The C-terminal Basic Tail of RhoG Assists the Guanine Nucleotide Exchange Factor Trio in Binding to Phospholipids

Abstract

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 ...

Kidins220/ARMS regulates Rac1-dependent neurite outgrowth by direct interaction with the RhoGEF Trio

“…Since the TrioGEF1 domain is able to activate both RhoG and Rac1 (Bellanger et al, 1998;Blangy et al, 2000;Chhatriwala et al, 2007), one attractive possibility is that it is the specific process that determines whether RhoG or Rac1 is activated. It was previously reported that RhoG, but not Rac1, mediates the membrane association of Trio, since Trio alone was not able to do so (Skowronek et al, 2004). In our system, Trio might be recruited to the membrane with the help of Kidins220 (Fig.…”

“…This leads to activation of Rac1 and might explain the enhancement of neurite outgrowth when Kidins220 and RhoG are overexpressed. Trio and RhoG can bind to a variety of phospholipids (PIP x ) (Skowronek et al, 2004;Ugolev et al, 2008). (B)The different phenotypes generated by the expression of the ankyrin-rich Kidins220 1-402 domain and the full-length protein is likely to be due to their different localisations.…”

The DH and PH Domains of Trio Coordinately Engage Rho GTPases for their Efficient Activation

“…43 RhoG is 80% homologous to Rac1, 65% identical, and the residues in nucleotide-depleted Rac1 that are buried in the interface with Trio are 100% identical in RhoG (data not shown). Consequently, mutants of Trio that have reduced exchange activity on Rac1 were also tested for activity on soluble, wild-type RhoG ( Figure 5(b)).…”

“…28; 42; 44 One notable exception to this generalization is the structure of Trio crystallized without bound GTPase. 43 However, in this case, the PH domain is locked into place by several crystal contacts.…”

Real-time NMR Study of Guanine Nucleotide Exchange and Activation of RhoA by PDZ-RhoGEF

“…The Trio PH domain promotes proper cellular membrane localization through interactions with phospholipids (24), whereas the Vav PH domain regulates the GEF activity of the DH domain through binding of phosphatidylinositides (25), and the Dbs PH domain assists the DH-associated domain in binding Rho GTPases (26,27). Based on the crystal structure of GEFGTPase complexes, the PH domains of some GEFs interact with the GTPase (e.g.…”