Genomic organization and expression profile of the small GTPases of the RhoBTB family in human and mouse

Members of the RhoBTB subfamily of Rho GTPases are present in vertebrates, Drosophila and Dictyostelium. RhoBTB proteins are characterized by a modular organization, consisting of a GTPase (guanosine triphosphatase) domain, a proline rich region, a tandem of two BTB (road-Complex, ramtrack, and ric à brac) domains and a C-terminal region of unknown function and might act as docking points for multiple components participating in signal transduction cascades. We have determined the genomic organization and the expression pattern of the three RHOBTB genes of human and mouse. The exon–intron organization of each gene is conserved in three vertebrate species (human, mouse and Fugu). RHOBTB1 and RHOBTB2 have a similar exon–intron organization and are closely related to the single gene encoding the RhoBTB orthologs of two insect species. By contrast, the exon–intron organization of RHOBTB3 differed substantially from that of the two other genes, indicating that this gene arose by a duplication event independent of the one that gave rise to RHOBTB1 and RHOBTB2. RHOBTB1 (located on chromosome 10) and RHOBTB3 (located on chromosome 5) appear ubiquitously expressed. However, they display a differential pattern of expression: RHOBTB1 showed high levels in stomach, skeletal muscle, placenta, kidney and testis, whereas RHOBTB3 was highly expressed in neural and cardiac tissues, pancreas, placenta and testis. RHOBTB2 (located on chromosome 8) showed much lower levels of expression than the other two human RHOBTB genes and it was most abundant in neural tissues. The expression patterns of the human and mouse genes were roughly comparable. All three genes were also detected in fetal tissues, and in a number of cell lines RHOBTB3 predominates. RHOBTB genes are upregulated in some cancer cell lines, suggesting that these proteins might participate in tumorigenesis.S.R. is the recipient of a fellowship from Fundación Ramón Areces and F.K. is the recipient of a fellowship from the Köln Fortune Program. This work was supported by the Deutsche Forschungsgemeinschaft (RI 1034/2) and by the Köln Fortune Program (Faculty of Medicine, University of Cologne).Peer Reviewe

RacG regulates morphology, phagocytosis, and chemotaxis

RacG is an unusual member of the complex family of Rho GTPases in Dictyostelium. We have generated a knockout (KO) strain, as well as strains that overexpress wild-type (WT), constitutively active (V12), or dominant negative (N17) RacG. The protein is targeted to the plasma membrane, apparently in a nucleotide-dependent manner, and induces the formation of abundant actin-driven filopods. RacG is enriched at the rim of the progressing phagocytic cup, and overexpression of RacG-WT or RacG-V12 induced an increased rate of particle uptake. The positive effect of RacG on phagocytosis was abolished in the presence of 50 μM LY294002, a phosphoinositide 3-kinase inhibitor, indicating that generation of phosphatidylinositol 3,4,5-trisphosphate is required for activation of RacG. RacG-KO cells showed a moderate chemotaxis defect that was stronger in the RacG-V12 and RacG-N17 mutants, in part because of interference with signaling through Rac1. The in vivo effects of RacG-V12 could not be reproduced by a mutant lacking the Rho insert region, indicating that this region is essential for interaction with downstream components. Processes like growth, pinocytosis, exocytosis, cytokinesis, and development were unaffected in Rac-KO cells and in the overexpressor mutants. In a cell-free system, RacG induced actin polymerization upon GTPγS stimulation, and this response could be blocked by an Arp3 antibody. While the mild phenotype of RacG-KO cells indicates some overlap with one or more Dictyostelium Rho GTPases, like Rac1 and RacB, the significant changes found in overexpressors show that RacG plays important roles. We hypothesize that RacG interacts with a subset of effectors, in particular those concerned with shape, motility, and phagocytosis

Defects in cytokinesis, actin reorganization and the contractile vacuole in cells deficient in RhoGDI

Rho GDP-dissociation inhibitors (RhoGDIs) modulate the cycling of Rho GTPases between active GTP-bound and inactive GDP-bound states. We identified two RhoGDI homologues in Dictyostelium. GDI1 shares 51–58% similarity to RhoGDIs from diverse species. GDI2 is more divergent (40–44% similarity) and lacks the N-terminal regulatory arm characteristic for RhoGDI proteins. Both are cytosolic proteins and do not relocalize upon reorganization of the actin cytoskeleton. Using a two-hybrid approach, we identified Rac1a/1b/1c, RacB, RacC and RacE as interacting partners for GDI1. Cells lacking GDI1 are multinucleate, grow slowly and display a moderate pinocytosis defect, but rates of phagocytosis are unaffected. Mutant cells present prominent actin-rich protrusions, and large vacuoles that are continuous with the contractile vacuole system. The actin polymerization response upon stimulation with cAMP was reduced, but the motile behavior toward the chemoattractant was unaffected. Our results indicate that GDI1 plays a central role in the regulation of signal transduction cascades mediated by Rho GTPases

Role of Rho-like proteins of the RhoBTB family in oncogenesis

Abstract of the 8th Joint Meeting of the Signal Transduction Society STS, Hilton Hotel, Weimar, 4–6 November 2004: Workshop E: “Interaction Domains and Signaling Complexes.Rho proteins have been implicated mainly in the regulation of the cytoskeleton, but also in vesicle trafficking,phagocytosis and transcriptional activation. They participate in tumorigenesis, where some of them are overfunc-tional. A subfamily of Rho GTPases, RhoBTB proteins, is represented by three isoforms in vertebrates: RhoBTB1,RhoBTB2 and RhoBTB3. They are around 600 amino acids long and consist of a GTPase domain, a prolin richregion, two BTB domains and a carboxyl-terminal region. The role of RhoBTB proteins is at present unknown.Tissue distribution studies by means of in situ hybridization detected the expression of RhoBTB1 and RhoBTB3specifically in vascular endothelial cells suggesting a role in angiogenesis. In addition, a role for RhoBTB2 as atumor suppressor has been proposed, and in fact we show that expression profiles of RhoBTB genes differbetween normal and tumoral human tissuesRhoBTB3 is targeted to Golgi membranes through a prenylation motif. RhoBTB1 and RhoBTB2 lack this motif,but localize at aggregates distinct from vesiclular structures like Golgi apparatus or lysosomes. We generatedtruncated contructs in order to identify the sequence responsible for targeting of RhoBTB1 and RhoBTB2 tothese aggregates.Recent studies demonstrate that BTB domain-containing proteins are involved in the formation of Cullin 3 depen-dent ubiquitin ligase complexes. Cullins are subunits of a prominent class of RING ubiquitin ligases. Using yeasttwo hybrid and coimmunoprecipitation studies we show that RhoBTB3 binds to Cullin 3 and might also be de-graded in the proteasomal apparatus. We propose a model in which RhoBTB proteins function as specific adap-tors for the degradation of unknown proteins involved in the control of cell proliferation and have identified poten-tial targets using a two-hybrid approach.Peer Reviewe