Substituting alanine for glycine at position 60 in v-HRas generated a dominant negative mutant that completely abolished the ability of v-H-Ras to transform NIH 3T3 cells and to induce germinal vesicle breakdown in Xenopus oocytes. The crystal structure of the GppNpbound form of RasG60A unexpectedly shows that the switch regions adopt an open conformation reminiscent of the structure of the nucleotide-free form of Ras in complex with Sos. Critical residues that normally stabilize the guanine nucleotide and the Mg 2؉ ion have moved considerably. Sos binds to RasG60A but is unable to catalyze nucleotide exchange. Our data suggest that the dominant negative effect observed for RasG60A⅐GTP could result from the sequestering of Sos in a non-productive Ras-GTP-guanine nucleotide exchange factor ternary complex.Ras is an essential component of signal transduction pathways that regulate growth, proliferation, differentiation, and apoptosis in response to the activation of membrane-bound receptors (1, 2). Dominant negative Ras mutants have been widely used to elucidate the role of Ras in a variety of signaling pathways. The asparagine for serine mutant at position 17, RasS17N, is probably the most frequently used dominant negative form of Ras (3) and the success of this mutant popularized the use of dominant negative mutants to study the signaling of other small GTPases. Despite this success, the exact molecular details by which dominant negative GTPases exert their inhibitory function are a matter of debate in the literature. It is widely accepted that RasS17N blocks the ability of endogenous Ras to function by sequestering and depleting the intracellular pool of available guanine nucleotide exchange factor (GEF), 1 thereby blocking the activation of endogenous Ras (4 -6). This argument is supported by the finding that overexpressing a dominant active form of Ras (e.g. RasG12V) or an activator domain usually abolishes the inhibitory effect of dominant negative Ras (3, 7). However, other explanations have been also proposed (8 -9) including low affinity of RasS17N for GTP and the inability of GTP to induce the RasS17N conformation necessary for binding and activating downstream effectors (4, 10). To complicate matters, the S17N mutant of Rap1A, which a priori should behave like RasS17N, is unable to inhibit the activation of Rap1A by its exchange factor, C3G, in vitro (11). Understanding at the molecular level how a dominant negative Ras functions should shed light on its cellular role and help in designing new tools to dissect the signaling of Ras and other small G-proteins. Because it inhibits the activation of endogenous Ras, dissecting the action of a dominant negative Ras should also better our understanding of the reaction of nucleotide exchange. So far, the structure of a dominant negative Ras complex is lacking in the literature.The substitution of alanine for glycine at position 60 in v-H-Ras, v-H-RasG60A, generated a dominant negative mutant that completely abolished the ability of v-H-Ras to transform NIH 3T3 ...

The multidomain protein Trio regulates among others neuronal outgrowth and axonal guidance in vertebrates and invertebrates. Trio contains two Dbl-homology/pleckstrin homology (DH/PH) tandem domains that activate several RhoGTPases. Here, we present the x-ray structure of the N-terminal DH/PH, hereafter TrioN, refined to 1.7-Å resolution. We show that the relative orientations of the DH and PH domains of TrioN and free Dbs are similar. However, this relative orientation is dissimilar to Dbs in the Dbs/Cdc42 structure. In vitro nucleotide exchange experiments catalyzed by TrioN show that RhoG is ϳ3؋ more efficiently exchanged than Rac and support the conclusion that RhoG is likely the downstream target of TrioN. Residues 54 and 69, which are not conserved between the two GTPases, are responsible for this specificity. Dot-blot assay reveals that the TrioN-PH domain does not detectably bind phosphatidylinositol 3,4-bisphosphate, PtdIns(3,4)P 2 , or other phospholipids. This finding is supported by our three-dimensional structure and affinity binding experiments. Interestingly, the presence of RhoG but not Rac or a C-terminal-truncated RhoG mutant allows TrioN to bind PtdIns(3,4)P 2 with a micromolar affinity constant. We conclude the variable C-terminal basic tail of RhoG specifically assists the recruitment of the TrioN-PH domain to specific membrane-bound phospholipids. Our data suggest a role for the phosphoinositide 3-kinase, PI 3-kinase, in modulating the Trio/RhoG signaling pathway.Reorganization of the actin cytoskeleton is an essential step accompanying changes in cell shape, cell migration, cell-cell adhesion, cell transformation, tumorigenesis, and other cellular processes (reviewed in Refs. 1-4). More than any other cell type, neurons rely on changes in their actin cytoskeleton for growth, guidance, and branching (reviewed in Refs. 5-8). It is now well established that members of the Rho family of small GTP-binding proteins, or RhoGTPases, are major regulators of the actomyosin machinery in a large number of cell types. RhoGTPases (Rho, Rac, Cdc42, and their isoforms) function as protein switches in response to the activation of cell surface receptors to activate various cellular processes including gene transcription and the formation of actin stress fibers, membrane ruffling, and filopodia (reviewed in Refs. 3, 4 and 9).One key step in the receptor/RhoGTPase signal pathway is the activation of the RhoGTPase. The activation step controls the intensity and the duration of the signal, and thus is subject to tight regulation. The activation step consists of switching the RhoGTPase from an inactive GDP-bound form to an active or GTP-bound form and is generally catalyzed by guanine nucleotide exchange factors (GEFs).1 Mammalian Rho-specific GEFs, or RhoGEFs, form a family of ϳ47 multidomain proteins and often contain multiple protein-protein or protein-phospholipid binding domains suggesting regulation by inter-or intramolecular interaction. RhoGEFs share a ϳ200 amino acid homology domain initially found in ...

Trio is a multidomain signaling protein that plays an important role in neurite outgrowth, axon guidance and skeletal muscle development. Trio contains two DH/PH tandem domains that respectively activate the small GTPases RhoG/Rac and RhoA. The N-terminal DH/PH domain, TrioN, crystallizes in space group P3(1)21, with one TrioN molecule in the asymmetric unit and diffracts to 1.7 A resolution. The unit-cell parameters are a = b = 99.5, c = 98.3 A, alpha = beta = 90, gamma = 120 degrees. A greater than 90% complete native data set has been collected and structure determination using the multiple isomorphous replacement (MIR) method is ongoing.

The Saccharomyces cerevisiae RGS protein Sst2p is involved in desensitization to pheromone and acts as a GTPase-activating protein for the Gα subunit Gpa1p. Other results indicate that Sst2p acts through Mpt5p and that this action occurs downstream of Fus3p and through Cln3p/Cdc28p. Our results indicate that the interaction of Sst2p with Mpt5p requires the N-terminal MPI (Mpt5p-interacting) domain of Sst2p and is independent of the C-terminal RGS domain. Overexpression of the MPI domain results in an Mpt5p-dependent increase in recovery from pheromone arrest. Overexpression of either intact Sst2p or the MPI domain leads to partial suppression of a gpa1 growth defect, and this suppression is dependent on Mpt5p, indicating that MPI function occurs downstream of Gpa1p and through Mpt5p. Combination of an mpt5 mutation with the GPA1G302S mutation, which uncouples Gpa1p from Sst2p, results in pheromone supersensitivity similar to the sst2 mutant, and promotion of recovery by overexpression of Sst2p is dependent on both Mpt5p and the Gpa1p interaction. These results indicate that Sst2p is a bifunctional protein and that the MPI domain acts through Mpt5p independently of the RGS domain. RGS family members from other fungi contain N-terminal domains with sequence similarity to the Sst2p MPI domain, suggesting that MPI function may be conserved.

No abstract