The Rho GTPases form a subgroup of the Ras superfamily of 20- to 30-kD GTP-binding proteins that have been shown to regulate a wide spectrum of cellular functions. These proteins are ubiquitously expressed across the species, from yeast to man. The mammalian Rho-like GTPases comprise at least 10 distinct proteins: RhoA, B, C, D, and E; Rac1 and 2; RacE; Cdc42Hs, and TC10. A comparison of the amino acid sequences of the Rho proteins from various species has revealed that they are conserved in primary structure and are 50%–55% homologous to each other. Like all members of the Ras superfamily, the Rho GTPases function as molecular switches, cycling between an inactive GDP-bound state and an active GTP-bound state. Until recently, members of the Rho subfamily were believed to be involved primarily in the regulation of cytoskeletal organization in response to extracellular growth factors. However, research from a number of laboratories over the past few years has revealed that the Rho GTPases play crucial roles in diverse cellular events such as membrane trafficking, transcriptional regulation, cell growth control, and development. Consequently, a major challenge has been to unravel the underlying molecular mechanisms by which the Rho GTPases mediate these various activities. Many targets of the Rho GTPases have now been identified and further characterization of some of them has provided major insights toward our understanding of Rho GTPase function at the molecular level. This review aims to summarize the general established principles about the Rho GTPases and some of the more recent exciting findings, hinting at novel, unanticipated functions of the Rho GTPases.

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Rho GTPases and signaling networks

Rho GTPases are molecular switches that regulate many essential cellular processes, including actin dynamics, gene transcription, cell-cycle progression and cell adhesion. About 30 potential effector proteins have been identified that interact with members of the Rho family, but it is still unclear which of these are responsible for the diverse biological effects of Rho GTPases. This review will discuss how Rho GTPases physically interact with, and regulate the activity of, multiple effector proteins and how specific effector proteins contribute to cellular responses. To date most progress has been made in the cytoskeleton field, and several biochemical links have now been established between GTPases and the assembly of filamentous actin. The main focus of this review will be Rho, Rac and Cdc42, the three best characterized mammalian Rho GTPases, though the genetic analysis of Rho GTPases in lower eukaryotes is making increasingly important contributions to this field.

Rho GTPases and their effector proteins.

Guanine nucleotide-exchange factors (GEFs) are directly responsible for the activation of Rho-family GTPases in response to diverse extracellular stimuli, and ultimately regulate numerous cellular responses such as proliferation, differentiation and movement. With 69 distinct homologues, Dbl-related GEFs represent the largest family of direct activators of Rho GTPases in humans, and they activate Rho GTPases within particular spatio-temporal contexts. The failure to do so can have significant consequences and is reflected in the aberrant function of Dbl-family GEFs in some human diseases.

GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors

Rho GTPases control many aspects of cell behavior through the regulation of multiple signal transduction pathways (Van Aelst and D’Souza-Schorey 1997; Hall 1998). Rho, Rac, and Cdc42 were first recognized in the early 1990s for their unique ability to induce specific filamentous actin structures in fibroblasts; stress fibers, lamellipodia/membrane ruffles, and filopodia, respectively (Hall 1998). Over the intervening years, evidence has accumulated to show that in all eukaryotic cells, Rho GTPases are involved in most, if not all, actin-dependent processes such as those involved in migration, adhesion, morphogenesis, axon guidance, and phagocytosis (Kaibuchi et al. 1999; Chimini and Chavrier 2000; Luo 2000). In addition to their well-established roles in controlling the actin cytoskeleton, Rho GTPases regulate the microtubule cytoskeleton, cell polarity, gene expression, cell cycle progression, and membrane transport pathways (Van Aelst and D’Souza-Schorey 1997; Daub et al. 2001; Etienne-Manneville and Hall 2001). With such a prominent role in so many aspects of cell biology, it is not surprising that they are themselves highly regulated. Like all GTPases, Rho proteins act as binary switches by cycling between an inactive (GDP-bound) and an active (GTP-bound) conformational state (Fig. 1; Van Aelst and D’Souza-Schorey 1997). The cell controls this switch by regulating the interconversion and accessibility of these two forms in a variety of ways. Guanine nucleotide exchange factors (GEFs) stimulate the exchange of GDP for GTP to generate the activated form, which is then capable of recognizing downstream targets, or effector proteins. GTPase activating proteins (GAPs) accelerate the intrinsic GTPase activity of Rho family members to inactivate the switch. Finally, guanine nucleotide dissociation inhibitors (GDIs) interact with the prenylated, GDP-bound form to control cycling between membranes and cytosol. In theory, activation of a Rho GTPase could occur through stimulation of a GEF or inhibition of a GAP. In practice, however, all the evidence points to GEFs being the critical mediators of Rho GTPase activation, and this paper reviews our present understanding of how they do this. Structural features

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Guanine nucleotide exchange factors for Rho GTPases: turning on the switch

Background The causes of intellectual disability remain largely unknown because of extensive clinical and genetic heterogeneity. Methods We evaluated patients with intellectual disability to exclude known causes of the disorder. We then sequenced the coding regions of more than 21,000 genes obtained from 100 patients with an IQ below 50 and their unaffected parents. A data-analysis procedure was developed to identify and classify de novo, autosomal recessive, and X-linked mutations. In addition, we used high-throughput resequencing to confirm new candidate genes in 765 persons with intellectual disability (a confirmation series). All mutations were evaluated by molecular geneticists and clinicians in the context of the patients' clinical presentation. Results We identified 79 de novo mutations in 53 of 100 patients. A total of 10 de novo mutations and 3 X-linked (maternally inherited) mutations that had been previously predicted to compromise the function of known intellectual-disability genes were found in 13 patients. Potentially causative de novo mutations in novel candidate genes were detected in 22 patients. Additional de novo mutations in 3 of these candidate genes were identified in patients with similar phenotypes in the confirmation series, providing support for mutations in these genes as the cause of intellectual disability. We detected no causative autosomal recessive inherited mutations in the discovery series. Thus, the total diagnostic yield was 16%, mostly involving de novo mutations. Conclusions De novo mutations represent an important cause of intellectual disability; exome sequencing was used as an effective diagnostic strategy for their detection. (Funded by the European Union and others.).

https://www.nejm.org/doi/pdf/10.1056/NEJMoa1206524

Diagnostic Exome Sequencing in Persons with Severe Intellectual Disability

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

https://www.pnas.org/content/pnas/93/11/5466.full.pdf

The Multidomain Protein Trio Binds the LAR Transmembrane Tyrosine Phosphatase, Contains a Protein Kinase Domain, and Has Separate Rac-Specific and Rho-Specific Guanine Nucleotide Exchange Factor Domains

Dbl-homology guanine nucleotide exchange factors (DH-GEFs) regulate actin cytoskeletal reorganization, cell adhesion, and gene transcription via activation of Rho GTPases. However, little is known about the physiological role of mammalian DH-GEFs during development. The DH-GEF family member Trio is of particular interest because it is a multifunctional protein possessing two GEF domains, as well as a protein serine/threonine kinase domain, and trio-like genes in Caenorhabditis elegans and Drosophila were shown to function in neural migration and axon guidance. To determine the role of Trio during mammalian development, we generated a mouse trio loss-of-function mutation (trio−/−). Trio function is essential during late embryonic development as genotype analysis indicated that trio−/− embryos died between embryonic day (E)-15.5 and birth, or shortly thereafter. In the trio−/− embryos, primary skeletal myofibers were relatively normal at E14.5, but by E18.5 highly unusual spherical myofibers accumulated. Trio deficiency may cause a defect in secondary myogenesis, as the appearance of the abnormal trio−/− skeletal myofibers temporally coincided with the onset of secondary myogenesis, and smaller secondary myofibers located adjacent to the primary myofibers were absent. The proliferation of trio−/− secondary myoblasts appeared normal, suggesting that Trio may regulate secondary myoblast alignment or fusion. trio−/− embryos also displayed aberrant organization in several regions within the brain, including the hippocampal formation and olfactory bulb. We thus conclude that Trio is essential for late embryonic development, and that Trio functions in fetal skeletal muscle formation and in the organization of neural tissues.

https://www.pnas.org/content/pnas/97/22/12074.full.pdf

Skeletal muscle deformity and neuronal disorder in Trio exchange factor-deficient mouse embryos.

Reorganization of the actin cytoskeleton is essential to numerous cellular processes including cell locomotion and cytokinesis. This actin remodeling is regulated in part by Rho family GTPases. Previous studies implicated Trio, a Dbl-homology guanine nucleotide exchange factor with two exchange factor domains, in regulating actin cytoskeleton reorganization, cell motility and cell growth via activation of Rho GTPases. Trio is essential for mouse embryonic development and Trio-deficiency is associated with abnormal skeletal muscle and neural tissue development. Furthermore, genetic analyses in Caenorhabditis elegans and Drosophila demonstrate a role for trio-like genes in cell migration and axon guidance. Herein we characterize a novel Trio-binding protein, Tara, that is comprised of an N-terminal pleckstrin homology domain and a C-terminal coiled-coil region. Trio and Tara associate as assessed by the yeast interaction-trap assays and mammalian co-immunoprecipitation studies. Ectopically expressed Tara localizes to F-actin in a periodic pattern that is highly similar to the pattern of myosin II. Furthermore, a direct interaction between Tara and F-actin is indicated by in vitro binding studies. Cells that transiently or stably overexpress Tara display an extensively flattened cell morphology with enhanced stress fibers and cortical F-actin. Tara expression does not alter the ability of the cell to attach or to initially spread, but rather increases cell spreading following these initial events. Tara stabilizes F-actin structures as indicated by the relative resistance of Tara-expressing cells to the F-actin destabilizer Latrunculin B. We propose that Tara regulates actin cytoskeletal organization by directly binding and stabilizing F-actin, and that the localized formation of Tara and Trio complexes functions to coordinate actin remodeling.

Tara, a novel F-actin binding protein, associates with the Trio guanine nucleotide exchange factor and regulates actin cytoskeletal organization

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

Trio amino-terminal guanine nucleotide exchange factor domain expression promotes actin cytoskeleton reorganization, cell migration and anchorage-independent cell growth.

Katja Seipel

Papers

The Multidomain Protein Trio Binds the LAR Transmembrane Tyrosine Phosphatase, Contains a Protein Kinase Domain, and Has Separate Rac-Specific and Rho-Specific Guanine Nucleotide Exchange Factor Domains

Skeletal muscle deformity and neuronal disorder in Trio exchange factor-deficient mouse embryos.

Tara, a novel F-actin binding protein, associates with the Trio guanine nucleotide exchange factor and regulates actin cytoskeletal organization

Trio amino-terminal guanine nucleotide exchange factor domain expression promotes actin cytoskeleton reorganization, cell migration and anchorage-independent cell growth.

The trio guanine nucleotide exchange factor is a RhoA target. Binding of RhoA to the trio immunoglobulin-like domain.