Regulation of Rho-Family GTPase Signaling by ARHGEF25 Proteins
Both GEFT and p63RhoGEF exhibit isoform specificity for the Rho-GTPases. p63RhoGEF appears to be ras homology gene family, member A (RhoA) specific, while GEFT has been shown to activate RhoA, ras-related C3 botulinum toxin substrate 1 (Rac1), and cell division cycle 42 (Cdc42) in a cell type–dependent manner. For instance, overexpression of GEFT in fibroblasts drives activation of Rac1 and Cdc42 leading to membrane protrusions (Guo et al. 2003); however, GEFT regulates gene expression and cell differentiation in skeletal muscle, neuronal cells, and lens epithelial cells through activation of multiple GTPases (Bryan et al. 2004, 2005, 2006; Mitchell et al. 2011). Rho-GTPase specificity of this nature is not at all surprising as similar isoform-specific regulation of Rho family members has been observed with the ARHGEF25 paralog MCF2 as well as other GEFs (Komai et al. 2002). p63RhoGEF induces RhoA-dependent stress fiber formation in fibroblasts and in H9C2 cardiac myoblasts (Souchet et al. 2002), while GEFT stimulates the formation of lamellipodia, actin microspikes, and filopodia, similar to overexpression of constitutively active Rac1 and Cdc42 mutants (Guo et al. 2003; Bryan et al. 2004, 2006). p63RhoGEF has been shown to promote serum response factor (SRF) activity through a RhoA-specific pathway, while GEFT reportedly modulates the activity and/or subcellular localization of a number of transcription factors including SRF, activating protein 1 (AP1), nuclear factor kappa beta (NFkappaβ), myogenic differentiation 1 (MyoD), peroxisome proliferator-activated receptor gamma (PPARγ), and activating transcription factor 2 (ATF2) through RhoA, Rac1, and Cdc42 signaling (Bryan et al. 2004, 2005). Moreover, overexpression of GEFT stimulates the activation of numerous downstream kinases including Rho-associated, coiled-coil containing protein kinase (ROCK), p21 protein–activated kinase 1 ( PAKI), p21 protein–activated kinase 5 (Pak5), mitogen-activated protein kinase 14 (MAPK14), mitogen-activated protein kinase 8 (MAPK8), and p42/44 MAPK (Bryan et al. 2004, 2005) and promotes crystallin gene expression in lens epithelial cells (Mitchell et al. 2011). p63RhoGEF is expressed most highly in the heart and brain (Souchet et al. 2002). The expression of GEFT is detected at the highest levels in the excitable tissues (brain, heart, and skeletal muscle), while lower levels of protein expression are observed in a number of diverse tissues (Guo et al. 2003). An extensive analysis of GEFT expression in the developing and adult eye was recently performed by Mitchell et al. (2011), detecting high levels of the protein in the neuroblastic layer and differentiating lens fibers of the late-stage mouse embryo, and in the postnatal corneal epithelium, lens epithelium, and throughout the retina.
ARHGEF25 Protein Regulation of Muscle Physiology
Both p63RhoGEF and GEFT appear to play important roles in skeletal, cardiac, and smooth muscle function. For instance, p63RhoGEF is expressed highly in cardiomyocytes, where it strongly colocalizes with myosin at the sarcomeric I-band (Souchet et al. 2002). These data suggest the p63RhoGEF may be connected directly or indirectly to actin thin filaments and may play an important role in cardiac muscle contraction. In vascular smooth muscle cells, knockdown of endogenous p63RhoGEF ablates angiotensin II-mediated proliferation, actin stress fiber formation, longitudinal focal adhesion arrangement, and peripheral distribution of vimentin (Wuertz et al. 2010). Moreover, Wuertz et al. demonstrated that depletion of endogenous p63RhoGEF ablates angiotensin II-induced smooth muscle contraction using an in vitro collagen matrix assay. GEFT is expressed highly in the embryonic mouse limb bud, during differentiation of skeletal muscle precursors, and in established adult skeletal muscle (Bryan et al. 2005). Moreover, GEFT expression in cardiotoxin-damaged skeletal muscle is detected at high levels during the late phases of tissue regeneration, suggesting a role in the differentiation phase rather than the proliferative phase of muscle regeneration. Subcellular fractionation reveals that GEFT is wholly cytoplasmic, and fluorescence staining demonstrates that GEFT strongly colocalizes to the actin cytoskeleton in both undifferentiated skeletal muscle precursors and in differentiated multinucleated myotubes. Exogenous overexpression of GEFT in skeletal muscle precursors leads to activation of RhoA, Rac1, and Cdc42 and their downstream effector proteins, and promotes myogenesis, inhibition of adipogenesis, and enhances in vivo tissue regeneration following skeletal muscle injury (Bryan et al. 2005).
ARHGEF25 Protein Regulation of Neuronal Physiology
p63RhoGEF expression has been detected in the cell bodies of astrocytes and oligodendrocytes localized in the cerebellar cortex (Souchet et al. 2002), while GEFT levels are observed throughout the adult brain, with prominent expression in the hippocampus, Purkinje cells, and granular region of the cerebellum (Bryan et al. 2005). Subcellularly, GEFT colocalizes to actin-rich regions, particularly those found in axons and dendrites (Bryan et al. 2006). Exogenous expression of GEFT promotes dendritic outgrown in cultured hippocampal neurons, resulting in a higher abundance and increased size of mature dendritic spines compared to the control (Bryan et al. 2005). GEFT activates RhoA, Rac1, and Cdc42 signaling in neuronal cells, and strongly enhances neurite outgrowth in neuroblastoma cells in a Pak1/Pak5-dependent manner, as well promotes axon and dendrite formation during neuronal cell differentiation (Bryan et al. 2005, 2006). Additionally, GEFT expression is highly upregulated during retinoic acid- or dibutyric cyclic adenosine monophosphate (cAMP)-induced neuronal cell differentiation (Bryan et al. 2006). During neuronal development, neural precursor cells migrate, differentiate, and extend axons and dendrites to specific regions to form synapses with appropriate target cells. Multiple GEFs have been implicated in neuronal morphonesis, growth cone guidance, and neuronal dendritic spines, and these data suggest that GEFT could play a strong role in neuronal guidance and pathfinding.
ARHGEF25 Protein Regulation of Ocular Development
High levels of Rho GTPase expression in the developing and adult eye suggest their importance in the morphogenesis and maintenance of ocular components (Mitchell et al. 2007). GEFT expression has been detected in mice as early as 9 days p.c. in the neuroepithelial tissue adjacent to the lumen of the optic vessel, and is notably expressed in differentiating lens fibers at later developmental stages (Mitchell et al. 2011). In the adult mouse eye, GEFT is observed in corneal and lens epithelium, and in the axons and cell bodies of the retinal ganglion cells of the optic nerve layer, the nerve fibers of the inner and outer plexiform layers, and the photoreceptor layer containing the rods and cones. In both in vitro and ex vivo systems, GEFT promotes lens epithelium differentiation through regulation of the expression of multiple lens crystallin and filensin genes through modulating the activation and subcellular localization of Rac1.
ARHGEF25 and Oncogenic Properties
In addition to normal physiological properties, GEFT has been shown to promote the oncogenic properties of transformed cells. The gene encoding GEFT was initially identified using an enhanced retroviral mutagen strategy selecting for foci-forming complementary DNAs (cDNAs) expressed in a human brain library (Liu et al. 2000), and its overexpression strongly induces foci-formation, proliferation, and migration in transformed fibroblasts (Guo et al. 2003). Moreover, expression of the ARHGEF25 gene is significantly elevated in approximately 4% of adenomas and carcinomas utilizing a large scale microarray study on 950 human tumor lines (https://array.nci.nih.gov/caarray/project/woost-00041).
Known Protein–Protein Interactions with ARHGEF Proteins
p63RhoGEF has been shown to physically interact with the activated G-protein coupled receptors (GPCR) G-alpha(q) and G-alpha11, but not with G-alpha12 or 13 (Lutz et al. 2005). This interaction occurs independently and in competition with the activation of the canonical G-alpha(q/11) effector phospholipase C beta, and strongly enhances the activity of p63RhoGEF. As has been reported for many GEF proteins, in the unbound state the C-terminal pleckstrin homology domain of p63RhoGEF folds over and autoinhibits its Dbl catalytic domain (Rojas et al. 2007; Lutz et al. 2007; Shankaranarayanan et al. 2010). This autoinhibition is relieved upon interaction of the GPCR with the highly conserved C-terminal extension of the p63RhoGEF PH domain, thus coupling GPCR stimulation to Rho-GTPase activation. Alternatively, p63RhoGEF can serve as an inhibitor of GPCR function by forming a stable complex with activated G-alpha16 to inhibit its activation of phospholipase beta2, Ras, and Stat3 activation via competitive inhibition (Yeung and Wong 2009).
Several inhibitors of p63RhoGEF activity have been recently identified. p63RhoGEF, G protein coupled receptor kinase 2 (GRK2), and RGS GTPase activating proteins form a ternary complex with G-alpha(q)-coupled receptors (Shankaranarayanan et al. 2008). RGS 2 and RGS4 serve as negative allosteric regulators of G-alpha(q) binding to p63RhoGEF, thus inhibiting RhoA activation and downstream signaling. Moreover, the mixed lineage kinase 3 ( MLK3), a MAPK3 protein that normally activates the JNK-dependent MAPK pathways, binds directly to p63RhoGEF and prevents its G-alpha(q)-mediated activation of Rho-signaling pathways (Swenson-Fields et al. 2008). GEFT was recently identified as a protein that colocalizes and directly interacts with the cytoplasmic region of the Popdc family integral membrane protein Bves (blood vessel epicardial substance) in cardiac, smooth, and skeletal muscle (Smith et al. 2008). This interaction inhibits GEFT activity and leads to a reduction in cell movement and an increase in cell roundness via blocking of Rac1 and Cdc42 activity.
The ARHGEF25 gene encodes for two key GEF isoforms, p63RhoGEF and GEFT. These proteins are responsible for modulation of the activity of Rho-GTPases and their downstream signaling pathways in muscle and neuronal cell lineages. Future studies will likely uncover more roles and signaling cross talk for these protein products in a variety of tissues.