Rho GDP Dissociation Inhibitors Research Articles

Proteome-wide Characterization and Pathophysiology Correlation in Non-ischemic Cardiomyopathies.

Although the clinical consequences of advanced heart failure (HF) may be similar across different etiologies of cardiomyopathies, their proteomic expression may show substantial differences in relation to underlying pathophysiology. We aimed to identify myocardial tissue-based proteomic characteristics and the underlying molecular pathophysiology in non-ischemic cardiomyopathy with different etiologies. Comparative extensive proteomic analysis of the myocardium was performed in nine patients with biopsy-proven non-ischemic cardiomyopathies (3 dilated cardiomyopathy [DCM], 2 hypertrophic cardiomyopathy [HCM], and 4 myocarditis) as well as five controls using tandem mass tags combined with liquid chromatography-mass spectrometry. Differential protein expression analysis, Gene Ontology (GO) analysis, and Ingenuity Pathway Analysis (IPA) were performed to identify proteomic differences and molecular mechanisms in each cardiomyopathy type compared to the control. Proteomic characteristics were further evaluated in accordance with clinical and pathological findings. The principal component analysis score plot showed that the controls, DCM, and HCM clustered well. However, myocarditis samples exhibited scattered distribution. IPA revealed the downregulation of oxidative phosphorylation and upregulation of the sirtuin signaling pathway in both DCM and HCM. Various inflammatory pathways were upregulated in myocarditis with the downregulation of Rho GDP dissociation inhibitors. The molecular pathophysiology identified by extensive proteomic analysis represented the clinical and pathological properties of each cardiomyopathy with abundant proteomes. Different etiologies of non-ischemic cardiomyopathies in advanced HF exhibit distinct proteomic expression despite shared pathologic findings. The benefit of tailored management strategies considering the different proteomic expressions in non-ischemic advanced HF requires further investigation.

RhoGDI1 regulates cell-cell junctions in polarized epithelial cells.

Cell-cell contact formation of polarized epithelial cells is a multi-step process that involves the co-ordinated activities of Rho family small GTPases. Consistent with the central role of Rho GTPases, a number of Rho guanine nucleotide exchange factors (GEFs) and Rho GTPase-activating proteins (GAPs) have been identified at cell-cell junctions at various stages of junction maturation. As opposed to RhoGEFs and RhoGAPs, the role of Rho GDP dissociation inhibitors (GDIs) during cell-cell contact formation is poorly understood. Here, we have analyzed the role of RhoGDI1/ARHGDIA, a member of the RhoGDI family, during cell-cell contact formation of polarized epithelial cells. Depletion of RhoGDI1 delays the development of linear cell-cell junctions and the formation of barrier-forming tight junctions. In addition, RhoGDI1 depletion impairs the ability of cells to stop migration in response to cell collision and increases the migration velocity of collectively migrating cells. We also find that the cell adhesion receptor JAM-A promotes the recruitment of RhoGDI1 to cell-cell contacts. Our findings implicate RhoGDI1 in various processes involving the dynamic reorganization of cell-cell junctions.

Characterization of myocardial proteomics in biopsy proven cardiomyopathies

Abstract Background Difference in proteomic expression according to the etiology of cardiomyopathies is not well known. Purpose We aimed to identify proteome based pathogenesis in patients with biopsy proven cardiomyopathies. Methods Comparative proteomic analysis of biopsy specimens were performed from 9 patients with cardiomyopathy (3 dilated cardiomyopathy (DCM), 2 hypertrophic cardiomyopathy (HCM) and 4 myocarditis) as well as 5 controls (normal endomyocardial biopsy specimen from one-year surveillance of heart transplant recipients) by tandem mass tag combined with liquid chromatography-mass spectrometry. Differential expression protein analysis, gene ontology analysis and Ingenuity pathway analysis were done to discover molecular mechanism for the differentially expressed proteins in each cardiomyopathy compared to the control. Results Differential expression protein analysis showed higher proportion of significantly increased proteins (Log2 fold change ≥1) in HCM and myocarditis, whereas higher proportion of significantly decreased proteins (Log2 fold change ≤−1) in DCM compared to controls (Figure). According to the gene ontology analysis, upregulation of neutrophil degranulation, and down-regulation of mitochondrial translation protein was noted in patients with DCM. In patients with HCM, platelet degranulation protein was increased, and mitochondrial ATP synthesis coupled electron transport, was decreased. In patients with myocarditis, neutrophil related proteins and calcium ion binding protein was increased, but muscle cell development, protein was decreased. Ingenuity pathway analysis revealed downregulation of oxidative phosphorylation and upregulation of sirtuin signalling pathway both in DCM and HCM. In myocarditis, various pathways related to inflammation were upregulated with only RHO GDP dissociation inhibitors downregulated. Conclusions This study showed that each cardiomyopathy exhibited different proteomic expression compared to normal heart. Further large detailed study is needed to understand the association between proteomic expression and disease pathophysiology. Funding Acknowledgement Type of funding sources: None.

Regulation of Rho GTPases by RhoGDIs in Human Cancers.

Rho GDP dissociation inhibitors (RhoGDIs) play important roles in various cellular processes, including cell migration, adhesion, and proliferation, by regulating the functions of the Rho GTPase family. Dissociation of Rho GTPases from RhoGDIs is necessary for their spatiotemporal activation and is dynamically regulated by several mechanisms, such as phosphorylation, sumoylation, and protein interaction. The expression of RhoGDIs has changed in many human cancers and become associated with the malignant phenotype, including migration, invasion, metastasis, and resistance to anticancer agents. Here, we review how RhoGDIs control the function of Rho GTPases by regulating their spatiotemporal activity and describe the regulatory mechanisms of the dissociation of Rho GTPases from RhoGDIs. We also discuss the role of RhoGDIs in cancer progression and their potential uses for therapeutic intervention.

Prognostic value of Rho GDP dissociation inhibitors in patients with hepatocellular carcinoma following liver transplantation.

Rho GDP dissociation inhibitors (GDIs) are pivotal regulators of Rho GTPases, which are essential for tumor progression, yet their role in hepatocellular carcinoma (HCC) remains poorly understood. The purpose of the present study was to assess the role of RhoGDIs in the invasiveness and migration of liver cancer, and to determine their clinical prognostic significances in HCC following liver transplantation (LT). In the present study, the expression of RhoGDIs was assessed using reverse transcription-quantitative polymerase chain reaction and confirmed by western-blot analysis and immunohistochemistry. Their prognostic values were also analyzed, and determined in patients treated with LT. In addition, the functions of RhoGDIs in liver cancer cell line were studied in vitro. As a result, the downregulation of RhoGDI1 and RhoGDI2 at mRNA and protein levels were detected in HCC when compared with that of adjacent noncancerous tissues (P<0.05). However, the level of RhoGDI3 was identified to be similar in tumor and para-carcinoma tissues. Additionally, Kaplan-Meier curves demonstrated that patients with lower expression of RhoGDI1 or RhoGDI2 exhibited significantly increased risk of tumor recurrence following LT (P=0.007 and P=0.006, respectively). Cox proportional hazards model analysis revealed that the decreased expression level of RhoGDI2 was an unfavorable independent prognostic factor (hazard ratio, 3.306; P=0.001). In vitro studies involving the silencing of RhoGDI1 or RhoGDI2 demonstrated a significant increase in the migratory and invasive ability of tumor cells upon the silencing of these genes. Results from the present study indicate that RhoGDI dysregulation is a frequent event in human HCC, and that it promotes cancer progression by stimulating cell migration and invasion. RhoGDI2 may be a prognostic biomarker for patients with HCC following LT, and act as a potential therapeutic target.

Rho GDIalpha Modulates Rabbit Trophoblast Stem Cell Survival and Migration.

Trophoblast stem cells differentiate into different trophoblast cell populations that are indispensable for successful pregnancy through interactions with the maternal uterine decidua. Rho GTPases play an important role in the regulation of trophoblast stem cell (TSC) self-renewal and differentiation; however, the role of Rho GDP-dissociation inhibitors (Rho GDIs) remains unclear. Here we report that overexpression of Rho GDIalpha resulted in rapid apoptosis of TSCs, while its knockdown promoted proliferation. Moreover, Rho GDIalpha knockdown also enhanced TSC invasion. Collectively, these results establish a potential mechanism whereby TSCs can balance growth and apoptosis, and thus ensure normal fetal development.

Positive regulation of Rho GTPase activity by RhoGDIs as a result of their direct interaction with GAPs.

BackgroundRho GTPases function as molecular switches in many different signaling pathways and control a wide range of cellular processes. Rho GDP-dissociation inhibitors (RhoGDIs) regulate Rho GTPase signaling and can function as both negative and positive regulators. The role of RhoGDIs as negative regulators of Rho GTPase signaling has been extensively investigated; however, little is known about how RhoGDIs act as positive regulators. Furthermore, it is unclear how this opposing role of GDIs influences the Rho GTPase cycle. We constructed ordinary differential equation models of the Rho GTPase cycle in which RhoGDIs inhibit the regulatory activities of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) by interacting with them directly as well as by sequestering the Rho GTPases. Using this model, we analyzed the role of RhoGDIs in Rho GTPase signaling.ResultsThe model constructed in this study showed that the functions of GEFs and GAPs are integrated into Rho GTPase signaling through the interactions of these regulators with GDIs, and that the negative role of GDIs is to suppress the overall Rho activity by inhibiting GEFs. Furthermore, the positive role of GDIs is to sustain Rho activation by inhibiting GAPs under certain conditions. The interconversion between transient and sustained Rho activation occurs mainly through changes in the affinities of GDIs to GAPs and the concentrations of GAPs.ConclusionsRhoGDIs positively regulate Rho GTPase signaling primarily by interacting with GAPs and may participate in the switching between transient and sustained signals of the Rho GTPases. These findings enhance our understanding of the physiological roles of RhoGDIs and Rho GTPase signaling.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-015-0143-5) contains supplementary material, which is available to authorized users.

Cyclic Strain-Induced Cytoskeletal Rearrangement of Human Periodontal Ligament Cells via the Rho Signaling Pathway

BackgroundAlthough mechanical stimulations are known have a significant impact on cytoskeletal rearrangement, little is known regarding the behavioral alteration of human periodontal ligament cells (hPDLCs) under cyclic strain. The aim of this study was to elucidate the role of the Rho signaling pathway on cyclic strain-induced cytoskeletal rearrangement of hPDLCs.MethodsHealthy hPDLCs obtained from teeth extracted for orthodontic purposes were subjected to cyclic strain with physiological loading (10%) at a frequency of 0.1 Hz for 6 h or 24 h using a FX-5000T system. Changes in cell morphology were examined by phase-contrast microscopy, while F-actin reorganization was observed by phalloidin staining and confocal microscopy. Protein expression was analyzed through western blot analysis.ResultsSignificant enhancement of cytoskeletal reorganization was observed following exposure to the cyclic strain. In addition, a significant increase was noted in the expression levels of GTP-Rho, Rho-associated protein kinase (ROCK) and p-cofilin, whereas the expression levels of Rho GDP dissociation inhibitors alpha (Rho-GDIa) were reduced in the hPDLCs, compared with the static control cells. More importantly, the Rock inhibitor Y-27632 suppressed cyclic strain-induced cytoskeletal rearrangement of hPDLCs. Additionally, Y-27632 and overexpression of Rho-GDIa were found to lower p-cofilin protein expressions under cyclic strain, while Rho-GDIa siRNA transfection had the opposite effect on the hPDLCs.ConclusionCyclic strain promotes cytoskeletal rearrangement of hPDLCs by downregulating the expression levels of Rho-GDIa and upregulating the expression levels of GTP-Rho, Rock and p-cofilin. These observations may provide valuable insight into understanding orthodontic tooth movement as well as alveolar bone remodeling.

Negative Charges in the Flexible N-Terminal Domain of Rho GDP-Dissociation Inhibitors (RhoGDIs) Regulate the Targeting of the RhoGDI–Rac1 Complex to Membranes

In its resting state, Rho GDP-dissociation inhibitor (RhoGDI) α forms a soluble cytoplasmic heterodimer with the GDP-bound form of Rac. Upon stimulation, the dissociation of RhoGDIα from the RhoGDIα-Rac complex is a mandatory step for Rac activation; however, this mechanism is poorly understood. In this study, we examined how the cytoplasm/membrane cycles of the RhoGDI-Rac complex are regulated, as well as where RhoGDI dissociates from the RhoGDI-Rac complex, during FcγR-mediated phagocytosis. The negatively charged and flexible N terminus (25 residues) of RhoGDIα, particularly its second negative amino acid cluster possessing five negatively charged amino acids, was a pivotal regulator in the cytoplasm/membrane cycles of the RhoGDI-Rac complex. We also found that RhoGDIα translocated to the phagosomes as a RhoGDIα-Rac1 complex, and this translocation was mediated by an interaction between the polybasic motif in the C terminus of Rac1 and anionic phospholipids produced on phagosomes, such as phosphatidic acid, that is, by a phagosome-targeting mechanism of Rac1. Thus, we demonstrated that the targeting/accumulation of the RhoGDIα-Rac1 complex to phagosomes is regulated by a balance between three factors: 1) the negatively charged and flexible N-terminal of RhoGDIα, 2) the binding affinity of RhoGDIα for Rac1, and 3) anionic phospholipids produced on phagosomes. Moreover, we demonstrated that the mechanism of targeting/accumulation of the RhoGDIα-Rac1 complex is also applicable for the RhoGDIβ-Rac1 complex.

Downregulation of RhoGDIα increased migration and invasion of ER+ MCF7 and ER− MDA-MB-231 breast cancer cells

Rho GDP dissociation inhibitors (RhoGDIs) can inhibit cell motility, invasion, and metastasis in cancer by inactivating the RhoGTPases. A member of RhoGDI family has been consistently shown to interact with estrogen receptor (ER), and change its transcriptional activity. ER is a receptor known to be inversely correlated with cell motility and invasion in breast cancer. The consequence of RhoGDIα activity on migration and invasion of ER+ and ER− breast cancers is not clear. The aim of our study was to investigate the possible opposing effect of RhoGDIα on the migration and invasion of ER+ MCF7 and ER− MDA-MB-231 breast cancer cells. RhoGDIα was downregulated using short interfering RNA (siRNA) and upregulated using GFP-tagged ORF clone of RhoGDIα, and their ability for migration and invasion was assayed using transwell chambers. It was found that the silencing of RhoGDIα in MCF7 and MDA-MB-231 cells significantly increased migration and invasion of these cells into the lower surface of porous membrane of the chambers. Overexpression of RhoGDIα in MCF7 cells suppressed their migration and invasion, but no significant effect was found on MDA-MB-231 cells. Our results indicate that the downregulation of RhoGDIα similarly affects the in vitro migration and invasion of ER+ MCF7 and ER− MDA-MB-231 cells. However, our assays are differently affected by the upregulation of RhoGDIα in these two cell lines and this may be due to the differences in ER expression, primary invasive ability and/or other molecules between these two cell line models which warrant further investigation.

Centrosomal localization of RhoGDIβ and its relevance to mitotic processes in cancer cells

Rho GDP-dissociation inhibitors (RhoGDIs) are regulators of Rho family GTPases. RhoGDIβ has been implicated in cancer progression, but its precise role remains unclear. We determined the subcellular localization of RhoGDIβ and examined the effects of its overexpression and RNAi knockdown in cancer cells. Immunofluorescence staining showed that RhoGDIβ localized to centrosomes in human cancer cells. In HeLa cells, exogenous GFP-tagged RhoGDIβ localized to centrosomes and its overexpression caused prolonged mitosis and aberrant cytokinesis in which the cell shape was distorted. RNAi knockdown of RhoGDIβ led to increased incidence of monopolar spindle mitosis resulting in polyploid cells. These results suggest that RhoGDIβ has mitotic functions, including regulation of cytokinesis and bipolar spindle formation. The dysregulated expression of RhoGDIβ may contribute to cancer progression by disrupting these processes.

Regulation of Neural Stem Cell Differentiation by Transcription Factors HNF4-1 and MAZ-1

Neural stem cells (NSCs) are promising candidates for a variety of neurological diseases due to their ability to differentiate into neurons, astrocytes, and oligodentrocytes. During this process, Rho GTPases are heavily involved in neuritogenesis, axon formation and dendritic development, due to their effects on the cytoskeleton through downstream effectors. The activities of Rho GTPases are controlled by Rho-GDP dissociation inhibitors (Rho-GDIs). As shown in our previous study, these are also involved in the differentiation of NSCs; however, little is known about the underlying regulatory mechanism. Here, we describe how the transcription factors hepatic nuclear factor (HNF4-1) and myc-associated zinc finger protein (MAZ-1) regulate the expression of Rho-GDIγ in the stimulation of NSC differentiation. Using a transfection of cis-element double-stranded oligodeoxynucleotides (ODNs) strategy, referred to as "decoy" ODNs, we examined the effects of HNF4-1 and MAZ-1 on NSC differentiation in the NSC line C17.2. Our results show that HNF4-1 and MAZ-1 decoy ODNs significantly knock down Rho-GDIγ gene transcription, leading to NSC differentiation towards neurons. We observed that HNF4-1 and MAZ-1 decoy ODNs are able enter to the cell nucleolus and specifically bind to their target transcription factors. Furthermore, the expression of Rho-GDIγ-mediated genes was identified, suggesting that the regulatory mechanism for the differentiation of NSCs is triggered by the transcription factors MAZ-1 and HNF4-1. These findings indicate that HNF4-1 and MAZ-1 regulate the expression of Rho-GDIγ and contribute to the differentiation of NSCs. Our findings provide a new perspective within regulatory mechanism research during differentiation of NSCs, especially the clinical application of transcription factor decoys in vivo, suggesting potential therapeutic strategies for neurodegenerative disease.

Coronin 1A promotes a cytoskeletal-based feedback loop that facilitates Rac1 translocation and activation

The activation of the Rac1 GTPase during cell signalling entails its translocation from the cytosol to membranes, release from sequestering Rho GDP dissociation inhibitors (RhoGDI), and GDP/GTP exchange. In addition to those steps, we show here that optimal Rac1 activation during cell signalling requires the engagement of a downstream, cytoskeletal-based feedback loop nucleated around the cytoskeletal protein coronin 1A and the Rac1 exchange factor ArhGEF7. These two proteins form a cytosolic complex that, upon Rac1-driven F-actin polymerization, translocates to juxtamembrane areas where it expands the pool of activated, membrane-bound Rac1. Such activity requires the formation of an F-actin/ArhGEF7-dependent physical complex of coronin 1A with Pak1 and RhoGDIα that, once assembled, promotes the Pak1-dependent dissociation of Rac1 from the Rac1/RhoGDIα complex and subsequent Rac1 activation. Genetic evidence demonstrates that this relay circuit is essential for generating sustained Rac1 activation levels during cell signalling.

Functional analysis of RhoGDI inhibitory activity on vacuole membrane fusion

RhoGDIs (Rho GDP-dissociation inhibitors) are the natural inhibitors of Rho GTPases. They interfere with Rho protein function by either blocking upstream activation or association with downstream signalling molecules. RhoGDIs can also extract membrane-bound Rho GTPases to form soluble cytosolic complexes. We have shown previously that purified yeast RhoGDI Rdi1p, can inhibit vacuole membrane fusion in vitro. In the present paper we functionally dissect Rdi1p to discover its mode of regulating membrane fusion. Overexpression of Rdi1p in vivo profoundly affected cell morphology including increased actin patches in mother cells indicative of polarity defects, delayed ALP (alkaline phosphatase) sorting and the presence of highly fragmented vacuoles indicative of membrane fusion defects. These defects were not caused by the loss of typical transport and fusion proteins, but rather were linked to the reduction of membrane localization and activation of Cdc42p and Rho1p. Subcellular fractionation showed that Rdi1p is predominantly a cytosolic monomer, free of bound Rho GTPases. Overexpression of endogenous Rdi1p, or the addition of exogenous Rdi1p, generated stable cytosolic complexes. Rdi1p structure-function analysis showed that membrane association via the C-terminal β-sheet domain was required for the functional inhibition of membrane fusion. Furthermore, Rdi1p inhibited membrane fusion through the binding of Rho GTPases independent from its extraction activity.

Ovarian clear cell carcinomas: RHO GTPases may contribute to explain their singular biologic behavior

Ovarian clear cell carcinoma is found more often confined to the ovary (stage I) than high-grade serous carcinoma. The RHO GTPase family of proteins is involved in tumor invasion and metastasis through regulation of the cytoskeleton. The expression of several RHO family genes, including RHOA, RHOC, CDC42, and 3 ARHGDIs (Rho GDP dissociation inhibitors), was studied by real-time polymerase chain reaction in 22 clear cell carcinomas and 31 high-grade serous carcinomas. Immunoreaction for p21-activated kinase 1 (a downstream effector of CDC42) was also investigated on 6 tissue microarrays containing 76 carcinomas (13 clear cell carcinomas and 63 high-grade serous carcinomas). Eight clear cell carcinomas (8/21; 38%) were at stage I, whereas only 3 high-grade serous carcinomas (3/31; 10%) were at stage I. Postoperatively, all patients were treated with taxane and cisplatinum or carboplatinum. ARHGDIA messenger RNA expression was higher in clear cell carcinomas than high-grade serous carcinomas (P = .07). In contrast, CDC42 messenger RNA levels were lower in clear cell carcinomas than high-grade serous carcinomas (P = .02). Immunoreaction for p21-activated kinase 1 was concordant with the results obtained by real-time polymerase chain reaction for CDC42. In clear cell carcinomas, RHOA and RHOC messenger RNA expression was lower in stage I than in advanced-stage tumors (P = .03 and P = .005, respectively). Furthermore, in high-grade serous carcinomas, RHOA expression was higher in patients who did not respond to chemotherapy (P = .04). ARHGDIA, CDC42, RHOA, and RHOC expression may contribute to explain the different stage at diagnosis of clear cell and high-grade serous carcinomas. RHOA may cause resistance of high-grade serous carcinoma to chemotherapy.

Expression profile of RhoGDI2 in lung cancers and role of RhoGDI2 in lung cancer metastasis

Rho GDP dissociation inhibitors (RhoGDIs) are important regulators of the GTP hydrolase activity and biological functions of Rho GTPases. RhoGDI2 has been shown to be a metastasis suppressor in bladder cancer and several other cancers. However, the underlying mechanism, effector targets, and the cognate biological functions of RhoGDI2 are not fully understood. To investigate the possible role of RhoGDI2 in lung cancer tumorigenesis and metastasis, the expression pattern of RhoGDI2 in various lung cancer tissue samples and lung cancer-derived cell lines were profiled at both mRNA and protein levels. Furthermore, possible interplay between PI3K/Akt/mTOR pathway activation/inhibition and RhoGDI2 signalling is examined in lung cancer-related cell lines. Our results suggest that RhoGDI2 is likely to be involved in lung tumor malignancy and metastasis.

RhoGDI signaling provides targets for cancer therapy

Rho GDP-Dissociation Inhibitors (RhoGDIs) are important regulators of the Rho family of small GTPases. The expression of RhoGDIs is altered in a variety of cancers and they have been shown to mediate several processes during tumorigenesis and cancer progression. Using examples of RhoGDI-mediated signaling and expression patterns in endothelial cells as well as pancreatic, breast, and bladder cancer, the multitude of potential cancer therapeutic targets presented by a better understanding of their function is illustrated. Several novel therapeutic strategies are proposed for intervening in RhoGDI signaling, and potential complications arising from their implementation are discussed.

Regulators of small GTPases.

Small GTPases are converted from the GDP-bound inactive form to the GTP-bound active form by a GDP/GTP exchange reaction which is regulated by GDP/GTP exchange proteins (GEPs). We have found both stimulatory and inhibitory GEPs, which we have named GDP dissociation stimulators (GDSs) and GDP dissociation inhibitors (GDIs) respectively. We have isolated Smg GDS, Rho GDI and Rab GDI, cloned them, and determined their primary structures. These GEPs are active on a group of small GTPases: Smg GDS on at least K-Ras, Rap1/Smg21, Rho and Rac; Rho GDI on at least Rho, Rac and Cdc42; Rab GDI on most of the Rab family members. These GEPs have an additional function, regulating the translocation of their substrate small GTPases between the membrane and the cytosol. The GEPs interact only with the post-translationally modified form of their substrate small GTPases.

Rho GDP dissociation inhibitors as potential targets for anticancer treatment

The Rho GDP dissociation inhibitors (RhoGDIs) are a major class of regulators of Rho GTPases and play essential roles in normal cell growth and malignant transformation. Although RhoGDIs are known to inhibit Rho activities, recent studies indicate that RhoGDIs can also act as positive regulators through their ability to target Rho GTPases to specific subcellular membranes or to protect the GTPases from degradation by caspases. RhoGDIs are aberrantly expressed in human tumors and this may contribute to Rho-induced cancer progression. This review will discuss the dual roles of RhoGDIs in the regulation of Rho GTPases, highlighting a possible role in regulating tumorigenicity. In addition, the potential for targeting RhoGDIs for anticancer therapy will be discussed.

RhoGDI-3, a promising system to investigate the regulatory function of rhoGDIs: uncoupling of inhibitory and shuttling functions of rhoGDIs

rhoGDIs (Rho GDP dissociation inhibitors) are postulated to regulate the activity and the localization of small G-proteins of the Rho family by a shuttling process involving extraction of Rho from donor membranes, formation of inhibitory cytosolic rhoGDI/Rho complexes, and delivery of Rho to target membranes. However, the role of rhoGDIs in site-specific membrane targeting or extraction of Rho is still poorly understood. We investigated here the in vivo functions of two mammalian rhoGDIs: the specific rhoGDI-3 and the well-studied rhoGDI-1 (rhoGDI) after structure-based mutagenesis. We identified two sites in rhoGDIs, forming conserved interactions with their Rho target, whose mutation results in the uncoupling of inhibitory and shuttling functions of rhoGDIs in vivo. Remarkably, these rhoGDI mutants were detected at Rho-induced membrane ruffles or protrusions, where they co-localized with RhoG or Cdc42, probably identifying for the first time the site of extraction of a Rho protein by a rhoGDI in vivo. We propose that these mutations act by modifying the steady-state kinetics of the shuttling process regulated by rhoGDIs, such that transient steps at the cell membranes now become detectable. They should provide valuable tools for future investigations of the dynamics of membrane extraction or delivery of Rho proteins and their regulation by cellular partners.