Cell wall remodeling and vesicle trafficking mediate the root clock in Arabidopsis.

In Arabidopsis thaliana, lateral roots initiate in a process preceded by periodic gene expression known as the root clock. We identified the vesicle-trafficking regulator GNOM and its suppressor, ADENOSINE PHOSPHATE RIBOSYLATION FACTOR GTPase ACTIVATION PROTEIN DOMAIN3, as root clock regulators. GNOM is required for the proper distribution of pectin, a mediator of intercellular adhesion, whereas the pectin esterification state is essential for a functional root clock. In sites of lateral root primordia emergence, both esterified and de-esterified pectin variants are differentially distributed. Using a reverse-genetics approach, we show that genes controlling pectin esterification regulate the root clock and lateral root initiation. These results indicate that the balance between esterified and de-esterified pectin states is essential for proper root clock function and the subsequent initiation of lateral root primordia.

Resveratrol Influences Pancreatic Islets by Opposing Effects on Electrical Activity and Insulin Release.

SCOPE: Resveratrol is suggested to improve glycemic control by activation of sirtuin 1 (SIRT1) and has already been tested clinically. Our investigation characterizes the targets of resveratrol in pancreatic beta cells and their contribution to short- and long-term effects on insulin secretion. METHODS AND RESULTS: Islets or beta cells are isolated from C57BL/6N mice. Electrophysiology is performed with microelectrode arrays and patch-clamp technique, insulin secretion and content are determined by radioimmunoassay, cAMP is measured by enzyme-linked immunosorbent assay, and cytosolic Ca2+ concentration by fluorescence methods. Resveratrol (25 µmol L-1 ) elevates [Ca2+ ]c and potentiates glucose-stimulated insulin secretion. These effects are associated with increased intracellular cAMP and are sensitive to the SIRT1 blocker Ex-527. Inhibition of EPAC1 by CE3F4 also abolishes the stimulatory effect of resveratrol. The underlying mechanism does not involve membrane depolarization as resveratrol even reduces electrical activity despite blocking KATP channels. Importantly, after prolonged exposure to resveratrol (14 days), the beneficial influence of the polyphenol on insulin release is lost. CONCLUSION: Resveratrol addresses multiple targets in pancreatic islets. Potentiation of insulin secretion is mediated by SIRT1-dependent activation of cAMP/EPAC1. Considering resveratrol as therapeutic supplement for patients with type 2 diabetes mellitus, the inhibitory influence on electrical excitability attenuates positive effects.

Epac activator critically regulates action potential duration by decreasing potassium current in rat adult ventricle.

Sympathetic stimulation is an important modulator of cardiac function via the classic cAMP-dependent signaling pathway, PKA. Recently, this paradigm has been challenged by the discovery of a family of guanine nucleotide exchange proteins directly activated by cAMP (Epac), acting in parallel to the classic signaling pathway. In cardiac myocytes, Epac activation is known to modulate Ca(2+) cycling yet their actions on cardiac ionic currents remain poorly characterized. This study attempts to address this paucity of information using the patch clamp technique to record action potential (AP) and ionic currents on rat ventricular myocytes. Epac was selectively activated by 8-CPT-AM (acetoxymethyl ester form of 8-CPT). AP amplitude, maximum depolarization rate and resting membrane amplitude were unaltered by 8-CPT-AM, strongly suggesting that Na(+) current and inward rectifier K(+) current are not regulated by Epac. In contrast, AP duration was significantly increased by 8-CPT-AM (prolongation of duration at 50% and 90% of repolarization by 41±10% and 43±8% respectively, n=11). L-type Ca(2+) current density was unaltered by 8-CPT-AM (n=16) so this cannot explain the action potential lengthening. However, the steady state component of K(+) current was significantly inhibited by 8-CPT-AM (-38±6%, n=15), while the transient outward K(+) current was unaffected by 8-CPT-AM. These effects were PKA-independent since they were observed in the presence of PKA inhibitor KT5720. Isoprenaline (100nM) induced a significant prolongation of AP duration, even in the presence of KT5720. This study provides the first evidence that the cAMP-binding protein Epac critically modulates cardiac AP duration by decreasing steady state K(+) current. These observations may be relevant to diseases in which Epac is upregulated, like cardiac hypertrophy.

Hyperdopaminergic modulation of inhibitory transmission is dependent on GSK-3ß signaling-mediated trafficking of GABAA receptors.

Cortical dopamine (DA) modulation of the gamma-amino butyric acid (GABA) system is closely associated with cognitive function and psychiatric disorders. We recently reported that the glycogen synthase kinase 3ß (GSK-3ß) pathway is required for hyperdopamine/D2 receptor-mediated inhibition of NMDA receptors in the prefrontal cortex. Here we explore whether or not GSK-3ß is also involved in dopaminergic modulation of GABAA receptor-mediated inhibitory transmission. We confirmed that DA induces a dose-dependent, bidirectional regulatory effect on inhibitory postsynaptic currents (IPSCs) in prefrontal neurons. The modulatory effects of DA were differentially affected by co-application of GSK-3ß inhibitors and different doses of DA. GSK-3ß inhibitors completely blocked high-dose (20 µM) DA-induced depressive effects on IPSCs but exhibited limited effects on the facilitating regulation of IPSC in low-dose DA (200 nM). We also confirmed that surface expressions of GABAA receptor ß2/3 subunits were significantly decreased by DA applied in cultured prefrontal neurons and in vivo administration of DA reuptake inhibitor. These effects were blocked by prior administration of GSK-3ß inhibitors. We explored DA-mediated regulation of GABAA receptor trafficking and exhibited the participation of brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2) or dynamin-dependent trafficking of GABAA receptors. Together, these data suggest that DA may act through different signaling pathways to affect synaptic inhibition, depending on the concentration. The GSK-3ß signaling pathway is involved in DA-induced decrease in BIG2-dependent insertion and an increase in the dynamin-dependent internalization of GABAA receptors, which results in suppression of inhibitory synaptic transmission.

Silencing of RhoA nucleotide exchange factor, ARHGEF3, reveals its unexpected role in iron uptake.

Genomewide association meta-analysis studies have identified > 100 independent genetic loci associated with blood cell indices, including volume and count of platelets and erythrocytes. Although several of these loci encode known regulators of hematopoiesis, the mechanism by which most sequence variants exert their effect on blood cell formation remains elusive. An example is the Rho guanine nucleotide exchange factor, ARHGEF3, which was previously implicated by genomewide association meta-analysis studies in bone cell biology. Here, we report on the unexpected role of ARHGEF3 in regulation of iron uptake and erythroid cell maturation. Although early erythroid differentiation progressed normally, silencing of arhgef3 in Danio rerio resulted in microcytic and hypochromic anemia. This was rescued by intracellular supplementation of iron, showing that arhgef3-depleted erythroid cells are fully capable of hemoglobinization. Disruption of the arhgef3 target, RhoA, also produced severe anemia, which was, again, corrected by iron injection. Moreover, silencing of ARHGEF3 in erythromyeloblastoid cells K562 showed that the uptake of transferrin was severely impaired. Taken together, this is the first study to provide evidence for ARHGEF3 being a regulator of transferrin uptake in erythroid cells, through activation of RHOA.

GNOM-LIKE 2, encoding an adenosine diphosphate-ribosylation factor-guanine nucleotide exchange factor protein homologous to GNOM and GNL1, is essential for pollen germination in Arabidopsis.

In flowering plants, male gametes are delivered to female gametophytes by pollen tubes. Although it is important for sexual plant reproduction, little is known about the genetic mechanism that controls pollen germination and pollen tube growth. Here we report the identification and characterization of two novel mutants, gnom-like 2-1 (gnl2-1) and gnl2-2 in Arabidopsis thaliana, in which the pollen grains failed to germinate in vitro and in vivo. GNL2 encodes a protein homologous to the adenosine diphosphate-ribosylation factor-guanine nucleotide exchange factors, GNOM and GNL1 that are involved in endosomal recycling and endoplasmic reticulum-Golgi vesicular trafficking. It was prolifically expressed in pollen grains and pollen tubes. The results of the present study suggest that GNL2 plays an important role in pollen germination.

High-throughput screening for small-molecule inhibitors of LARG-stimulated RhoA nucleotide binding via a novel fluorescence polarization assay.

Guanine nucleotide exchange factors (GEFs) stimulate guanine nucleotide exchange and the subsequent activation of Rho-family proteins in response to extracellular stimuli acting upon cytokine, tyrosine kinase, adhesion, integrin, and G-protein-coupled receptors (GPCRs). Upon Rho activation, several downstream events occur, such as morphological and cytoskeletal changes, motility, growth, survival, and gene transcription. The leukemia-associated RhoGEF (LARG) is a member of the regulators of G-protein signaling homology domain (RH) family of GEFs originally identified as a result of chromosomal translocation in acute myeloid leukemia. Using a novel fluorescence polarization guanine nucleotide-binding assay using BODIPY-Texas Red-GTPgammaS (BODIPY-TR-GTPgammaS), the authors performed a 10,000-compound high-throughput screen for inhibitors of LARG-stimulated RhoA nucleotide binding. Five compounds identified from the high-throughput screen were confirmed in a nonfluorescent radioactive guanine nucleotide-binding assay measuring LARG-stimulated [( 35)S] GTPgammaS binding to RhoA, thus ruling out nonspecific fluorescent effects. All 5 compounds selectively inhibited LARG-stimulated RhoA [( 35)S] GTPgammaS binding but had little to no effect on RhoA or Galpha( o) [(35)S] GTPgammaS binding. Therefore, these 5 compounds should serve as promising starting points for the development of small-molecule inhibitors of LARG-mediated nucleotide exchange as both pharmacological tools and therapeutics. In addition, the fluorescence polarization guanine nucleotide-binding assay described here should serve as a useful approach for both high-throughput screening and general biological applications.

General receptor for phosphoinositides 1, a novel repressor of thyroid hormone receptor action that prevents deoxyribonucleic acid binding.

Thyroid hormone receptors (TRs) bind to response elements (TREs) located in the promoter region of target genes and modulate their transcription. The effects of TRs require the presence of coregulators that act as adaptor molecules between TRs and complexes that are involved in chromatin remodeling or that directly contact the basal transcription machinery. Using the yeast two-hybrid system, we identified a new interacting partner for TRs: GRP1 (general receptor for phosphoinositides-1), a nucleotide exchange factor, which had never been shown to interact with nuclear receptors. We reconfirmed the interaction between TRs and GRP1 in yeast and glutathione-S-transferase pull-down assays, and determined the areas of TRs and GRP1 involved in the interaction. Coimmunoprecipitation studies demonstrated that the interaction between GRP1 and TRs takes place in the cytoplasm and the nucleus of mammalian cells. To assess functional consequences of the interaction, we used transient transfection of CV-1 cells with TR and GRP1 expression vectors and luciferase reporter genes. On positive TREs, GRP1 decreased activation by 45-60%. On the negative TREs it increased repression by blunting the activation in the absence of T3, except for TRbeta2, which was not affected. Using EMSA, we have determined that addition of GRP1 diminishes the formation of TR/TR homodimers and TR/retinoid X receptor heterodimers on TREs, which could explain the effect of GRP1 on transcription. Furthermore, protein interaction assays using increasing concentrations of double-stranded TREs show a dose-dependent decrease of the interaction between GRP1 and TRs. The homo/heterodimers formed by TRs and retinoic X receptor-alpha were not influenced by the presence of GRP1, also suggesting that GRP1 interferes directly with DNA binding. Taken together, these data provide evidence that GRP1 is a new corepressor for TRs, which modulates both positive and negative regulation by T3 by decreasing TR-complex formation on TREs.

Calcium channel function regulated by the SH3-GK module in beta subunits.

beta subunits of voltage-gated calcium channels (VGCCs) regulate channel trafficking and function, thereby shaping the intensity and duration of intracellular changes in calcium. beta subunits share limited sequence homology with the Src homology 3-guanylate kinase (SH3-GK) module of membrane-associated guanylate kinases (MAGUKs). Here, we show biochemical similarities between beta subunits and MAGUKs, revealing important aspects of beta subunit structure and function. Similar to MAGUKs, an SH3-GK interaction within beta subunits can occur both intramolecularly and intermolecularly. Mutations that disrupt the SH3-GK interaction in beta subunits alter channel inactivation and can inhibit binding between the alpha(1) and beta subunits. Coexpression of beta subunits with complementary mutations in their SH3 and GK domains rescues these deficits through intermolecular beta subunit assembly. In MAGUKs, the SH3-GK module controls protein scaffolding. In beta subunits, this module regulates the inactivation of VGCCs and provides an additional mechanism for tuning calcium responsiveness.

Src kinase regulates the activation of a novel FGD-1-related Cdc42 guanine nucleotide exchange factor in the signaling pathway from the endothelin A receptor to JNK.

Small GTPases act as binary switches by cycling between an inactive (GDP-bound) and an active (GTP-bound) state. Upon stimulation with extracellular signals, guanine-nucleotide exchange factors (GEFs) stimulate the exchange of GDP to GTP to shift toward the active forms of small GTPases, recognizing the downstream targets. Here we show that KIAA0793, containing substantial sequence homology with the catalytic Dbl homology domain of the faciogenital dysplasia gene product (FGD1), is a specific GEF for Cdc42. We, therefore, tentatively named it FRG (FGD1-related Cdc42-GEF). Src kinase directly phosphorylates and activates FRG, as Vav family GEFs. Additionally, FRG is involved in the signaling pathway from the endothelin A receptor to c-Jun N-terminal kinase, resulting in the inhibition of cell motility. These results suggest that FRG is a member of Cdc42-GEF and plays an important role in the signaling pathway downstream of G protein-coupled receptors.

Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly.

Vertebrate-striated muscle is assumed to owe its remarkable order to the molecular ruler functions of the giant modular signaling proteins, titin and nebulin. It was believed that these two proteins represented unique results of protein evolution in vertebrate muscle. In this paper we report the identification of a third giant protein from vertebrate muscle, obscurin, encoded on chromosome 1q42. Obscurin is approximately 800 kD and is expressed specifically in skeletal and cardiac muscle. The complete cDNA sequence of obscurin reveals a modular architecture, consisting of >67 intracellular immunoglobulin (Ig)- or fibronectin-3-like domains with multiple splice variants. A large region of obscurin shows a modular architecture of tandem Ig domains reminiscent of the elastic region of titin. The COOH-terminal region of obscurin interacts via two specific Ig-like domains with the NH(2)-terminal Z-disk region of titin. Both proteins coassemble during myofibrillogenesis. During the progression of myofibrillogenesis, all obscurin epitopes become detectable at the M band. The presence of a calmodulin-binding IQ motif, and a Rho guanine nucleotide exchange factor domain in the COOH-terminal region suggest that obscurin is involved in Ca(2+)/calmodulin, as well as G protein-coupled signal transduction in the sarcomere.