EphA receptors regulate growth cone dynamics through the novel guanine nucleotide exchange factor ephexin

Eph receptors transduce short-range repulsive signals for axon guidance by modulating actin dynamics within growth cones. We report the cloning and characterization of ephexin, a novel Eph receptor-interacting protein that is a member of the Dbl family of guanine nucleotide exchange factors (GEFs) for Rho GTPases. Ephrin-A stimulation of EphA receptors modulates the activity of ephexin leading to RhoA activation, Cdc42 and Rac1 inhibition, and cell morphology changes. In addition, expression of a mutant form of ephexin in primary neurons interferes with ephrin-A-induced growth cone collapse. The association of ephexin with Eph receptors constitutes a molecular link between Eph receptors and the actin cytoskeleton and provides a novel mechanism for achieving highly localized regulation of growth cone motility.     

Eph/ephrin signaling in morphogenesis, neural development and plasticity

Ephrins are cell-surface-tethered ligands for Eph receptors, the largest family of receptor tyrosine kinases. During development, the Eph/ephrin cell communication system appears to influence cell behavior such as attraction/repulsion, adhesion/de-adhesion and migration, thereby influencing cell fate, morphogenesis and organogenesis. During adulthood, the Eph/ephrin system continues to play roles in tissue plasticity, for example in shaping dendritic spines during neuronal plasticity. Mechanistically, Eph-ephrin repulsive behavior appears to require ligand-receptor internalization and signaling to Rho GTPases.     

Eph receptor-ephrin bidirectional signals that target Ras and Rho proteins

The ability of cells to respond to their surrounding environment and relay signals to the cell interior is essential for numerous processes during the development and maintenance of tissues. Eph receptors and their membrane-bound ligands, the ephrins, are unique in the receptor tyrosine kinase family in that their signaling is bidirectional, through both the receptor and the ligand. Eph receptors and ephrins are essential for a variety of biological processes, and play a particularly important role in regulating cell shape and cell movement. Recent data have linked Eph receptor-ephrin signaling complexes to the Ras and Rho families of small molecular weight GTPases and also to heterotrimeric G proteins. Understanding the signaling networks involved is an important step to understand the molecular basis for normal and defective cell-cell communication through Eph receptors and ephrins.     

Bait and switch: synthetic GEFs divert an input signal to diverse cellular responses

The Rho family GTPases regulate many cellular functions including the organization, mechanics, and dynamics of the actin cytoskeleton. Re-engineering the highly conserved autoinhibitory structure of GEFs, Yeh et al. (2007) generated novel synthetic proteins that regulated Rho GTPase signaling and cell morphology, highlighting the power of protein module engineering.     

A novel signaling intermediate, SHEP1, directly couples Eph receptors to R-Ras and Rap1A.

The Eph family of receptor tyrosine kinases has been implicated in many developmental patterning processes, including cell segregation, cell migration, and axon guidance. The cellular components involved in the signaling pathways of the Eph receptors, however, are incompletely characterized. Using a yeast two-hybrid screen, we have identified a novel signaling intermediate, SHEP1 (SH2 domain-containing Eph receptor-binding protein 1), which is expressed in the embryonic and adult brain. SHEP1 contains an Src homology 2 domain that binds to a conserved tyrosine-phosphorylated motif in the juxtamembrane region of the EphB2 receptor and may itself be a target of EphB2 kinase activity, since it becomes heavily tyrosine-phosphorylated in cells expressing activated EphB2. SHEP1 also contains a domain similar to Ras guanine nucleotide exchange factor domains and binds to the GTPases R-Ras and Rap1A, but not Ha-Ras or RalA. Thus, SHEP1 directly links activated, tyrosine-phosphorylated Eph receptors to small Ras superfamily GTPases.     

Sizes of interface residues account for cross‐class binding affinity patterns in Eph receptor–ephrin families

Eph receptors comprise the largest known family of receptor tyrosine kinases in mammals. They bind members of a second family, the ephrins. As both Eph receptors and ephrins are membrane bound, interactions permit unusual bidirectional cell–cell signaling. Eph receptors and ephrins each form two classes, A and B, based on sequences, structures, and patterns of affinity: Class A Eph receptors bind class A ephrins, and class B Eph receptors bind class B ephrins. The only known exceptions are the receptor EphA4, which can bind ephrinB2 and ephrinB3 in addition to the ephrin‐As (Bowden et al., Structure 2009;17:1386–1397); and EphB2, which can bind ephrin‐A5 in addition to the ephrin‐Bs (Himanen et al., Nat Neurosci 2004;7:501–509). A crystal structure is available of the interacting domains of the EphA4‐ephrin B2 complex (wwPDB entry 2WO2) (Bowden et al., Structure 2009;17:1386–1397). In this complex, the ligand‐binding domain of EphA4 adopts an EphB‐like conformation. To understand why other cross‐class EphA receptor–ephrinB complexes do not form, we modeled hypothetical complexes between (1) EphA4–ephrinB1, (2) EphA4–ephrinB3, and (3) EphA2–ephrinB2. We identify particular residues in the interface region, the size variations of which cause steric clashes that prevent formation of the unobserved complexes. The sizes of the sidechains of residues at these positions correlate with the pattern of binding affinity. Proteins 2014; 82:349–353. © 2013 Wiley Periodicals, Inc.     

Beta2-adrenergic receptor/cyclic adenosine monophosphate (cAMP) leads to JNK activation through Rho family small GTPases.

Gi- and Gq-coupled G protein-coupled receptors (GPCRs) have been shown to activate c-Jun N-terminal kinase (JNK), a subfamily of mitogen-activated protein kinases (MAPKs), through Rho family small GTPases in mammalian cells. We investigated the signaling pathway linking the Gs-coupled beta2-adrenergic receptor with JNK, using smooth muscle DDT1 MF-2 cells, which natively express the beta2-adrenergic receptor. Stimulation of the beta2-adrenergic receptor activated JNK in a time-dependent manner, and a cell-permeable cyclic adenosine monophosphate analogue (8-Br-cAMP) activated JNK. The beta2-adrenergic receptor- or 8-Br-cAMP-induced activation of JNK required Rho family small GTPases. Also, the beta2-adrenergic receptor or 8-Br-cAMP induced activation of Rho family small GTPases. These results demonstrate that the beta2-adrenergic receptor/cAMP leads to JNK activation through Rho family small GTPases in DDT1 MF-2 cells. Activation of Rho family small GTPases may provide a common step in GPCR-mediated JNK activation.     

Rho GTPases in neuronal morphogenesis

The Rho family of small GTPases act as intracellular molecular switches that transduce signals from extracellular stimuli to the actin cytoskeleton and the nucleus. Recent evidence implicates Rho GTPases in the regulation of neuronal morphogenesis, including migration, polarity, axon growth and guidance, dendrite elaboration and plasticity, and synapse formation. Signalling pathways from membrane receptors to Rho GTPases and from Rho GTPases to the actin cytoskeleton are beginning to be discovered. Mutations in these signalling pathways have been reported in human neurological diseases, which underscores their importance in the development and function of the nervous system.     

Dialogue between RhoA/ROCK and members of the Par complex in cell polarity

Cell polarity is essential for many biological processes and is regulated by conserved protein complexes, including the Par complex, Rho GTPases, and their regulators. In this issue of Developmental Cell, studies by Nakayama et al. and Zhang and Macara examine how interplay between Rho GTPases and the Par complex control polarized cell migration and dendritic spine morphogenesis in alternate ways.     

Galphaq-dependent activation of mitogen-activated protein kinase kinase 4/c-Jun N-terminal kinase cascade.

G-protein-coupled receptors (GPCRs) typically activate c-Jun N-terminal kinase (JNK) through the G protein betagamma subunit (Gbetagamma), in a manner dependent on Rho family small GTPases, in mammalian cells. Here we show that JNK activation by the prototypic Gq-coupled alpha1B-adrenergic receptor is mediated by the alpha subunit of Gq (Galphaq), not by Gbetagamma, using a transient transfection system in human embryonic kidney cells. JNK activation by the alpha1B-adrenergic receptor/Galphaq was selectively mediated by mitogen-activated protein kinase kinase 4 (MKK4), but not MKK7. Also, MKK4 activation by the alpha1B-adrenergic receptor/Galphaq required c-Src and Rho family small GTPases. Furthermore, activation of the alpha1B-adrenergic receptor stimulated JNK activity through Src family tyrosine kinases and Rho family small GTPases in hamster smooth muscle cells that natively express the alpha1B-adrenergic receptor. Together, these results suggest that the alpha1B-adrenergic receptor/Galphaq may up-regulate JNK activity through a MKK4 pathway dependent on c-Src and Rho family small GTPases in mammalian cells.     

I’m coming to GEF you: Regulation of RhoGEFs during cell migration

Cell migration is a highly regulated multistep process that requires the coordinated regulation of cell adhesion, protrusion, and contraction. These processes require numerous protein–protein interactions and the activation of specific signaling pathways. The Rho family of GTPases plays a key role in virtually every aspect of the cell migration cycle. The activation of Rho GTPases is mediated by a large and diverse family of proteins; the guanine nucleotide exchange factors (RhoGEFs). GEFs work immediately upstream of Rho proteins to provide a direct link between Rho activation and cell–surface receptors for various cytokines, growth factors, adhesion molecules, and G protein-coupled receptors. The regulated targeting and activation of RhoGEFs is essential to coordinate the migratory process. In this review, we summarize the recent advances in our understanding of the role of RhoGEFs in the regulation of cell migration.     

EphA4 catalytic activity causes inhibition of RhoA GTPase in Xenopus laevis embryos

The Eph family of receptor tyrosine kinases is involved in limiting cell and tissue interactions via a repulsive mechanism. The mechanism of repulsion involves reorganizing the actin cytoskeleton, but little is known of the molecular components that connect the receptor to the actin cytoskeleton. Recent studies in retinal ganglion cells have demonstrated that EphA4 activates the small GTPase Rho. We have investigated the involvement of Rho in signaling downstream from EphA4. As a model system, we have used a chimeric receptor called EPP that we express and activate in early Xenopus embryos. Previous studies demonstrated that EPP activation leads to loss of cell-cell adhesion and change in cell shape, plus loss of aspects of cell polarity in epithelial cells, such as apical microvilli and the apical/basolateral boundary. In this study, we show that injecting inhibitors of Rho GTPases into early Xenopus embryos produces a phenotype very similar to that resulting from EPP activation. More importantly, expression of a constitutively active form of Xenopus RhoA (XRhoA) concurrent with activated EPP rescued embryos from the loss of cell-cell adhesion and change in cell shape associated with EPP. These data argue that, in contrast to the case in retinal ganglion cells, EphA4 in early Xenopus embryos acts to inhibit RhoA, suggesting that this receptor may regulate Rho differently (and therefore affect the cytoskeleton differently) in neuronal and non-neuronal cells. Furthermore, overexpression of ephexin, a novel guanine nucleotide exchange factor for Rho family GTPases, also blocks EPP-induced dissociation. This suggests that EphA4, which has been demonstrated to activate ephexin in cultured neuronal cells, may also target Rho GTPase via an ephexin-independent pathway.     

Rho family GTPases as key regulators for neuronal network formation

Rho family GTPases act as transducers of signals from extracellular stimuli to the cytoskeleton and gene expression. Their actions are temporal and spatial determinants for cellular functions. The cellular functions of Rho family GTPases have been studied in fibroblasts and endothelial cells, and recent advances have revealed their roles in the regulation of neuronal network formation, including migration, neurite outgrowth, polarity, axon guidance, dendrite maturation and synapse formation. In addition, a significant number of X-linked mental retardation genes have been shown to encode components directly involved in signal transduction pathways of Rho family GTPases, underscoring the view that Rho family GTPases essentially participate in the neuronal network formation. In this review, we will overview current understanding of the functions of Rho family GTPases in neuronal network formation.     

Clostridial toxins: Molecular probes of Rho-dependent signaling and apoptosis

The Rho family small GTPases are members of the Ras superfamily of small GTPases. Rho proteins were first determined to act as key regulators of many types of actin cytoskeletal-dependent cellular functions. Recent work by several investigators indicates that Rho GTPases are also critical modulators of several important intracellular and nuclear signal transduction pathways. Certain clostridial toxins and exoenzymes covalently modify, and thereby inactivate, specific types of Rho family GTPases. As such, these microbial enzymes have proven invaluable in helping to identify structural and functional attributes of Rho GTPases.     

Two poles and a compass

Rho GTPases control fundamental aspects of neutrophil chemotaxis: establishment of front and back and orientation toward the chemoattractant. Two reports in this issue show that activated Cdc42 at the leading edge helps orient the cell's axis in a signaling complex with G beta gamma, PAK1, and PIX alpha; while Rho, activated via G alpha 13, mediates formation of the uropod, which then interacts by mutual negative feedback with the front to reinforce polarization (Li et al., 2003 [this issue of Cell]; Xu et al., [this issue of Cell]).     

The Small GTPases Rho and Rac Are Required for the Establishment of Cadherin-dependent Cell–Cell Contacts

Cadherins are calcium-dependent cell–cell adhesion molecules that require the interaction of the cytoplasmic tail with the actin cytoskeleton for adhesive activity. Because of the functional relationship between cadherin receptors and actin filament organization, we investigated whether members of the Rho family of small GTPases are necessary for cadherin adhesion. In fibroblasts, the Rho family members Rho and Rac regulate actin polymerization to produce stress fibers and lamellipodia, respectively. In epithelial cells, we demonstrate that Rho and Rac are required for the establishment of cadherin-mediated cell–cell adhesion and the actin reorganization necessary to stabilize the receptors at sites of intercellular junctions. Blocking endogenous Rho or Rac selectively removed cadherin complexes from junctions induced for up to 3 h, while desmosomes were not perturbed. In addition, withdrawal of cadherins from intercellular junctions temporally precedes the removal of CD44 and integrins, other microfilament-associated receptors. Our data showed that the concerted action of Rho and Rac modulate the establishment of cadherin adhesion: a constitutively active form of Rac was not sufficient to stabilize cadherindependent cell–cell contacts when endogenous Rho was inhibited. Upon induction of calcium-dependent intercellular adhesion, there was a rapid accumulation of actin at sites of cell–cell contacts, which was prevented by blocking cadherin function, Rho or Rac activity. However, if cadherin complexes are clustered by specific antibodies attached to beads, actin recruitment to the receptors was perturbed by inhibiting Rac but not Rho. Our results provide new insights into the role of the small GTPases in the cadherin-dependent cell– cell contact formation and the remodelling of actin filaments in epithelial cells.     

ARAP3 is a PI3K- and rap-regulated GAP for RhoA

Rho and Arf family small GTPases are well-known regulators of cellular actin dynamics. We recently identified ARAP3, a member of the ARAP family of dual GTPase activating proteins (GAPs) for Arf and Rho family GTPases, in a screen for PtdIns(3,4,5)P(3) binding proteins. PtdIns(3,4,5)P(3) is the lipid product of class I phosphoinositide 3OH-kinases (PI3Ks) and is a signaling molecule used by growth factor receptors and integrins in the regulation of cell dynamics. We report here that as a Rho GAP, ARAP3 prefers RhoA as a substrate and that it can be activated in vitro by the direct binding of Rap proteins to a neighbouring Ras binding domain (RBD). This activation by Rap is GTP dependent and specific for Rap versus other Ras family members. We found no evidence for direct regulation of ARAP3's Rho GAP activity by PtdIns(3,4,5)P(3) in vitro, but PI3K activity was required for activation by Rap in a cellular context, suggesting that PtdIns(3,4,5)P(3)-dependent translocation of ARAP3 to the plasma membrane may be required for further activation by Rap. Our results indicate that ARAP3 is a Rap-effector that plays an important role in mediating PI3K-dependent crosstalk between Ras, Rho, and Arf family small GTPases.     

Semaphorin-mediated axonal guidance via Rho-related G proteins.

For many growing axons, interaction with an extracelluar Semaphorin signal leads to growth cone collapse and axon repulsion. Semaphorin receptors composed of Neuropilins and Plexins transduce extracellular cues into changes in the growth cone actin cytoskeleton. The data implicating Rho family G proteins in Semaphorin signaling and in other axon guidance events are considered here. Recent work makes it clear that Rac1 is required for this process. In particular, there is intriguing new evidence that the Plexin receptors communicate directly with members of the Rho family GTPases, although uncertainties remain concerning how Plexins alter Rac1 function. The CRMP (collapsin response mediator protein) family is also required for Plexin-based Semaphorin signaling, and new data demonstrate direct links to Rho and Rac1-based signaling.