergent extension. Because microtubule depolymerization elicits striking effects on actin-based protrusions, the role of Rho-family GTPases was tested. The effects of nocodazole are partially rescued using dominant negative Rho, Rho-kinase inhibitor, or constitutively active Rac, suggesting that microtubules regulate small GTPases, possibly via a guanine-nucleotide exchange factor. Full-length XLfc, a microtubule-binding Rho-GEF, was cloned. Nucleotide exchange activity of XLfc is required for nocodazole-mediated inhibition of convergent extension; constitutively active XLfc recapitulates the effects of microtubule depolymerization. Morpholino knockdown of XLfc abrogates the ability of nocodazole to inhibit convergent extension. Therefore, it is believed that XLfc is a crucial regulator of cell morphology during convergent extension, and microtubules limit its activity through binding to the lattice (Kwan, 2005).

Human ARHGEF11, a PDZ-domain-containing Rho guanine nucleotide exchange factor (RhoGEF), has been studied primarily in tissue culture, where it exhibits transforming ability, associates with and modulates the actin cytoskeleton, regulates neurite outgrowth, and mediates activation of Rho in response to stimulation by activated Gα12/13 or Plexin B1. The fruit fly homolog, RhoGEF2, interacts with heterotrimeric G protein subunits to activate Rho, associates with microtubules, and is required during gastrulation for cell shape changes that mediate epithelial folding. This study reports functional characterization of a zebrafish homolog of ARHGEF11 that is expressed ubiquitously at blastula and gastrula stages and is enriched in neural tissues and the pronephros during later embryogenesis. Similar to its human homolog, zebrafish Arhgef11 stimulates actin stress fiber formation in cultured cells, whereas overexpression in the embryo of either the zebrafish or human protein impairs gastrulation movements. Loss-of-function experiments utilizing a chromosomal deletion that encompasses the arhgef11 locus, and antisense morpholino oligonucleotides designed to block either translation or splicing, produces embryos with ventrally-curved axes and a number of other phenotypes associated with ciliated epithelia. Arhgef11-deficient embryos often exhibit altered expression of laterality markers, enlarged brain ventricles, kidney cysts, and an excess number of otoliths in the otic vesicles. Although cilia form and are motile in these embryos, polarized distribution of F-actin and Na+/K+-ATPase in the pronephric ducts is disturbed. These studies in zebrafish embryos have identified new, essential roles for this RhoGEF in ciliated epithelia during vertebrate development (Panizzi, 2007).

RhoGEFs are specialized proteins that directly bind to and activate Rho family GTPases in response to upstream regulatory signals, thus linking extracellular signals with intracellular responses. At least 70 RhoGEFs have been identified in the human genome, many of which have homologs in other vertebrate and invertebrate species. Most of these RhoGEFs possess both Dbl-homology (DH) and Pleckstrin-homology (PH) domains in tandem. These domains often interact specifically with the target GTPase(s) and constitute the functional nucleotide exchange subunit. In addition to the DH and PH domains, many RhoGEFs possess domains that modulate their functions and connect Rho to a variety of signaling pathways. The regulator of G protein-coupled signaling (RGS) domains link G protein-coupled signaling to Rho by binding the alpha subunits of activated heterotrimeric G proteins to RhoGEFs. The family of RGS-domain-containing RhoGEFs includes ARHGEF1 (p115RhoGEF), ARHGEF12 (Leukemia Associated RhoGEF, LARG) and ARHGEF11 (KIAA0380, PDZ-RhoGEF). Both ARHGEF11 and ARHGEF12 also contain a PDZ (PSD-95/DLG/ZO1) domain, which has been shown to interact with the C-terminus of the Semaphorin receptor, Plexin B1. Through this interaction, Semaphorin 4D stimulation of Plexin B1 activates Rho signaling pathways and influences axon guidance. These studies demonstrate that activation of Rho via ARHGEF11 and ARHGEF12 can be modulated through both the PDZ and RGS domains (Panizzi, 2007).

Thus far, human ARHGEF11 and its closely related family member, ARHGEF12, have been studied primarily in cell culture, where they activate Rho and promote reorganization of the actin cytoskeleton in response to stimulation by heterotrimeric G protein subunits Gα12/13. In addition to the work illuminating the interactions and roles of the conserved domains of ARHGEF11, further studies have delineated other regions of the protein that may be important for its function. One such region is the C-terminus, which may interact with p-21 activated kinase 4 (PAK4), or homo- or heterodimerize with the C-terminus of another ARHGEF11 or ARHGEF12 molecule. Moreover, a small region of ARHGEF11 between the RGS and DH domains has been shown to interact with the actin cytoskeleton. These reported interactions and functions were determined using yeast and mammalian cell culture systems, where both ARHGEF11 and ARHGEF12 exhibit very similar activities. Interestingly, there is evidence from mouse studies suggesting that the expression profiles of these two proteins are somewhat different, with ARHGEF11 detected predominantly in neural tissues and ARHGEF12 found in both neural and non-neural tissues. Therefore, although ARHGEF11 and ARHGEF12 seem to have mostly redundant activities in cultured cells, these functions may be modulated differently and in a tissue-specific manner (Panizzi, 2007).

Most of the knowledge of in vivo roles for ARHGEF11 has come from work conducted with Drosophila RhoGEF2, which contains all the major domains and displays significant sequence similarity with ARHGEF11. RhoGEF2 was first identified in a screen for Rho signaling pathway components, and was further shown to control cell shape changes during gastrulation. This RhoGEF also associates with microtubules via the plus-end-binding protein, EB1, and regulates actomyosin contraction in the epithelia of the developing embryo. Additionally, apical distribution of RhoGEF2 and other Rho activators together with the basolateral distribution of Rho inhibitors are required to modulate actin via Rho during cell invagination and lumen formation for proper development of the spiracle in the fruit fly (Panizzi, 2007 and references therein).

Studies of these vertebrate and invertebrate PDZ-domain-containing RhoGEFs suggest they may act similarly to regulate the cytoskeleton by modulating Rho in response to G protein-coupled signaling, possibly while associating with actin and/or microtubules. Despite all the information garnered from cultured cells and the D. melanogaster system, the roles of ARHGEF11 in developing and adult vertebrates remain to be elucidated. This study uses the zebrafish model, which is particularly amenable to in vivo analysis, to assess the functions of a zebrafish homolog of human ARHGEF11 during vertebrate development. Employing several loss-of-function approaches, new and unanticipated roles have been identified for this vertebrate RhoGEF in processes involving ciliated epithelia, including establishment of left-right asymmetry, formation of otoliths in the otic vesicle, and development of the pronephros (Panizzi, 2007).

Formation of the mitotic cleavage furrow is dependent upon both microtubules and activity of the small GTPase RhoA. GEF-H1 is a microtubule-regulated exchange factor that couples microtubule dynamics to RhoA activation. GEF-H1 localized to the mitotic apparatus in HeLa cells, particularly at the tips of cortical microtubules and the midbody, and perturbation of GEF-H1 function induced mitotic aberrations, including asymmetric furrowing, membrane blebbing, and impaired cytokinesis. The mitotic kinases Aurora A/B and Cdk1/Cyclin B phosphorylate GEF-H1, thereby inhibiting GEF-H1 catalytic activity. Dephosphorylation of GEF-H1 occurs just prior to cytokinesis, accompanied by GEF-H1-dependent GTP loading on RhoA. Using a live cell biosensor, distinct roles have been demonstrated for GEF-H1 and Ect2 in regulating Rho activity in the cleavage furrow, with GEF-H1 catalyzing Rho activation in response to Ect2-dependent localization and initiation of cell cleavage. These results identify a GEF-H1-dependent mechanism to modulate localized RhoA activation during cytokinesis under the control of mitotic kinases (Birkenfeld, 2007).

The guanine nucleotide exchange factor p63RhoGEF is an effector of the heterotrimeric guanine nucleotide-binding protein (G protein) Galphaq and thereby links Galphaq-coupled receptors (GPCRs) to the activation of the small-molecular-weight G protein RhoA. The crystal structure of the Galphaq-p63RhoGEF-RhoA complex was determined, detailing the interactions of Galphaq with the Dbl and pleckstrin homology (DH and PH) domains of p63RhoGEF. These interactions involve the effector-binding site and the C-terminal region of Galphaq and appear to relieve autoinhibition of the catalytic DH domain by the PH domain. Trio, Duet, and p63RhoGEF are shown to constitute a family of Galphaq effectors that appear to activate RhoA both in vitro and in intact cells. It is proposed that this structure represents the crux of an ancient signal transduction pathway that is expected to be important in an array of physiological processes (Lutz, 2007).

The Rho GTPases-Rho, Rac, and Cdc42-regulate the dynamics of F-actin (filamentous actin) and myosin-2 with considerable subcellular precision. Consistent with this ability, active Rho and Cdc42 occupy mutually exclusive zones during single-cell wound repair and asymmetric cytokinesis, suggesting the existence of mechanisms for local crosstalk, but how local Rho GTPase crosstalk is controlled is unknown. Using a candidate screen approach for Rho GTPase activators (guanine nucleotide exchange factors; GEFs) and Rho GTPase inactivators (GTPase-activating proteins; GAPs), Abr, a protein with both GEF and GAP activity, was found to regulate Rho and Cdc42 during single-cell wound repair. Abr is targeted to the Rho activity zone via active Rho. Within the Rho zone, Abr promotes local Rho activation via its GEF domain and controls local crosstalk via its GAP domain, which limits Cdc42 activity within the Rho zone. Depletion of Abr attenuates Rho activity and wound repair. Abr is the first identified Rho GTPase regulator of single-cell wound healing. Its novel mode of targeting by interaction with active Rho allows Abr to rapidly amplify local increases in Rho activity using its GEF domain while its ability to inactivate Cdc42 using its GAP domain results in sharp segregation of the Rho and Cdc42 zones. Similar mechanisms of local Rho GTPase activation and segregation enforcement may be employed in other processes that exhibit local Rho GTPase crosstalk (Vaughan, 2011).

Table of contents

Rho1: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.