Modulation of RhoA-Rho kinase-mediated Ca2+ sensitization of rabbit myometrium during pregnancy - role of Rnd3.

During pregnancy, the uterus undergoes major functional and structural remodelling. It is well known that during the major part of pregnancy, the myometrium normally remains relatively quiescent but is able to generate powerful contractions at the time of parturition. However, the intracellular molecular events regulating myometrial contractility during pregnancy still remain poorly understood. We applied differential gene expression screening using cDNA array technology to probe myometrium samples from non-pregnant and mid-pregnant (15 days) rabbits. Among the differentially expressed genes, the farnesylated small G-protein of the Rho family, Rnd3, was found to be upregulated (3.6-fold) at mid-pregnancy. Upregulation of Rnd3 was confirmed at the protein level by a 3.4-fold increase in Rnd3 expression in mid-pregnant myometrium. Measurements of contractile properties of beta-escin permeabilized smooth muscle strips revealed that the upregulation of Rnd3 correlated with an inhibition of RhoA-Rho kinase-mediated Ca2+ sensitization at mid-pregnancy. Treatment of muscle strips from mid-pregnant myometrium with the farnesyl-transferase inhibitor manumycin A (10 muM) led to the recovery of RhoA-Rho kinase-dependent Ca2+ sensitization. At late pregnancy (31 days), upregulation of RhoA and Rho kinase expression was associated with an increase in Ca2+ sensitivity of contractile proteins that was inhibited by the Rho kinase inhibitor Y-27632 (10 muM). These data thus demonstrate the time-dependent regulation of the RhoA-Rho kinase-mediated Ca2+ sensitization during the course of pregnancy. The depression of this mechanism at mid-pregnancy followed by its constitutive activation near term is associated with a co-ordinated modulation of Rnd3, RhoA and Rho kinase expression. The RhoA-Rho kinase signalling pathway and its regulators might thus represent potential targets for the development of new treatments for pre-term labour.

Membrane targeting: what a difference a G makes.

Pleckstrin homology domains are modular domains that direct membrane targeting of their host proteins by binding to polyphosphoinositides; recent results have increased our appreciation of how some of these domains actually bind 3-phosphoinositides, and along the way thrown up some unexpected observations.

Induced expression of Rnd3 is associated with transformation of polarized epithelial cells by the Raf-MEK-extracellular signal-regulated kinase pathway.

Madin-Darby canine kidney (MDCK) epithelial cells transformed by oncogenic Ras and Raf exhibit cell multilayering and alterations in the actin cytoskeleton. The changes in the actin cytoskeleton comprise a loss of actin stress fibers and enhanced cortical actin. Using MDCK cells expressing a conditionally active form of Raf, we have explored the molecular mechanisms that underlie these observations. Raf activation elicited a robust increase in Rac1 activity consistent with the observed increase in cortical actin. Loss of actin stress fibers is indicative of attenuated Rho function, but no change in Rho-GTP levels was detected following Raf activation. However, the loss of actin stress fibers in Raf-transformed cells was preceded by the induced expression of Rnd3, an endogenous inhibitor of Rho protein function. Expression of Rnd3 alone at levels equivalent to those observed following Raf transformation led to a substantial loss of actin stress fibers. Moreover, cells expressing activated RhoA failed to multilayer in response to Raf. Pharmacological inhibition of MEK activation prevented all of the biological and biochemical changes described above. Consequently, the data are consistent with a role for induced Rnd3 expression downstream of the Raf-MEK-extracellular signal-regulated kinase pathway in epithelial oncogenesis.

P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptors are coupled to Rho and Rho kinase activation in vascular myocytes.

In the cardiovascular system, activation of ionotropic (P2X receptors) and metabotropic (P2Y receptors) P2 nucleotide receptors exerts potent and various responses including vasodilation, vasoconstriction, and vascular smooth muscle cell proliferation. Here we examined the involvement of the small GTPase RhoA in P2Y receptor-mediated effects in vascular myocytes. Stimulation of cultured aortic myocytes with P2Y receptor agonists induced an increase in the amount of membrane-bound RhoA and stimulated actin cytoskeleton organization. P2Y receptor agonist-induced actin stress fiber formation was inhibited by C3 exoenzyme and the Rho kinase inhibitor Y-27632. Stimulation of actin cytoskeleton organization by extracellular nucleotides was also abolished in aortic myocytes expressing a dominant negative form of RhoA. Extracellular nucleotides induced contraction and Y-27632-sensitive Ca(2+) sensitization in aortic rings. Transfection of Swiss 3T3 cells with P2Y receptors showed that Rho kinase-dependent actin stress fiber organization was induced in cells expressing P2Y(1), P2Y(2), P2Y(4), or P2Y(6) receptor subtypes. Our data demonstrate that P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptor subtypes are coupled to activation of RhoA and subsequently to Rho-dependent signaling pathways.

LdARL-3A, a Leishmania promastigote-specific ADP-ribosylation factor-like protein, is essential for flagellum integrity.

The small G protein-encoding LdARL-3A gene, a homologue of the human ARL-3 gene, was isolated from Leishmania donovani, and its protein product characterised. It is unique in the Leishmania genome and expressed only in the extracellular promastigote insect form, but not in the intracellular amastigote mammalian form, as shown by northern blots and western blots developed with a specific anti-C terminus immune serum. Indirect immunofluorescence microscopy revealed distinct labelled spots regularly distributed on the plasma membrane, including the part lining the flagellum and the flagellar pocket. By transfection experiments, it was found that wild-type LdARL-3A-overexpressing promastigotes reached higher densities in culture, but released significantly less secreted acid phosphatase in the extracellular medium than the parental strain. When LdARL-3A blocked under the GDP-bound 'inactive' form or with an inactivated potential myristoylation site was overexpressed, the cells displayed an apparent wild-type phenotype, but died earlier in the stationary phase; in contrast to parental cells, they showed a diffuse pattern of fluorescence labelling in the cytoplasm and on the cell membrane. Strikingly, when a constitutively 'active' form of LdARL-3A (blocked under the GTP-bound form) was overexpressed, the promastigotes were immobile with a very short flagellum, a slow growth rate and a low level of acid phosphatase secretion; the length of the flagellum was inversely proportional to mutant protein expression. We concluded that LdARL-3A could be an essential gene involved in flagellum biogenesis; it may provide new approaches for control of the parasite at the insect stage.

Cyclic GMP-dependent protein kinase signaling pathway inhibits RhoA-induced Ca2+ sensitization of contraction in vascular smooth muscle.

The potent vasodilator action of cyclic GMP-dependent protein kinase (cGK) involves decreasing the Ca(2+) sensitivity of contraction of smooth muscle via stimulation of myosin light chain phosphatase through unknown mechanisms (Wu, X., Somlyo, A. V., and Somlyo, A. P. (1996) Biochem. Biophys. Res. Commun. 220, 658-663). Myosin light chain phosphatase activity is controlled by the small GTPase RhoA and its target Rho kinase. Here we demonstrate cGMP effects mediated by cGK that inhibit RhoA-dependent Ca(2+) sensitization of contraction of blood vessels and actin cytoskeleton organization in cultured vascular myocytes. Ca(2+) sensitization and actin organization were inhibited by both 8-bromo-cGMP and sodium nitroprusside (SNP). SNP also caused translocation of activated RhoA from the membrane to the cytosol. SNP-induced actin disassembly was lost in vascular myocytes in culture after successive passages but was restored by transfection of cells with cGK I. Furthermore, cGK phosphorylated RhoA in vitro, and addition of cGK I inhibited RhoA-induced Ca(2+) sensitization in permeabilized smooth muscle. 8-Bromo-cGMP-induced actin disassembly was inhibited in vascular myocytes expressing RhoA(Ala-188), a mutant that could not be phosphorylated. Collectively, these results indicate that cGK phosphorylates and inhibits RhoA and suggest that the consequent inhibition of RhoA-induced Ca(2+) sensitization and actin cytoskeleton organization contributes to the vasodilator action of nitric oxide.

Diaphanous-related formins bridge Rho GTPase and Src tyrosine kinase signaling.

We have examined the role of the mouse Diaphanous-related formin (DRF) Rho GTPase binding proteins, mDia1 and mDia2, in cell regulation. The DRFs are required for cytokinesis, stress fiber formation, and transcriptional activation of the serum response factor (SRF). 'Activated' mDia1 and mDia2 variants, lacking their GTPase binding domains, cooperated with Rho-kinase or ROCK to form stress fibers but independently activated SRF. Src tyrosine kinase associated and co-localized with the DRFs in endosomes and in mid-bodies of dividing cells. Inhibition of Src also blocked cytokinesis, SRF induction by activated DRFs, and cooperative stress fiber formation with active ROCK. Our results show that the DRF proteins couple Rho and Src during signaling and the regulation of actin dynamics.

Interaction of the Grb7 adapter protein with Rnd1, a new member of the Rho family.

Grb7 is a member of a family of molecular adapters which are able to contribute positively but also negatively to signal transduction and whose precise roles remain obscure. Rnd1 is a member of the Rho family, but, as opposed to usual GTPases, it is constitutively bound to GTP. We show here that Rnd1 and Grb7 interact, in two-hybrid assays, in vitro, and in pull-down experiments performed with SK-BR3, a breast cancer cell line that overexpresses Grb7. This interaction involves switch II loop of Rnd1, a region crucial for guanine nucleotide exchange in all GTPases, and a Grb7 SH2 domain, a region crucial for Grb7 interaction with several activated receptors. The contribution of the interaction between Rnd1 and Grb7 to their respective functions and properties is discussed.

GEFs: structural basis for their activation of small GTP-binding proteins.

Small GTP-binding proteins of the Ras superfamily function as molecular switches in fundamental events such as signal transduction, cytoskeleton dynamics and intracellular trafficking. Guanine-nucleotide-exchange factors (GEFs) positively regulate these GTP-binding proteins in response to a variety of signals. GEFs catalyze the dissociation of GDP from the inactive GTP-binding proteins. GTP can then bind and induce structural changes that allow interaction with effectors. Representative structures of four main classes of exchange factors have been described recently and, in two cases, structures of the GTP-binding protein-GEF complex have been solved. These structures, together with biochemical studies, have allowed a deeper understanding of the mechanisms of activation of Ras-like GTP-binding proteins and suggested how they might represent targets for therapeutic intervention.

The Rho-related protein Rnd1 inhibits Ca2+ sensitization of rat smooth muscle.

1. The small GTP-binding Rho proteins are involved in the agonist-induced Ca2+ sensitization of smooth muscle. The action and the expression of Rnd1, a new member of the Rho protein family constitutively bound to GTP, has been studied in rat smooth muscle. 2. Recombinant prenylated Rnd1 (0.01-0.1 mg ml-1) dose dependently inhibited carbachol- and GTPgammaS-induced Ca2+ sensitization in beta-escin-permeabilized ileal smooth muscle strips but had no effect on the tension at submaximal [Ca2+] (pCa 6.3). Rnd1 inhibited GTPgammaS-induced tension without shifting the dose-response curves to GTPgammaS. 3. pCa-tension relationships were not modified by Rnd1 and the rise in tension induced through the inhibition of myosin light chain phosphatase by calyculin A was not affected by Rnd1. 4. The Ca2+ sensitization induced by recombinant RhoA was completely abolished when RhoA and Rnd1 were applied together. 5. Rnd1 was expressed at a low level in membrane fractions prepared from intestinal or arterial smooth muscles. The expression of Rnd1 was strongly increased in ileal and aortic smooth muscle from rats treated with progesterone or oestrogen. Progesterone-treated ileal muscle strips showed a decrease in agonist-induced Ca2+ sensitization. 6. The present study shows that (i) Rnd1 inhibits agonist- and GTPgammaS-induced Ca2+ sensitization of smooth muscle by specifically interfering with a RhoA-dependent mechanism and (ii) an increase in Rnd1 expression may account, at least in part, for the steroid-induced decrease in agonist-induced Ca2+ sensitization.

Overexpression of the ARF1 exchange factor ARNO inhibits the early secretory pathway and causes the disassembly of the Golgi complex.

The small GTPase ARF1 is a key regulator of intracellular membrane traffic. In its active, GTP-bound form, ARF1 is associated with Golgi membranes and promotes the recruitment of the cytosolic coat protein complex, which will result in membrane budding and vesicle formation. ARNO (ARF nucleotide site opener) has been shown to act in vitro as a GTP exchange factor for ARF1. Here, we have investigated the function of ARNO in vivo. By immunofluorescence and cell fractionation, ARNO was found to be mostly cytosolic in HeLa cells. Its overexpression led to a strong inhibition of the secretion of SEAP (secreted form of alkaline phosphatase). Newly synthesized SEAP failed to acquire endoglycosidase H resistance, indicating a block in the early secretory pathway. This effect on secretion was accompanied by a disassembly of the Golgi complex and a redistribution of Golgi resident proteins into the endoplasmic reticulum (ER). On the other hand, ARNO overexpression did not affect the early endocytic pathway. These results show that ARNO functions in vivo in Golgi to ER transport. Its behavior is then consistent with ARNO being an exchange factor for ARF1.

ARNO3, a Sec7-domain guanine nucleotide exchange factor for ADP ribosylation factor 1, is involved in the control of Golgi structure and function.

Budding of transport vesicles in the Golgi apparatus requires the recruitment of coat proteins and is regulated by ADP ribosylation factor (ARF) 1. ARF1 activation is promoted by guanine nucleotide exchange factors (GEFs), which catalyze the transition to GTP-bound ARF1. We recently have identified a human protein, ARNO (ARF nucleotide-binding-site opener), as an ARF1-GEF that shares a conserved domain with the yeast Sec7 protein. We now describe a human Sec7 domain-containing GEF referred to as ARNO3. ARNO and ARNO3, as well as a third GEF called cytohesin-1, form a family of highly related proteins with identical structural organization that consists of a central Sec7 domain and a carboxy-terminal pleckstrin homology domain. We show that all three proteins act as ARF1 GEF in vitro, whereas they have no effect on ARF6, an ARF protein implicated in the early endocytic pathway. Substrate specificity of ARNO-like GEFs for ARF1 depends solely on the Sec7 domain. Overexpression of ARNO3 in mammalian cells results in (i) fragmentation of the Golgi apparatus, (ii) redistribution of Golgi resident proteins as well as the coat component beta-COP, and (iii) inhibition of SEAP transport (secreted form of alkaline phosphatase). In contrast, the distribution of endocytic markers is not affected. This study indicates that Sec7 domain-containing GEFs control intracellular membrane compartment structure and function through the regulation of specific ARF proteins in mammalian cells.

A glutamic finger in the guanine nucleotide exchange factor ARNO displaces Mg2+ and the beta-phosphate to destabilize GDP on ARF1.

The Sec7 domain of the guanine nucleotide exchange factor ARNO (ARNO-Sec7) is responsible for the exchange activity on the small GTP-binding protein ARF1. ARNO-Sec7 forms a stable complex with the nucleotide-free form of [Delta17]ARF1, a soluble truncated form of ARF1. The crystal structure of ARNO-Sec7 has been solved recently, and a site-directed mutagenesis approach identified a hydrophobic groove and an adjacent hydrophilic loop as the ARF1-binding site. We show that Glu156 in the hydrophilic loop of ARNO-Sec7 is involved in the destabilization of Mg2+ and GDP from ARF1. The conservative mutation E156D and the charge reversal mutation E156K reduce the exchange activity of ARNO-Sec7 by several orders of magnitude. Moreover, [E156K]ARNO-Sec7 forms a complex with the Mg2+-free form of [Delta17]ARF1-GDP without inducing the release of GDP. Other mutations in ARNO-Sec7 and in [Delta17]ARF1 suggest that prominent hydrophobic residues of the switch I region of ARF1 insert into the groove of the Sec7 domain, and that Lys73 of the switch II region of ARF1 forms an ion pair with Asp183 of ARNO-Sec7.

A new member of the Rho family, Rnd1, promotes disassembly of actin filament structures and loss of cell adhesion.

Members of the Rho GTPase family regulate the organization of the actin cytoskeleton in response to extracellular growth factors. We have identified three proteins that form a distinct branch of the Rho family: Rnd1, expressed mostly in brain and liver; Rnd2, highly expressed in testis; and Rnd3/RhoE, showing a ubiquitous low expression. At the subcellular level, Rnd1 is concentrated at adherens junctions both in confluent fibroblasts and in epithelial cells. Rnd1 has a low affinity for GDP and spontaneously exchanges nucleotide rapidly in a physiological buffer. Furthermore, Rnd1 lacks intrinsic GTPase activity suggesting that in vivo, it might be constitutively in a GTP-bound form. Expression of Rnd1 or Rnd3/RhoE in fibroblasts inhibits the formation of actin stress fibers, membrane ruffles, and integrin-based focal adhesions and induces loss of cell-substrate adhesion leading to cell rounding (hence Rnd for "round"). We suggest that these proteins control rearrangements of the actin cytoskeleton and changes in cell adhesion.

Structure of the Sec7 domain of the Arf exchange factor ARNO.

Small G proteins switch from a resting, GDP-bound state to an active, GTP-bound state. As spontaneous GDP release is slow, guanine-nucleotide-exchange factors (GEFs) are required to promote fast activation of small G proteins through replacement of GDP with GTP in vivo. Families of GEFs with no sequence similarity to other GEF families have now been assigned to most families of small G proteins. In the case of the small G protein Arf1, the exchange of bound GDP for GTP promotes the coating of secretory vesicles in Golgi traffic. An exchange factor for human Arf1, ARNO, and two closely related proteins, named cytohesin 1 and GPS1, have been identified. These three proteins are modular proteins with an amino-terminal coiled-coil, a central Sec7-like domain and a carboxy-terminal pleckstrin homology domain. The Sec7 domain contains the exchange-factor activity. It was first found in Sec7, a yeast protein involved in secretion, and is present in several other proteins, including the yeast exchange factors for Arf, Geal and Gea2. Here we report the crystal structure of the Sec7 domain of human ARNO at 2 A resolution and the identification of the site of interaction of ARNO with Arf.

ARF-independent inhibition of the carbachol-induced contractions by brefeldin A in intestinal smooth muscle.

The aim of this study was to determine whether an ADP ribosylation factor (ARF)-regulated pathway is involved in the carbachol-induced contraction in rat intestinal smooth muscle. Brefeldin A, a known inhibitor of the guanine nucleotide exchange activity on ARF, reversibly inhibited the carbachol-induced contraction in intact ileal muscle strips, whereas the carbachol- and guanosine 5'-O-(3-thiotriphosphate)-induced increases in the Ca2+ sensitivity of myofilaments in beta-escin-permeabilized strips were not affected. The high-K(+)-induced contraction in intact strips was also inhibited by brefeldin A. In isolated ileal myocytes, brefeldin A inhibited the Ca2+ channel current, indicating that the inhibitory effect of brefeldin A in intact cells is related to an inhibition of voltage-dependent Ca2+ channels. Furthermore, the loading of permeabilized strips with the combination of the recombinant fully myristoylated ARF1, the guanine nucleotide exchange factor ARNO, and guanosine 5'-triphosphate did not change the tone at constant pCa (6.45) and did not modify the carbachol- and guanosine 5'-O-(3-thiotriphosphate)-induced Ca2+ sensitization. Taken together, these findings suggest that an ARF-dependent pathway is not involved in the carbachol-induced contraction.

Role of protein-phospholipid interactions in the activation of ARF1 by the guanine nucleotide exchange factor Arno.

Arno is a 47-kDa human protein recently identified as a guanine nucleotide exchange factor for ADP ribosylation factor 1 (ARF1) with a central Sec7 domain responsible for the exchange activity and a carboxyl-terminal pleckstrin homology (PH) domain (Chardin, P., Paris, S., Antonny, B., Robineau, S., Béraud-Dufour, S., Jackson, C. L., and Chabre, M. (1996) Nature 384, 481-484). Binding of the PH domain to phosphatidylinositol 4,5-bisphosphate (PIP2) greatly enhances Arno-mediated activation of myristoylated ARF1. We show here that in the absence of phospholipids, Arno promotes nucleotide exchange on [Delta17]ARF1, a soluble mutant of ARF1 lacking the first 17 amino acids. This reaction is unaffected by PIP2, which suggests that the PIP2-PH domain interaction does not directly regulate the catalytic activity of Arno but rather serves to recruit Arno to membranes. Arno catalyzes the release of GDP more efficiently than that of GTP from [Delta17]ARF1, and a stable complex between Arno Sec7 domain and nucleotide-free [Delta17]ARF1 can be isolated. In contrast to [Delta17]ARF1, full-length unmyristoylated ARF1 is not readily activated by Arno in solution. Its activation requires the presence of phospholipids and a reduction of ionic strength and Mg2+ concentration. PIP2 is strongly stimulatory, indicating that binding of Arno to phospholipids is involved, but in addition, electrostatic interactions between phospholipids and the amino-terminal portion of unmyristoylated ARF1GDP seem to be important. We conclude that efficient activation of full-length ARF1 by Arno requires a membrane surface and two distinct protein-phospholipid interactions: one between the PH domain of Arno and PIP2, and the other between amino-terminal cationic residues of ARF1 and anionic phospholipids. The latter interaction is normally induced by insertion of the amino-terminal myristate into the bilayer but can also be artificially facilitated by decreasing Mg2+ and salt concentrations.

Toxin-induced activation of the G protein p21 Rho by deamidation of glutamine.

Pathogenic Escherichia coli are responsible for a variety of diseases, including diarrhoea, haemolytic uraemic syndrome, kidney infection, septicaemia, pneumonia and meningitis. Toxins called cytotoxic necrotizing factors (CNFs) are among the virulence factors produced by uropathogenic (CNF1) or enteropathogenic (CNF2) E. coli strains that cause diseases in humans and animals, respectively. CNFs induce an increase in the content of actin stress fibres and focal contacts in cultured cells. Effects of CNFs on the actin cytoskeleton correlated with a decrease in the electrophoretic mobility of the GTP-binding protein Rho and indirect evidence indicates that CNF1 might constitutively activate Rho. Here we show that CNF1 catalyses the deamidation of a glutamine residue at position 63 of Rho, turning it into glutamic acid, which inhibits both intrinsic GTP hydrolysis and that stimulated by its GTPase-activating protein (GAP). Thus, this deamidation of glutamine 63 by CNF1 leads to the constitutive activation of Rho, and induces the reorganization of actin stress fibres. To our knowledge, CNF1 is the first example of a bacterial toxin acting by deamidation of a specific target protein.