Rho Family of Guanine Exchange Factors (GEFs) in Cellular Activation
Who’s Dancing? And With Whom?
Over the past decade, an increasing appreciation has developed for the role of small GTP-binding proteins (GTPases) as critical molecular switches. Small GTPases function as important regulators of multiple cellular processes by transducing signals from extracellular stimuli to intracellular effector pathways. The three primary members of the Rho family of GTPases include Rho, Rac, and Cdc42, whose collective activation is most closely linked to actin cytoskeletal rearrangement (see review1). Classically, Rho activation promotes the assembly of contractile actomyosin stress fibers, Rac activation produces lamellipodia and membrane ruffles, and Cdc42 induces filopodia formation. Because they regulate essential cellular processes such as cell shape, migration, transcription, vesicle trafficking, and barrier function, these proteins represent important potential targets for therapeutic intervention in a diverse array of human diseases. As a result, Rho GTPase regulatory pathways have been the focus of intensive investigation.
Rho GTPases are inactive when GDP-bound and active when GTP-bound. Cycling between these states is controlled by three known classes of regulatory proteins: GTPase-activating proteins (GAPs), guanine nucleotide dissociation inhibitors (GDIs), and guanine nucleotide exchange factors (GEFs). GAPs and GDIs negatively regulate Rho GTPase activation by promoting the GDP-bound state. Conversely, activation of the Rho family is directly controlled by the GEFs, which stimulate the exchange of GDP for GTP (see Figure). 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.2 A growing number of Rho family GEFs have recently been identified, with a majority as members of the Dbl family of GEF proteins containing a Dbl homology (DH) domain (named for the dbl oncogene product identified in a diffuse B-cell lymphoma) and a pleckstrin homology (PH) domain located just C-terminal of the DH site.2 Functionally, the DH domain is responsible for catalyzing guanine exchange activity while the PH domain appears to be involved in intracellular localization of the GEF protein. Approximately 40 of these Dbl family GEFs have now been identified, with specific GEFs exhibiting individual Rho GTPase and/or cell-specific activity. For example, p115RhoGEF/Lsc specifically activates RhoA, and FRG appears to be specific for Cdc42, whereas other Dbl GEFs are known to exhibit the capacity to activate additional Rho family proteins.2,3⇓ When one considers the possible interactions between these 40 GEFs combined with the approximately 20 Rho GTPases present in human cells (plus the additional regulation provided by GAPs and GDIs), the impressive complexity of these signaling pathways is readily apparent.
In this issue of Circulation Research, Niu and colleagues4 have added significant clarity to this dizzying array of potential “Rho dancing partners” by characterizing the recently identified p114RhoGEF. Like all Dbl family members, p114RhoGEF contains tandemly linked DH and PH domains, but interestingly, has very little sequence homology with the better characterized Rho-specific p115RhoGEF despite their similar molecular weights.5 Utilizing deletion construct analysis in a series of experiments designed to outline the functional role of p114RhoGEF in fibroblasts, the authors demonstrate lysophosphatidic acid (LPA) engagement of G protein–coupled receptors releases Gβγ subunits to stimulate p114RhoGEF, culminating in Rho protein activation and subsequent downstream effects (see Figure). This Gβγ subunit stimulation of p114RhoGEF contrasts with the Gα induction seen in p115RhoGEF activation.6 The precise molecular mechanism through which these heterotrimeric G protein subunits stimulate GEF activity, however, remains unclear.
Although the initial report of p114RhoGEF was described as a Rho-specific activator,5 Niu et al4 now convincingly demonstrate Rac GTPase activation as well. While this apparent discrepancy may reflect differences in the cell type studied (NIH3T3 fibroblasts compared with HEK-293 cells) or sensitivities of the protocols used to assay Rho family protein activation, it appears clear that overexpression of p114RhoGEF in this study induced generation of reactive oxygen species through NADPH oxidase activation, a process stimulated by Rac.7 Interestingly, and certainly perplexing, overexpression of p114RhoGEF also produced significant Rho activation leading to stress fiber formation and cell rounding. Similar to other reported coactivating GEFs for Rho and Rac,2 p114RhoGEF provides a potential modulator through which Rho and Rac downstream effects can be coordinated. The dynamic interaction of these two pathways is of particular importance in endothelial cell systems and the regulation of vascular permeability, since Rho activation produces stress fiber formation, cellular contraction, and increased endothelial cell (EC) permeability whereas Rac activation in ECs produces cytoskeletal changes associated with reduced vascular leak.8,9⇓ LPA has been reported to have both barrier-stabilizing10 as well as barrier-disrupting11 effects on cultured ECs, suggesting that differences in cell type–specific expression of p114RhoGEF and/or LPA concentration may partially account for the specific Rac- or Rho-associated barrier-regulatory phenotype produced by LPA stimulation. Better understanding of these complex regulatory interactions may help improve our ability to modulate vascular permeability in a clinically relevant and beneficial way.
The question as to which dancing partners (GEFs) interact with specific Rho family GTPases is critical to our understanding of individual cellular responses to extracellular stimuli. The intricate balance among GDIs, GAPs, and GEFs in their control of Rho family protein activation becomes more complicated as additional members of each of these groups are identified and characterized. However, because these pathways play essential roles in vital, yet diverse, cellular processes such as motility, vascular permeability, and transcription, these layers of complexity provide multiple potential therapeutic targets for intervention in human disease. The results of the study by Niu and colleagues4 shed new light on these signaling pathways and move us a little closer to understanding their integration. The regulation of Rho family protein activation continues to be an area of active, dance-provoking research.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
- ↵Hall A. Rho GTPases and the actin cytoskeleton. Science. 1998; 279: 509–514.
- ↵Miyamoto Y, Yamauchi J, Itoh H. Src kinase regulates the activation of a novel FGD-1-related Cdc42 guanine nucleotide exchange factor in the signaling pathway from the endothelin A receptor to JNK. J Biol Chem. 2003; 278: 29890–29900.
- ↵Niu J, Profirovic J, Pan H, Vaiskunaite R, Voyno-Yasenetskaya T. G protein βγ subunits stimulate p114RhoGEF, a guanine nucleotide exchange factor for RhoA and Rac1: regulation of cell shape and reactive oxygen species production. Circ Res. 2003; 93: 848–856.
- ↵Blomquist A, Schworer G, Schablowski H, Psoma A, Lehnen M, Jakobs KH, Rumenapp U. Identification and characterization of a novel Rho-specific guanine nucleotide exchange factor. Biochem J. 2000; 352 (pt 2): 319–325.
- ↵Hart MJ, Jiang X, Kozasa T, Roscoe W, Singer WD, Gilman AG, Sternweis PC, Bollag G. Direct stimulation of the guanine nucleotide exchange activity of p115 RhoGEF by Gα13. Science. 1998; 280: 2112–2114.
- ↵Dudek SM, Garcia JG. Cytoskeletal regulation of pulmonary vascular permeability. J Appl Physiol. 2001; 91: 1487–1500.
- ↵English D, Kovala AT, Welch Z, Harvey KA, Siddiqui RA, Brindley DN, Garcia JG. Induction of endothelial cell chemotaxis by sphingosine 1-phosphate and stabilization of endothelial monolayer barrier function by lysophosphatidic acid, potential mediators of hematopoietic angiogenesis. J Hematother Stem Cell Res. 1999; 8: 627–634.