The Minimal Autoinhibited Unit of the Guanine Nucleotide Exchange Factor Intersectin

Intersectin-1L is a member of the Dbl homology (DH) domain guanine nucleotide exchange factors (GEF) which control Rho-family GTPase signaling. Intersectin-1L is a GEF that is specific for Cdc42. It plays an important role in endocytosis, and is regulated by several partners including the actin regulator N-WASP. Intact intersectin-1L shows low Cdc42 exchange activity, although the isolated catalytic DH domain shows high activity. This finding suggests that the molecule is autoinhibited. To investigate the mechanism of autoinhibition we have constructed a series of domain deletions. We find that the five SH3 domains of intersectin are important for autoinhibition, with the fifth domain (SH3(E)) being sufficient for the bulk of the autoinhibitory effect. This SH3 domain appears to primarily interact with the DH domain. We have determined the crystal structure of the SH3(E)-DH domain construct, which shows a domain swapped arrangement in which the SH3 from one monomer interacts with the DH domain of the other monomer. Analytical ultracentrifugation and gel filtration, however, show that under biochemical concentrations, the construct is fully monomeric. Thus we propose that the actual autoinhibited structure contains the related intramolecular SH3(E)-DH interaction. We propose a model in which this intramolecular interaction may block or distort the GTPase binding region of the DH domain.     

Activation of ARF6 by ARNO stimulates epithelial cell migration through downstream activation of both Rac1 and phospholipase D

Migration of epithelial cells is essential for tissue morphogenesis, wound healing, and metastasis of epithelial tumors. Here we show that ARNO, a guanine nucleotide exchange factor for ADP-ribosylation factor (ARF) GTPases, induces Madin-Darby canine kidney epithelial cells to develop broad lamellipodia, to separate from neighboring cells, and to exhibit a dramatic increase in migratory behavior. This transition requires ARNO catalytic activity, which we show leads to enhanced activation of endogenous ARF6, but not ARF1, using a novel pulldown assay. We further demonstrate that expression of ARNO leads to increased activation of endogenous Rac1, and that Rac activation is required for ARNO-induced cell motility. Finally, ARNO-induced activation of ARF6 also results in increased activation of phospholipase D (PLD), and inhibition of PLD activity also inhibits motility. However, inhibition of PLD does not prevent activation of Rac. Together, these data suggest that ARF6 activation stimulates two distinct signaling pathways, one leading to Rac activation, the other to changes in membrane phospholipid composition, and that both pathways are required for cell motility.     

Remodeling of the Actin Cytoskeleton Is Coordinately Regulated by Protein Kinase C and the ADP-Ribosylation Factor Nucleotide Exchange Factor ARNO

ARNO is a member of a family of guanine-nucleotide exchange factors with specificity for the ADP-ribosylation factor (ARF) GTPases. ARNO possesses a central catalytic domain with homology to yeast Sec7p and an adjacent C-terminal pleckstrin homology (PH) domain. We have previously shown that ARNO localizes to the plasma membrane in vivo and efficiently catalyzes ARF6 nucleotide exchange in vitro. In addition to a role in endocytosis, ARF6 has also been shown to regulate assembly of the actin cytoskeleton. To determine whether ARNO is an upstream regulator of ARF6 in vivo, we examined the distribution of actin in HeLa cells overexpressing ARNO. We found that, while expression of ARNO leads to disassembly of actin stress fibers, it does not result in obvious changes in cell morphology. However, treatment of ARNO transfectants with the PKC agonist phorbol 12-myristate 13-acetate results in the dramatic redistribution of ARNO, ARF6, and actin into membrane protrusions resembling lamellipodia. This process requires ARF activation, as actin rearrangement does not occur in cells expressing a catalytically inactive ARNO mutant. PKC phosphorylates ARNO at a site immediately C-terminal to its PH domain. However, mutation of this site had no effect on the ability of ARNO to regulate actin rearrangement, suggesting that phosphorylation of ARNO by PKC does not positively regulate its activity. Finally, we demonstrate that an ARNO mutant lacking the C-terminal PH domain no longer mediates cytoskeletal reorganization, indicating a role for this domain in appropriate membrane localization. Taken together, these data suggest that ARNO represents an important link between cell surface receptors, ARF6, and the actin cytoskeleton.     

The Rho-specific Guanine Nucleotide Exchange Factor Dbs Regulates Breast Cancer Cell Migration

Dbs is a Rho-specific guanine nucleotide exchange factor (RhoGEF) that regulates neurotrophin-3-induced cell migration in Schwann cells. Here we report that Dbs regulates cell motility in tumor-derived, human breast epithelial cells through activation of Cdc42 and Rac1. Cdc42 and Rac1 are activated in T47D cells that stably express onco- or proto-Dbs, and activation is dependent upon growth of the cells on collagen I. Transient suppression of expression of Cdc42 or Rac1 by small interfering RNAs attenuates Dbs-enhanced motility. Both onco- and proto-Dbs-enhanced motility correlates with an increase in tyrosine phosphorylation of focal adhesion kinase on Tyr-397 and p130^Cas on Tyr-410 and an increase in the abundance of the Crk·p130^Cas complex. Suppression of expression of Cdc42 or its effector, Ack1, reduces tyrosine phosphorylation of focal adhesion kinase and p130^Cas and disrupts the Crk·p130^Cas complex. We further determined that suppression of expression of Cdc42, Ack1, p130^Cas, or Crk reduces Rac1 activation and cell motility in Dbs-expressing cells to a level comparable with that in vector cells. Therefore, a cascade of activation of Cdc42 and Rac1 by Dbs through the Cdc42 effector Ack1 and the Crk·p130^Cas complex is established. Suppression of the expression of endogenous Dbs reduces cell motility in both T47D cells and MDA-MB-231 cells, which correlates with the down-regulation of Cdc42 activity. This suggests that Dbs activates Cdc42 in these two human breast cancer cell lines and that the normal function of Dbs may be required to support cell movement.Rho GTPases are a subfamily of the Ras superfamily of small signaling molecules that are widely expressed in mammalian cells (1). RhoA, Cdc42, and Rac1 are the most extensively studied members of the Rho GTPase family, and each plays a prominent and discrete role in cell migration (2, 3). Cdc42 promotes the formation of filopodia and is required to establish cell polarity (3–5); Rac1 promotes the formation of lamellipodia at the leading edge of motile cells (6), and RhoA promotes the formation of stress fibers which generate the traction forces needed to retract the cell tail and move the cell body beyond the leading edge (7, 8). Consistent with this important role in cell motility, RhoA, Cdc42, and Rac1 are often overexpressed in human tumors including breast, lung, and colon (9), and overexpression of constitutively active RhoA, Cdc42, or Rac1 increases cell migration and invasion (2, 10, 11).The spatiotemporal regulation of Rho GTPase activity is tightly controlled by three classes of proteins. Rho-specific guanine nucleotide exchange factors (RhoGEFs)2 activate Rho proteins by facilitating the exchange of GDP for GTP; Rho GTPase-activating proteins (RhoGAPs) stimulate the intrinsic rate of hydrolysis of Rho proteins, thus converting them into their inactive state; Rho-specific guanine nucleotide dissociation inhibitors (RhoGDIs) compete with RhoGEFs for binding to GDP-bound Rho proteins and sequester Rho in the inactive state (12).Dbs was identified in the screen for proteins whose overexpression cause malignant growth in murine fibroblasts (13, 14). The full-length Dbs protein (proto-Dbs) is a RhoGEF family member which contains multiple recognizable domains (Fig. 1A) including a Sec14-like domain, spectrin-like repeats, a RhoGEF domain (includes a DH and PH domain), and an SH3 domain (13). The original oncogenic version of Dbs that was identified (amino acid residues 525–1097; designated onco-Dbs) contains the RhoGEF domain alone. When expressed in murine fibroblasts, the transforming and catalytic activity of Dbs is subject to autoinhibition that is mediated by the NH₂-terminal Sec14 domain (15). Although the endogenous function of Dbs is not known, recent studies suggest that Dbs and the Rac-specific exchange factor Tiam1 regulate neurotrophin-stimulated cell migration in Schwann cells through activation of Cdc42 and Rac1, respectively (16, 17).Open in a separate windowFIGURE 1.Onco-Dbs and proto-Dbs induce cell migration in tumor-derived breast epithelial cells. A, domain structure of the onco-Dbs and proto-Dbs proteins (Sec14 = Sec14-like domain; Spec = Spectrin-like repeats; DH = Dbl homology domain; PH = pleckstrin homology domain; SH3 = Src homology 3 domain). B, stable expression of HA-epitope-tagged onco-Dbs (M_r = 65) and proto-Dbs (M_r = 129 kDa) was confirmed by Western blot using an anti-HA antibody. Three independent sets of cell lines were generated. C, T47D cells stably expressing vector (Vec), onco-Dbs, or proto-Dbs were compared in a transwell motility assay on filters pre-coated with collagen I. The motility of cells stably expressing onco-Dbs or proto-Dbs is expressed relative to that of cells stably expressing vector. Data are represented as the mean ± S.D. of three independent experiments performed in triplicate. D, T47D cells stably expressing vector, onco-Dbs, or proto-Dbs were cultured to monolayer on dishes coated with poly-l-lysine or collagen I, as indicated. Cells were serum-starved overnight, and then the surface of the plate was scraped. Migration of cells at the wound edge was monitored and photographed at 18 h. Representative images are shown. E, growth curves of T47D cells stably expressing vector, onco-Dbs, or proto-Dbs. Cells were cultured in triplicate on poly-l-lysine (filled symbols) or on dishes pre-coated with collagen I (open symbols) and counted on the indicated days. Data shown are representative of three independent experiments.Conversion of Rho proteins to their active GTP-bound state allows them to interact with effector signaling molecules. Ack1 is a nonreceptor-tyrosine kinase that binds to active Cdc42 but not Rac1 or RhoA (18, 19). Activated Ack1 is overexpressed in primary tumors and cancer cell lines and has been implicated in cancer metastasis (20). Recent studies have identified a signaling complex that regulates the motility of human breast epithelial cells that contains Cdc42, Ack1, p130^Cas, and Crk (21). Ack1 and p130^Cas interact through their respective SH3 domains, and Ack1 phosphorylates p130^Cas in a collagen I-dependent manner. p130^Cas was first identified as a hyperphosphorylated adapter protein in cells transformed by v-Src and v-Crk (22, 23). Further studies showed that p130^Cas is associated with both cellular Src and Crk in a tyrosine phosphorylation-dependent manner (24, 25). Focal adhesion kinase (FAK) binds to the NH₂ terminus of p130^Cas and phosphorylates the COOH terminus in a region that is involved in p130^Cas binding to Src (26). The binding of Crk to p130^Cas recruits binding partners to the SH3 domain of Crk, including C3G and DOCK180, which activate Rap1 and Rac1, respectively (27–31). Thus, formation of the Crk·p130^Cas complex is considered to be a molecular switch that can induce cell migration by activating Rac1 (32).Here we show that both proto-Dbs and onco-Dbs increase cell migration in human breast adenocarcinoma cells in a collagen I-dependent manner. Increased motility is dependent upon the activation of Rac1 and Cdc42 and is mediated by the assembly of Crk·p130^Cas complexes. Suppression of endogenous Dbs expression in human tumor-derived breast epithelial cells limits cell motility, suggesting that Dbs may be a critical regulator of cell behavior in breast cancer.     

The Adenomatous Polyposis Coli-associated Guanine Nucleotide Exchange Factor Asef Is Involved in Angiogenesis

Mutation of the tumor suppressor adenomatous polyposis coli (APC) is a key early event in the development of most colorectal tumors. APC promotes degradation of β-catenin and thereby negatively regulates Wnt signaling, whereas mutated APCs present in colorectal tumor cells are defective in this activity. APC also stimulates the activity of the guanine nucleotide exchange factor Asef and regulates cell morphology and migration. Truncated mutant APCs constitutively activate Asef and induce aberrant migration of colorectal tumor cells. Furthermore, we have recently found that Asef and APC function downstream of hepatocyte growth factor and phosphatidylinositol 3-kinase. We show here that Asef is required for basic fibroblast growth factor- and vascular endothelial growth factor-induced endothelial cell migration. We further demonstrate that Asef is required for basic fibroblast growth factor- and vascular endothelial growth factor-induced microvessel formation. Furthermore, we show that the growth as well as vascularity of subcutaneously implanted tumors are markedly impaired in Asef^−/− mice compared with wild-type mice. Thus, Asef plays a critical role in tumor angiogenesis and may be a promising target for cancer chemotherapy.     

Regulation of Immature Dendritic Cell Migration by RhoA Guanine Nucleotide Exchange Factor Arhgef5

There are a large number of Rho guanine nucleotide exchange factors, most of which have no known functions. Here, we carried out a short hairpin RNA-based functional screen of Rho-GEFs for their roles in leukocyte chemotaxis and identified Arhgef5 as an important factor in chemotaxis of a macrophage phage-like RAW264.7 cell line. Arhgef5 can strongly activate RhoA and RhoB and weakly RhoC and RhoG, but not Rac1, RhoQ, RhoD, or RhoV, in transfected human embryonic kidney 293 cells. In addition, Gβγ interacts with Arhgef5 and can stimulate Arhgef5-mediated activation of RhoA in an in vitro assay. In vivo roles of Arhgef5 were investigated using an Arhgef-5-null mouse line. Arhgef5 deficiency did not affect chemotaxis of mouse macrophages, T and B lymphocytes, and bone marrow-derived mature dendritic cells (DC), but it abrogated MIP1α-induced chemotaxis of immature DCs and impaired migration of DCs from the skin to lymph node. In addition, Arhgef5 deficiency attenuated allergic airway inflammation. Therefore, this study provides new insights into signaling mechanisms for DC migration regulation.Leukocyte chemotaxis underlies leukocyte migration, infiltration, trafficking, and homing that are not only important for normal leukocyte functions, but also have a important role in inflammation-related diseases. Leukocyte chemotaxis is regulated by leukocyte chemoattractants that include bacterial by-products such as formylmethionylleucylphenylalanine, complement proteolytic fragments such as C5a, and the superfamily of chemotactic cytokines, chemokines. These chemoattractants bind to their specific cell G protein-coupled receptors and are primarily coupled to the G_i family of G proteins to regulate leukocyte chemotaxis. Previous studies have established that the Rho family of small GTPases regulates leukocyte migration (1, 2). Rac, Cdc42, and RhoA are the three best studied Rho small GTPases. In myeloid cells, Cdc42 regulates directionality by directing where F-actin and lamellipodia are formed, and Rac regulates F-actin formation in the lamellipodia, which provides a driving force for cell motility (3–6). On the other hand, RhoA regulates the formation and contractility of the actomyosin structure at the back that provides a pushing force (5, 7). Rho guanine nucleotide exchange factors (GEF)³ are key regulators for the activity of these small GTPases. GEFs activate small GTPases by promoting the loading of GTP to the small GTPases, a rate-limiting step in GTPase regulation (8–11). Previous biochemical and genetic studies have revealed how Cdc42 and Rac may be regulated by chemokine receptors in leukocytes. Chemokine receptors can regulate Cdc42 via a Rho-GEF PIXα, which is regulated by Gβγ from the G_i proteins via the interactions between Gβγ and Pak1 and between Pak1 and PIXα in myeloid cells 12. On the other hand, in neutrophils chemokine receptors regulate Rac2 via another Rho-GEF P-Rex1, which is directly regulated by Gβγ (13–15). Two Rho-GEFs have been implicated in regulation of RhoA in neutrophils. GEF115 was found in the leading edges of polarized mouse neutrophils, whereas PDZ Rho-GEF was found in the uropods of differentiated HL-60 cells. Both Rho-GEFs were believed to mediate pertussis toxin-resistant activation of RhoA in these cells. However, a significant portion of RhoA activity in leukocytes are pertussis toxin-sensitive, which is presumably regulated by the α and/or βγ subunits from the G_i proteins. The signaling mechanism for this pertussis toxin-sensitive RhoA regulation by chemokine receptors remains largely elusive.Molecular cloning and genomic sequencing have identified more than 70 Rho-GEFs in mammals (16–20). Many of these Rho-GEFs have been shown to activate RhoA in in vitro and overexpression assays (16–20). However, it is not known if any of them regulate RhoA in vivo, we have found that PIXα is a specific GEF for Cdcd42 in neutrophils (12) despite its potent activity on Rac in in vitro and overexpression assays (21, 22). Therefore, we used a siRNA-based loss of function screen in an attempt to identify the GEFs that regulate myeloid cell migration and RhoA activity. One of the candidates, Arhgef5, was found to be directly activated by Gβγ to regulate RhoA and has an important role in immature DC migration. In addition, Arhgef5 deficiency attenuated allergic airway inflammation in a mouse model.     

Intermediates in the Guanine Nucleotide Exchange Reaction of Rab8 Protein Catalyzed by Guanine Nucleotide Exchange Factors Rabin8 and GRAB

Small G-proteins of the Ras superfamily control the temporal and spatial coordination of intracellular signaling networks by acting as molecular on/off switches. Guanine nucleotide exchange factors (GEFs) regulate the activation of these G-proteins through catalytic replacement of GDP by GTP. During nucleotide exchange, three distinct substrate·enzyme complexes occur: a ternary complex with GDP at the start of the reaction (G-protein·GEF·GDP), an intermediary nucleotide-free binary complex (G-protein·GEF), and a ternary GTP complex after productive G-protein activation (G-protein·GEF·GTP). Here, we show structural snapshots of the full nucleotide exchange reaction sequence together with the G-protein substrates and products using Rabin8/GRAB (GEF) and Rab8 (G-protein) as a model system. Together with a thorough enzymatic characterization, our data provide a detailed view into the mechanism of Rabin8/GRAB-mediated nucleotide exchange.     

The Guanine Nucleotide Exchange Factor SWAP-70 Modulates the Migration and Invasiveness of Human Malignant Glioma Cells

The malignant glioma is the most common primary human brain tumor. Its tendency to invade away from the primary tumor mass is considered a leading cause of tumor recurrence and treatment failure. Accordingly, the molecular pathogenesis of glioma invasion is currently under investigation. Previously, we examined a gene expression array database comparing human gliomas to nonneoplastic controls and identified several Rac guanine nucleotide exchange factors with differential expression. Here, we report that the guanine nucleotide exchange factor SWAP-70 has increased expression in malignant gliomas and strongly correlates with lowered patient survival. SWAP-70 is a multifunctional signaling protein involved in membrane ruffling that works cooperatively with activated Rac. Using a glioma tissue microarray, we validated that SWAP-70 demonstrates higher expression in malignant gliomas compared with low-grade gliomas or nonneoplastic brain tissue. Through immunofluorescence, SWAP-70 localizes to membrane ruffles in response to the growth factor, epidermal growth factor. To assess the role of SWAP-70 in glioma migration and invasion, we inhibited its expression withsmall interfering RNAs and observed decreased glioma cell migration and invasion. SWAP-70 overexpression led to increased levels of active Rac even in low-serum conditions. In addition, when SWAP-70 was overexpressed in glioma cells, we observed enhanced membrane ruffle formation followed by increased cellmigration and invasiveness. Taken together, our findings suggest that the guanine nucleotide exchange factor SWAP-70 plays an important role in the migration and invasion of human gliomas into the surrounding tissue.     

Structural Basis for the Dual Substrate Specificity of DOCK7 Guanine Nucleotide Exchange Factor

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Multisite Phosphorylation of the Guanine Nucleotide Exchange Factor Cdc24 during Yeast Cell Polarization

Background

Cell polarization is essential for processes such as cell migration and asymmetric cell division. A common regulator of cell polarization in most eukaryotic cells is the conserved Rho GTPase, Cdc42. In budding yeast, Cdc42 is activated by a single guanine nucleotide exchange factor, Cdc24. The mechanistic details of Cdc24 activation at the onset of yeast cell polarization are unclear. Previous studies have suggested an important role for phosphorylation of Cdc24, which may regulate activity or function of the protein, representing a key step in the symmetry breaking process.

Methodology/Principal Findings

Here, we directly ask whether multisite phosphorylation of Cdc24 plays a role in its regulation. We identify through mass spectrometry analysis over thirty putative in vivo phosphorylation sites. We first focus on sites matching consensus sequences for cyclin-dependent and p21-activated kinases, two kinase families that have been previously shown to phosphorylate Cdc24. Through site-directed mutagenesis, yeast genetics, and light and fluorescence microscopy, we show that nonphosphorylatable mutations of these consensus sites do not lead to any detectable consequences on growth rate, morphology, kinetics of polarization, or localization of the mutant protein. We do, however, observe a change in the mobility shift of mutant Cdc24 proteins on SDS-PAGE, suggesting that we have indeed perturbed its phosphorylation. Finally, we show that mutation of all identified phosphorylation sites does not cause observable defects in growth rate or morphology.

Conclusions/Significance

We conclude that lack of phosphorylation on Cdc24 has no overt functional consequences in budding yeast. Yeast cell polarization may be more tightly regulated by inactivation of Cdc42 by GTPase activating proteins or by alternative methods of Cdc24 regulation, such as conformational changes or oligomerization.     

The Putative Guanine Nucleotide Exchange Factor RicA Mediates Upstream Signaling for Growth and Development in Aspergillus

Heterotrimeric G proteins (G proteins) govern growth, development, and secondary metabolism in various fungi. Here, we characterized ricA, which encodes a putative GDP/GTP exchange factor for G proteins in the model fungus Aspergillus nidulans and the opportunistic human pathogen Aspergillus fumigatus. In both species, ricA mRNA accumulates during vegetative growth and early developmental phases, but it is not present in spores. The deletion of ricA results in severely impaired colony growth and the total (for A. nidulans) or near (for A. fumigatus) absence of asexual sporulation (conidiation). The overexpression (OE) of the A. fumigatus ricA gene (AfricA) restores growth and conidiation in the ΔAnricA mutant to some extent, indicating partial conservation of RicA function in Aspergillus. A series of double mutant analyses revealed that the removal of RgsA (an RGS protein of the GanB Gα subunit), but not sfgA, flbA, rgsB, or rgsC, restored vegetative growth and conidiation in ΔAnricA. Furthermore, we found that RicA can physically interact with GanB in yeast and in vitro. Moreover, the presence of two copies or OE of pkaA suppresses the profound defects caused by ΔAnricA, indicating that RicA-mediated growth and developmental signaling is primarily through GanB and PkaA in A. nidulans. Despite the lack of conidiation, brlA and vosA mRNAs accumulated to normal levels in the ΔricA mutant. In addition, mutants overexpressing fluG or brlA (OEfluG or OEbrlA) failed to restore development in the ΔAnricA mutant. These findings suggest that the commencement of asexual development requires unknown RicA-mediated signaling input in A. nidulans.     

A Novel Guanine Nucleotide Exchange Factor,MYOGEF, is Required for Cytokinesis

The cleavage furrow is created by an actomyosin contractile ring that isregulated by small GTPase proteins such as Rac1 and RhoA. Guanine nucleotideexchange factors (GEFs) are positive regulators of the small GTPase proteins andhave been implicated as important factors in regulating cytokinesis. However, it isstill unclear how GEFs regulate the contractile ring during cytokinesis inmammalian cells. Here we report that a novel GEF, which is termed MyoGEF(myosin-interacting GEF), interacts with nonmuscle myosin II and exhibits activitytoward RhoA. MyoGEF and nonmuscle myosin II colocalize to the cleavage furrowin early anaphase cells. Disruption of MyoGEF expression in U2OS cells by RNAinterference (RNAi) results in the formation of multinucleated cells. These resultssuggest that MyoGEF, RhoA, and nonmuscle myosin II act as a functional unit atthe cleavage furrow to advance furrow ingression during cytokinesis.     

Kinetic Analysis of the Guanine Nucleotide Exchange Activity of TRAPP, a Multimeric Ypt1p Exchange Factor

TRAPP complexes, which are large multimeric assemblies that function in membrane traffic, are guanine nucleotide exchange factors (GEFs) that activate the Rab GTPase Ypt1p. Here we measured rate and equilibrium constants that define the interaction of Ypt1p with guanine nucleotide (guanosine 5'-diphosphate and guanosine 5'-triphosphate/guanosine 5′-(β,γ-imido)triphosphate) and the core TRAPP subunits required for GEF activity. These parameters allowed us to identify the kinetic and thermodynamic bases by which TRAPP catalyzes nucleotide exchange from Ypt1p. Nucleotide dissociation from Ypt1p is slow (∼ 10^− 4 s^− 1) and accelerated > 1000-fold by TRAPP. Acceleration of nucleotide exchange by TRAPP occurs via a predominantly Mg²⁺-independent pathway. Thermodynamic linkage analysis indicates that TRAPP weakens nucleotide affinity by < 80-fold and vice versa, in contrast to most other characterized GEF systems that weaken nucleotide binding affinities by 4-6 orders of magnitude. The overall net changes in nucleotide binding affinities are small because TRAPP accelerates both nucleotide binding and dissociation from Ypt1p. Weak thermodynamic coupling allows TRAPP, Ypt1p, and nucleotide to exist as a stable ternary complex, analogous to strain-sensing cytoskeleton motors. These results illustrate a novel strategy of guanine nucleotide exchange by TRAPP that is particularly suited for a multifunctional GEF involved in membrane traffic.     

ARF6 activation by Galpha q signaling: Galpha q forms molecular complexes with ARNO and ARF6

G protein-coupled receptors (GPCRs) are widely expressed hepta-helical receptors with tightly regulated pleiotropic effects. ADP-Ribosylation Factor 6 (ARF6) plays an important role in GPCR trafficking and is the subject of intense research. However, the mechanisms underlying activation and regulation of ARF6 by GPCRs are poorly characterized. Here we report that Galpha(q) signaling leads to the activation of ARF6. Stimulation of the TPbeta receptor triggered ARF6 activation which was completely inhibited by the RGS domain of GRK2 known to specifically bind and sequester Galpha(q). Co-immunoprecipitation studies revealed that ARNO (a guanine nucleotide exchange factor for ARF6) and ARF6 formed complexes preferentially with activated Galpha(q) compared to non-activated Galpha(q). Formation of the Galpha(q) complexes with ARNO and ARF6 was detected early and was optimal after 30 min of receptor stimulation corresponding with the profile of ARF6 activation. Interestingly, binding experiments using purified proteins showed that Galpha(q) interacted directly with ARNO. Galpha(q)-dependent TPbeta receptor-mediated activation of ARF6 resulted in phosphoinositol-4,5-bisphosphate production which was potently inhibited by dominant negative mutants of ARNO and ARF6. Furthermore, our data show that the expression of ARNO and ARF6 promoted, whereas dominant negative mutants of these proteins inhibited the internalization of the TPbeta receptor. This further elucidates our previous data on the PLCbeta- and PKC-independent mechanism involved in Galpha(q)-mediated internalization of the TPbeta receptor. Taken altogether, our results support a novel model where activated Galpha(q) forms molecular complexes with ARNO and ARF6, possibly through a direct interaction with ARNO, leading to ARF6 activation.     

Identification of the Catalytic Site of Phospholipase D2 (PLD2) Newly Described Guanine Nucleotide Exchange Factor Activity

We have demonstrated that phospholipase D2 (PLD2) is a guanine nucleotide exchange factor (GEF) for Rac2 and determined the PLD2 domains and amino acid site(s) responsible for its GEF activity. Experiments using GST fusion proteins or GST-free counterparts, purified proteins revealed that the PX domain is sufficient to exert GEF activity similar to full-length PLD2. The PLD2-GEF catalytic site is formed by a hydrophobic pocket of residues Phe-107, Phe-129, Leu-166, and Leu-173, all of which are in the PX domain. A nearby Arg-172 is also important in the overall activity. PX mutants altering any of those five amino acids fail to have GEF activity but still bind to Rac2, while their lipase activity was mostly unaffected. In addition to the PX domain, a region in the pleckstrin homology domain (Ile-306–Ala-310) aids in the PX-mediated GEF activity by providing a docking site to hold Rac2 in place during catalysis. We conclude that PLD2 is a unique GEF, with the PX being the major catalytic domain for its GEF activity, whereas the pleckstrin homology domain assists in the PX-mediated activity. The physiological relevance of this novel GEF in cell biology is demonstrated here in chemotaxis and phagocytosis of leukocytes, as the specific PX and PH mutants abolished cell function. Thus, this study reveals for the first time the catalytic site that forms the basis for the mechanism behind the GEF activity of PLD2.     

Identification of a Novel Actin-Binding Domain within the Rho Guanine Nucleotide Exchange Factor TEM4

Spatio-temporal activation of Rho GTPases is essential for their function in a variety of biological processes and is achieved in part by regulating the localization of their activators, the Rho guanine nucleotide exchange factors (RhoGEFs). In this study, we provide the first characterization of the full-length protein encoded by RhoGEF TEM4 and delineate its domain structure, catalytic activity, and subcellular localization. First, we determined that TEM4 can stimulate guanine nucleotide exchange on RhoA and the related RhoB and RhoC isoforms. Second, we determined that TEM4, like other Dbl RhoGEFs, contains a functional pleckstrin homology (PH) domain immediately C-terminal to the catalytic Dbl homology (DH) domain. Third, using immunofluorescence analysis, we showed that TEM4 localizes to the actin cytoskeleton through sequences in the N-terminus of TEM4 independently of the DH/PH domains. Using site-directed mutagenesis and deletion analysis, we identified a minimal region between residues 81 and 135 that binds directly to F-actin and has an ～90-fold higher affinity for ATP-loaded F-actin. Finally, we demonstrated that a single point mutation (R130D) within full-length TEM4 abolishes actin binding and localization of TEM4 to the actin cytoskeleton, as well as dampens the in vivo activity of TEM4 towards RhoC. Taken together, our data demonstrate that TEM4 contains a novel actin binding domain and binding to actin is essential for TEM4 subcellular localization and activity. The unique subcellular localization of TEM4 suggests a spatially-restricted activity and expands the diversity of mechanisms by which RhoGEF function can be regulated.     

Expression Pattern and Localization Dynamics of Guanine Nucleotide Exchange Factor RIC8 during Mouse Oogenesis

Targeting of G proteins to the cell cortex and their activation is one of the triggers of both asymmetric and symmetric cell division. Resistance to inhibitors of cholinesterase 8 (RIC8), a guanine nucleotide exchange factor, activates a certain subgroup of G protein α-subunits in a receptor independent manner. RIC8 controls the asymmetric cell division in Caenorhabditis elegans and Drosophila melanogaster, and symmetric cell division in cultured mammalian cells, where it regulates the mitotic spindle orientation. Although intensely studied in mitosis, the function of RIC8 in mammalian meiosis has remained unknown. Here we demonstrate that the expression and subcellular localization of RIC8 changes profoundly during mouse oogenesis. Immunofluorescence studies revealed that RIC8 expression is dependent on oocyte growth and cell cycle phase. During oocyte growth, RIC8 is abundantly present in cytoplasm of oocytes at primordial, primary and secondary preantral follicle stages. Later, upon oocyte maturation RIC8 also populates the germinal vesicle, its localization becomes cell cycle dependent, and it associates with chromatin and the meiotic spindle. After fertilization, RIC8 protein converges to the pronuclei and is also detectable at high levels in the nucleolus precursor bodies of both maternal and paternal pronucleus. During first cleavage of zygote RIC8 localizes in the mitotic spindle and cell cortex of forming blastomeres. In addition, we demonstrate that RIC8 co-localizes with its interaction partners Gα_i1/2:GDP and LGN in meiotic/mitotic spindle, cell cortex and polar bodies of maturing oocytes and zygotes. Downregulation of Ric8 by siRNA leads to interferred translocation of Gα_i1/2 to cortical region of maturing oocytes and reduction of its levels. RIC8 is also expressed at high level in female reproductive organs e.g. oviduct. Therefore we suggest a regulatory function for RIC8 in mammalian gametogenesis and fertility.