Search

Heterotrimeric G‐proteins are composed of α, β, and γ subunits, and function as signal transducers. Critical roles of the α‐subunits of Gi/o family heterotrimeric G‐proteins, Gαi2, and Gαo1, have so far been reported in brain development and neurodevelopmental disorders. In this study, we tried to clarify the role of Gαi1, α‐subunit of another Gi/o family member Gi1, during corticogenesis, based on the recent identification of its gene abnormalities in neurodevelopmental disorders. In western blot analyses, Gαi1 was found to be expressed in mouse brain in a developmental stage‐dependent manner. Morphological analyses revealed that Gαi1 was broadly distributed in cerebral cortex with relatively high expression in the ventricular zone (VZ) at embryonic day (E) 14. Meanwhile, Gαi1 was enriched in membrane area of yet unidentified early mitotic cells in the VZ and the marginal zone at E14. Acute knockdown of Gαi1 with in utero electroporation in cerebral cortex caused cell cycle elongation of the neural progenitor cells and promoted their cell cycle exit. Gαi1‐deficient cortical neurons also exhibited delayed radial migration during corticogenesis, with abnormally elongated leading processes and hampered nucleokinesis. In addition, silencing of Gαi1 prevented basal dendrite development. The migration and dendritic phenotypes were at least partially rescued by an RNAi‐resistant version of Gαi1. Collectively, these results strongly suggest a crucial role of Gi1 in cortical development, and disturbance of its function may cause deficits in synaptic network formation, leading to neurodevelopmental disorders. [ABSTRACT FROM AUTHOR]

Copyright of Mycosystema is the property of Mycosystema Editorial Board and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Acetylcholine activation of α7 nicotinic acetylcholine receptors (α7 nAChRs) in microglia attenuates neuroinflammation and regulates TNF-α release. We used lipopolysaccharide to model inflammation in the microglial cell line EOC20 and examined signaling by the α7 nAChR. Co-immunoprecipitation experiments confirm that α7 nAChRs bind heterotrimeric G proteins in EOC20 cells. Interaction with Gαi mediates α7 nAChR signaling via enhanced intracellular calcium release and a decrease in cAMP, p38 phosphorylation, and TNF-α release. These α7 nAChR effects were blocked by the inhibition of Gαi signaling via pertussis toxin, PLC activity with U73122, and α7 nAChR channel activity with the selective antagonist α-bungarotoxin. Moreover, α7 nAChR signaling in EOC20 cells was significantly diminished by the expression of a dominant-negative α7 nAChR, α7345-8A, shown to be impaired in G protein binding. These findings indicate an essential role for G protein coupling in α7 nAChR function in microglia leading to the regulation of inflammation in the nervous system. [ABSTRACT FROM AUTHOR]

Transmission ratio distortion ( TRD) is the departure from the expected Mendelian ratio in offspring, a poorly investigated biological phenomenon in livestock species. Given the current availability of specific parametric methods for the analysis of segregation data, this study focused on the screening of TRD in 602 402 single nucleotide polymorphisms covering all autosomal chromosomes in seven Spanish beef cattle breeds. On average, 0.13% ( n = 786) and 0.01% ( n = 29) of genetic markers evidenced sire- or dam-specific TRD respectively. There were no single nucleotide polymorphisms accounting for both sire- and dam-specific TRD at the same time, and only one marker (rs43147474) accounted for (sire-specific) TRD in all seven breeds. It must be noted that rs43147474 is located in the fourth intronic region of the GTP-binding protein 10 gene, and this locus has been previously linked to the maintenance of mitochondria and nucleolar architectures. Alternatively, other candidate genes surround this hot-spot for sire-specific TRD in the cattle genome, and they are related to embryonic and postnatal lethality as well as prostate cancer, among others. This research characterized the distribution of TRD in the bovine genome, highlighting heterogeneous results when comparing across breeds. [ABSTRACT FROM AUTHOR]

We analyzed the role of a heterotrimeric G-protein, Gi2, in the development of the cerebral cortex. Acute knockdown of the α-subunit (Gαi2) with in utero electroporation caused delayed radial migration of excitatory neurons during corticogenesis, perhaps because of impaired morphology. The migration phenotype was rescued by an RNAi-resistant version of Gαi2. On the other hand, silencing of Gαi2 did not affect axon elongation, dendritic arbor formation or neurogenesis at ventricular zone in vivo. When behavior analyses were conducted with acute Gαi2-knockdown mice, they showed defects in social interaction, novelty recognition and active avoidance learning as well as increased anxiety. Subsequently, using whole-exome sequencing analysis, we identified a de novo heterozygous missense mutation (c.680C>T; p.Ala227Val) in the GNAI2 gene encoding Gαi2 in an individual with periventricular nodular heterotopia and intellectual disability. Collectively, the phenotypes in the knockdown experiments suggest a role of Gαi2 in the brain development, and impairment of its function might cause defects in neuronal functions which lead to neurodevelopmental disorders. [ABSTRACT FROM AUTHOR]

Yeast GTP-binding protein (YPT1 protein) has been reported to function in the early stages of the secretory pathway. Particularly, YPT1 protein is observed to regulate both the endoplasmic reticulum-to-Golgi transport and the autophagy. Therefore, the YPT1 protein overexpressed in yeast vacuoles is expected to enhance antimicrobial and anticancer activity. The enhancement of yeast vacuolar activity under the overexpression of YPT1 was evaluated by the analysis of lysozyme activity, antimicrobial activity against Escherichia coli and Staphylococcus aureus, and MTT assay against HeLa cell lines. Additionally, the rise in concentration of some important proteinases inside the vacuole, such as proteinase A, proteinase B, and vacuolar carboxypeptidase Y (CPY) were also recorded using a 2DE technique. All results imply YPT1 involvement in the recruitment of some specific proteinases into vacuoles, which leads to the enhancement of vacuolar activity. Since these there proteinases belong to the CPY pathway, YPT1 is even believed to up-regulate this trafficking pathway in yeast cells. Future studies, however, should be carried out to discover the mechanisms that allow YPT1 to recruit these proteins into yeast vacuoles. [ABSTRACT FROM AUTHOR]

Plant-parasitic nematodes cause significant damage to major crops throughout the world. The small number of genes conferring natural plant resistance and the limitations of chemical control require the development of new protective strategies. RNA interference or the inducible over-expression of nematicidal genes provides an environment-friendly approach to this problem. Candidate genes include NGB, which encodes a small GTP-binding protein, and NAB/ ERabp1, which encodes an auxin-binding protein, which were identified as being up-regulated in tomato roots in a transcriptome screen of potato cyst nematode ( Globodera rostochiensis) feeding sites. Real-time reverse transcription-polymerase chain reaction ( RT-PCR) and in situ hybridization confirmed the localized up-regulation of these genes in syncytia and surrounding cells following nematode infection. Gene-silencing constructs were introduced into tomato, resulting in a 20%-98% decrease in transcription levels. Nematode infection tests conducted on transgenic plants showed 57%-82% reduction in the number of G. rostochiensis females in vitro and 30%-46% reduction in pot trials. Transmission electron microscopy revealed a deterioration of cytoplasm, and degraded mitochondria and plastids, in syncytia induced in plants with reduced NAB/ ERabp1 expression. Cytoplasm in syncytia induced in plants with low NGB expression was strongly electron translucent and contained very few ribosomes; however, mitochondria and plastids remained intact. Functional impairments in syncytial cytoplasm of silenced plants may result from NGB's role in ribosome biogenesis; this was confirmed by localization of yellow fluorescent protein ( YFP)-labelled NGB protein in nucleoli and co-repression of NGB in plants with reduced NAB/ ERabp1 expression. These results demonstrate that NGB and NAB/ ERabp1 play important roles in the development of nematode-induced syncytia. [ABSTRACT FROM AUTHOR]

Key message: Isolation, cloning, and expression characterization of an ARF gene from S. alterniflora, demonstrating its involvement in abiotic stress tolerance. Abstract: Adenosine diphosphate-ribosylation factors (ARFs) are small guanine nucleotide-binding proteins that play an important role in intracellular protein trafficking necessary for undertaking multiple physiological functions in plant growth and developmental processes. However, little is known about the mechanism of ARF functioning at the molecular level, as well as its involvement in abiotic stress tolerance. In this study, we demonstrated the direct involvement of an ARF gene SaARF from a grass halophyte Spartina alterniflora in abiotic stress adaptation for the first time. SaARF, which encodes a protein with predicted molecular mass of 21 kDa, revealed highest identity with ARF of Oryza sativa. The SaARF gene is transcriptionally regulated by salt, drought, cold, and ABA in the leaves and roots of S. alterniflora. Arabidopsis plants overexpressing SaARF showed improved seed germination and survival of seedlings under salinity stress. Similarly, SaARF transgenic Arabidopsis plants were more tolerant to drought stress, compared to wild-type plants, by maintaining chlorophyll synthesis, increasing osmolyte synthesis, and stabilizing membrane integrity. Oxidative damage due to moisture stress in transgenic Arabidopsis was also reduced possibly by activating antioxidant genes, AtSOD1 and AtCAT. Our results suggest that enhanced drought and salinity tolerance conferred by the SaARF gene may be due to its role in mediating multiple abiotic stress tolerance mechanisms. [ABSTRACT FROM AUTHOR]

Septins form oligomeric complexes consisting of septins from different subgroups, which form filaments that are involved in a number of biological processes. They are GTP-binding proteins that contain all the necessary elements to perform the general GDP-to-GTP conformational switch. It is however unclear whether or not such a switch is important for the dynamics of septin filaments. Here we investigate the complex GTPase reaction of members of each of the four human septin groups, which is dominated by the stability of dimer formation via the nucleotide binding or so-called G-interface. The results also show that the actual hydrolysis reaction is very similar for three septin groups in the monomeric state while the Sept6 has no GTPase activity. Sept7, the only member of the Sept7 subgroup, forms a very tight G-interface dimer in the GDP-bound state. Here we show that the stability of the interface is dramatically decreased by exchanging GDP with a nucleoside triphosphate, which is believed to influence filament formation and dynamics via Sept7. [ABSTRACT FROM AUTHOR]

Rab3D is a low molecular weight GTP-binding protein that associates with secretory granules in exocrine cells. AR42J cells are derived from rat pancreatic exocrine tumor cells and develop an acinar cell-like phenotype when treated with dexamethasone (Dex). In the present study, we examined the role of Rab3D in Dex-treated AR42J cells. Rab3D expression and localization were analyzed by subcellular fractionation and immunoblotting. The role of Rab3D was examined by overexpressing myc-labeled wild-type-Rab3D and a constitutively active form of Rab3D (Rab3D-Q81L) in AR42J cells. We found that Rab3D is predominantly membrane-associated in AR42J cells and co-localizes with zymogen granules (ZG). Following CCK-8-induced exocytosis, amylase-positive ZGs appeared to move towards the periphery of the cell and co-localization between Rab3D and amylase was less complete when compared to basal conditions. Overexpression of WT, but not mutant Rab3D, resulted in an increase in cellular amylase levels. Overexpression of mutant and WT Rab3D did not affect granule morphology, CCK-8-induced secretion, long-term (48 hr) basal amylase release or granule density. We conclude that Rab3D is not involved in agonist-induced exocytosis in AR42J cells. Instead, Rab3D may regulate amylase content in these cells. [ABSTRACT FROM AUTHOR]

Background: Rab proteins constitute a large family of monomeric GTP-binding proteins that regulate intracellular vesicle transport. Several Rab proteins, including rab31, have been shown to affect cancer progression and are related with prognosis in various types of cancer including breast cancer. Recently, the gene encoding rab31 was found to be overexpressed in estrogen receptor-positive breast cancer tissue. In a previous study we found a significant association of high rab31 mRNA expression with poor prognosis in node-negative breast cancer patients. In the present study, we aimed to investigate the impact of rab31 (over)-expression on important aspects of tumor progression in vitro and in vivo. Methods: Breast cancer cells displaying low (MDA-MB-231) or no (CAMA-1) endogenous rab31 expression were stably transfected with a rab31 expression plasmid. Batch-transfected cells as well as selected cell clones, expressing different levels of rab31 protein, were analyzed with regard to proliferation, cell adhesion, the invasive capacity of tumor cells, and in vivo in a xenograft tumor model. Polyclonal antibodies directed to recombinantly expressed rab31 were generated and protein expression analyzed by immunohistochemistry, Western blot analysis, and a newly developed sensitive ELISA. Results: Elevated rab31 protein levels were associated with enhanced proliferation of breast cancer cells. Interestingly, weak to moderate overexpression of rab31 in cell lines with no detectable endogenous rab31 expression was already sufficient to elicit distinct effects on cell proliferation. By contrast, increased expression of rab31 in breast cancer cells led to reduced adhesion towards several extracellular matrix proteins and decreased invasive capacity through MatrigelTM. Again, the rab31-mediated effects on cell adhesion and invasion were dose-dependent. Finally, in a xenograft mouse model, we observed a significantly impaired metastatic dissemination of rab31 overexpressing MDA-MB-231 breast cancer cells to the lung. Conclusions: Overexpression of rab31 in breast cancer cells leads to a switch from an invasive to a proliferative phenotype as indicated by an increased cell proliferation, reduced adhesion and invasion in vitro, and a reduced capacity to form lung metastases in vivo. [ABSTRACT FROM AUTHOR]

Brain tissue contains multiple forms of Phospholipase A2 (PLA2) whose activities are involved in intracellular and intercellular signalling related to normal functions such as long-term potentiation, neurotransmitter release, cell growth and differentiation. Among them, we focused on regulatory mechanism of cPLA2α (Group IVA cytosolic PLA2) in brain tissue. In the present study, we report the identification of a cPLA2-activating protein (cPLAP) in the bovine brain. cPLAP activity appeared as two major peaks with molecular masses of 200 and 42 kDa in a Superose 12 gel filtration FPLC column. The 42-kDa form of cPLAP, designated cPLAPγ, was further purified using a Mono S FPLC column to near homogeneity and characterized to as a GTP-binding protein (G protein). Metabolic labelling and immunoprecipitation studies revealed that cPLAPγ associates with cPLA2 in vitro and co-immunoprecipitates with [35S]-cPLA2. Notably, cPLAPγ rendered cPLA2 fully activated at submicromolar concentrations of Ca2+. These results suggest that cPLAPγ may act as a G protein, activating cPLA2α prior to reaching full intracellular Ca2+ concentrations. [ABSTRACT FROM PUBLISHER]

Septins constitute a family of conserved guanine nucleotide binding proteins found in a wide range of organisms from fungi to mammals. Members of the family share a canonical G-domain with N- and C-terminal extensions. G-domains assemble into hetero-oligomeric complexes which form non-polarised filaments or rings. Linear filaments are formed between the G-domains using either the guanine nucleotide binding site (G interface) or N- and C-terminal extensions (NC interface). Sept7 is a unique among the 13 human septins in that it occupies the ends of hexameric building blocks which assemble into non-polarised filaments. To gain insight into its particular properties we performed structural and biochemical studies on Sept7. We solved the crystal structure of a Sept7 dimer in the GDP-bound state. The structure and biochemistry of Sept7 provide new insights into the dynamics of the G interface and outline the differences in the properties of Sept7 compared to the members of group 2 septins. [ABSTRACT FROM AUTHOR]

cDNA clone designated arf1 was isolated from a physic nut ( Jatropha curcas L.) endosperm cDNA library which encodes a small GTP-binding protein and has significant homology to ADP-ribosylation factors (ARF) in plants, animals and microbes. The cDNA contains an open reading frame that encodes a polypeptide of 181 amino acids with a calculated molecular mass of 20.7 kDa. The deduced amino acid sequence showed high homology to known ARFs from other organisms. The products of the arf1 obtained by overexpression in E. coli revealed the specific binding activity toward GTP. The expression of arf1 was observed in flowers, roots, stems and leaves as analyzed by RT-PCR, and its transcriptional level was highest in flowers. In particular, the accumulation of arf1 transcripts was different under various environmental stresses in seedlings. The results suggest that arf1 plays distinct physiological roles in Jatropha curcas cells. [ABSTRACT FROM AUTHOR]

Sensitivity of the adenylyl cyclase signaling system (ACSS) to polypeptide hormones and biogenic amines is studied in testes and ovaries of rats after the 2- and 4-day fasting as compared with control animals. In tissues of the fasted rats there is shown a decrease in the basal activity of adenylyl cyclase (AC) and of the basal level of the GTP binding of heterotrimeric G proteins. An increase in duration of fasting from 2 to 4 days led to intensification of these changes. In the fasted rats, the AC-stimulating effects of chorionic gonadotropin, PACAP-38, and isoproterenol, realized via G protein of stimulatory type are enhanced, whereas the inhibitory effects of somatostatin on the AC activity realized via G protein of the inhibitory type are reduced. In testes of the fasted rats the stimulating effect of serotonin on AC via both types of G proteins increased, whereas the inhibitory effects of the hormone decreased. Thus, under conditions of fasting, in rat testes and ovaries the ACSS sensitivity to regulatory effects of hormones is changing: their stimulatory effects are increased, while its inhibitory effects, on the contrary, are decreased. We suggest that these changes are one of the key mechanisms of adaptation of organism to deficiency of nutritional resources to be aimed at intensifying the tissues catabolic processes, preferably lypolysis. [ABSTRACT FROM AUTHOR]

Sensitivity of the adenylyl cyclase signaling system (ACSS) to polypeptide hormones and biogenic amines is studied in testes and ovaries of rats after the 2- and 4-day fasting as compared with control animals. In tissues of the fasted rats there is shown a decrease in the basal activity of adenylyl cyclase (AC) and of the basal level of the GTP binding of heterotrimeric G proteins. An increase in duration of fasting from 2 to 4 days led to intensification of these changes. In the fasted rats, the AC-stimulating effects of chorionic gonadotropin, PACAP-38, and isoproterenol, realized via G protein of stimulatory type are enhanced, whereas the inhibitory effects of somatostatin on the AC activity realized via G protein of the inhibitory type are reduced. In testes of the fasted rats the stimulating effect of serotonin on AC via both types of G proteins increased, whereas the inhibitory effects of the hormone decreased. Thus, under conditions of fasting, in rat testes and ovaries the ACSS sensitivity to regulatory effects of hormones is changing: their stimulatory effects are increased, while its inhibitory effects, on the contrary, are decreased. We suggest that these changes are one of the key mechanisms of adaptation of organism to deficiency of nutritional resources to be aimed at intensifying the tissues catabolic processes, preferably lypolysis. [ABSTRACT FROM AUTHOR]

Abstract: Mono-glucosylation of (H/K/N)Ras by Clostridium sordellii lethal toxin (TcsL) blocks critical survival signaling pathways, resulting in apoptotic cell death. One yet unsolved problem in studies on TcsL is the lack of a method allowing the specific detection of (H/K/N)Ras glucosylation. In this study, we identify the Ras(Mab 27H5) antibody as a glucosylation-sensitive antibody capable for the immunoblot detection of (H/K/N)Ras glucosylation in TcsL-treated cells. Alternative Ras antibodies including the K-Ras(Mab F234) antibody or the v-H-Ras(Mab Y13-159) antibody recognize Ras proteins regardless of glucosylation. (H/K)Ras are further shown to be more efficaciously glucosylated by TcsL than Rac1 in rat basophilic leukemia cells as well as in a cell-free system. Structured summary: MINT-7261742: TcsL (uniprotkb:Q46342) enzymaticly reacts (MI:0414) H-RAS (uniprotkb:P01112) by enzymatic studies (MI:0415) MINT-7261729: TcsL (uniprotkb:Q46342) enzymaticly reacts (MI:0414) Rac1 (uniprotkb:P63000) by enzymatic studies (MI:0415) MINT-7261772: TcsL (uniprotkb:Q46342) enzymaticly reacts (MI:0414) K-RAS (uniprotkb:P01116) by enzymatic studies (MI:0415) MINT-7261784: TcsL (uniprotkb:Q46342) enzymaticly reacts (MI:0414) N-RAS (uniprotkb:P01111) by enzymatic studies (MI:0415) [Copyright &y& Elsevier]

Septins constitute a group of GTP-binding proteins involved in cytokinesis and other essential cellular functions. They form heterooligomeric complexes that polymerize into nonpolar filaments and are dynamic during different stages of the cell cycle. Posttranslational modifications and interacting partners are widely accepted regulators of septin filament function, but the contribution of nucleotide is undefined due to a lack of detailed structural information. Previous low-resolution structures showed that the G domain assembles into a linear polymer with 2 different interfaces involving the N and C termini and the G binding sites. Here we report the crystal structure of SEPT2 bound to GppNHp at 2.9 Å resolution. GTP binding induces conformational changes in the switch regions at the G interfaces, which are transmitted to the N-terminal helix and also affect the NC interface. Biochemical studies and sequence alignment suggest that a threonine, which is conserved in certain subgroups of septins, is responsible for GTP hydrolysis. Although this threonine is not present in yeast CDC3 and CDC11, its mutation in CDC10 and CDC12 induces temperature sensitivity. Highly conserved contact residues identified in the G interface are shown to be necessary for Cdc3-10, but not Cdc11-12, heterodimer formation and cell growth in yeast. Based on our findings, we propose that GTP binding/hydrolysis and the nature of the nucleotide influence the stability of interfaces in heterooligomeric and polymeric septins and are required for proper septin filament assembly/disassembly. These data also offer a first rationale for subdividing human septins into different functional subgroups. [ABSTRACT FROM AUTHOR]

DRGs are very highly conserved GTP-binding proteins. All eukaryotes contain DRG1 and DRG2 orthologs. Arabidopsis has three DRGs: AtDRG1 (At4g39520), AtDRG2 (At1g17470), and AtDRG3 (At1g72660). DRG2 and DRG3 encode proteins that are 95% identical; identity between DRG1 and DRG2/3 is 55%. The focus of this article is expression of Arabidopsis DRGs. DRG1 and DRG2 promoter-GUS constructs showed similar spatial expression in seedlings and mature organs, but gene-specific differences were noted. Quantitative real-time PCR experiments indicated similar levels of DRG1 and DRG2 mRNA accumulation in most tissues. DRG3 transcripts were very low in all tissues. Heat stress at 37°C led to a 10-fold increase in DRG1 transcripts and a 1000-fold increase in DRG3 transcripts. DRG1 antibodies recognized a 43-kD protein, and DRG2 antibodies recognized bands at 30, 43, and 45 kD. Plants were exposed to stresses (salt, heat, cold, UV light, osmotic, and other stresses) and examined by Western blotting. Only heat stress caused detectable changes. Heat did not affect DRG1, but DRG2 and a 72-kD protein recognized by DRG2 antibodies both increased. The modest changes in DRG mRNA and protein levels seen here suggest that other types of regulation, such as altered subcellular localization, may be important for their cellular functions. [ABSTRACT FROM AUTHOR]

Background: Basic secretagogues of connective tissue mast cells act as receptor mimetic agents that trigger mast cells by activating G proteins. This leads to simultaneous propagation of two signaling pathways: one that culminates in exocytosis, while the other involves protein tyrosine phosphorylation and leads to release of arachidonic acid metabolites. We have previously shown that introduction of a peptide that comprises the C-terminal end of Gαi3 into permeabilized mast cells inhibits basic secretagogue-induced exocytosis [Aridor et al., Science 1993;262:1569–1572]. We investigated whether cell-permeable peptides, composed of the C-terminus of Gαi3 fused with importation sequences, affect mast cell function. Methods: Following preincubation with the fused peptides, rat peritoneal mast cells were activated by compound 48/80 and analyzed for histamine and prostaglandin D2 release and protein tyrosine phosphorylations. Results: We demonstrate that out of three importation sequences tested only Gαi3 peptide fused with the Kaposi fibroblast growth factor importation sequence (ALL1) inhibited release of histamine. ALL1 as well as a cell-permeable peptide that corresponds to Gαi2 also blocked compound 48/80-stimulated protein tyrosine phosphorylation, though the latter did not block histamine release. ALL1 effect was G protein-specific, as it was incapable of blocking protein tyrosine phosphorylation stimulated by pervanadate. Conclusion: ALL1, a transducible Gαi3-corresponding peptide, blocks the two signaling pathways in mast cells: histamine release and protein tyrosine phosphorylation. Cell permeable peptides that block these two signaling cascades may constitute a novel approach for preventing the onset of the allergic reaction. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Kir/Gem, together with Rad, Rem and Rem2, is a member of the RGK small GTP-binding protein family. These multifunctional proteins regulate voltage-gated calcium channel (VGCC) activity and cell-shape remodeling. Calmodulin and 14-3-3 binding modulate the functions of RGK proteins. Intriguingly, abolishing the binding of calmodulin or calmodulin and 14-3-3 results in nuclear accumulation of RGK proteins. Under certain conditions, the Cavβ3-subunit of VGCCs can be translocated into the nucleus along with the RGK proteins, resulting in channel inactivation. The mechanism by which nuclear localization of RGK proteins is accomplished and regulated, however, is unknown. Here, we identify specific nuclear localization signals (NLS) in Kir/Gem that are both required and sufficient for nuclear transport. Importin α5 binds to Kir/Gem, and its depletion using RNA interference impairs nuclear translocation of this RGK protein. Calmodulin and predicted phosphorylations on serine residues within or in the vicinity of a C-terminal bipartite NLS regulate nuclear translocation by interfering with the association between importinα 5 and Kir/Gem. These predicted phosphorylations, however, do not affect Kir/Gem-mediated calcium channel downregulation but rather, as shown in the accompanying paper (Mahalakshmi RN, Ng MY, Guo K, Qi Z, Hunziker W, Béguin P. Nuclear localization of endogenous RGK proteins and modulation of cell shape remodeling by regulated nuclear transport. Traffic 2007; doi:10.1111/j.1600-0854.2007.00599.x), interfere with cell-shape remodeling. [ABSTRACT FROM AUTHOR]

The members of the RGK small GTP-binding protein family, Kir/Gem, Rad, Rem and Rem2, are multifunctional proteins that regulate voltage-gated calcium channel activity and cell shape remodeling. Calmodulin (CaM) or CaM 14-3-3 are regulators of RGK functions and their association defines the subcellular localization of RGK proteins. Abolition of CaM association results in the accumulation of RGK proteins in the nucleus, whereas 14-3-3 binding maintains them in the cytoplasm. Kir/Gem possesses nuclear localization signals (NLS) that mediate nuclear accumulation through an importin α5-dependent pathway (see Mahalakshmi RN, Nagashima K, Ng MY, Inagaki N, Hunziker W, Béguin P. Nuclear transport of Kir/Gem requires specific signals and importin α5 and is regulated by Calmodulin and predicted service phosphorylations. Traffic 2007; doi: 10.1111/j.1600-0854.2007.00598.x). Because the extent of nuclear localization depends on the RGK protein and the cell type, the mechanism and regulation of nuclear transport may differ. Here, we extend our analysis to the other RGK members and show that Rem also binds importin α5, whereas Rad associates with importins α3, α5 and β through three conserved NLS. Predicted phosphorylation of a serine residue within the bipartite NLS affects, as observed for Kir/Gem, nuclear accumulation of Rem, but not that of Rad or Rem2. We also identify an additional regulatory phosphorylation for all RGK proteins that prevents binding of 14-3-3 and thereby interferes with their cytosolic relocalization by 14-3-3. Functionally, nuclear localization of RGK proteins contributes to the suppression of RGK-mediated cell shape remodeling. Importantly, we show that endogenous RGK proteins are localized predominantly in the nucleus of individual cells of the brain cortex ‘ in situ’ as well as in primary hippocampal cells, indicating that transport between the nucleus and their site of action in the cytoplasm (i.e., cytoskeleton, endoplasmic reticulum or plasma membrane) is of physiological relevance for the regulation of RGK protein function. [ABSTRACT FROM AUTHOR]

Abstract: In the yeast Saccharomyces cerevisiae the genes involved in galactose metabolism (GAL1,7,10) are transcriptionally activated more than a 1000-fold in the presence of galactose as the sole carbon source in the culture media. In the present work, we monitored the activity of the GAL10 gene promoter in different Ras-cAMP genetic backgrounds. We demonstrate that overexpression of C-terminus of the nucleotide exchange factor Cdc25p stimulates GAL10 transcription in yeast strains carrying the contemporary deletion of both RAS genes. Moreover, the deletion of the chromosomal CDC25 gene provokes impaired growth on galactose based media in yeast strain lacking both RAS genes and adenylate cyclase (whose viability is assured by the presence of the Bcy1-11 allele). Surprisingly, reconstitution of the Ras-pathway inhibits GAL10-promoter activation. Activation of GAL10 gene promoter is indeed possible in the presence of Ras protein but only in strains with chromosomal deletion of adenylate cyclase. These results indicate a dual role of Ras-pathway on galactose metabolism and suggest that Cdc25p has a Ras-independent role in cellular metabolism. [Copyright &y& Elsevier]

Copyright of Biochemistry & Cell Biology is the property of Canadian Science Publishing and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Using a subtracted Xenopus cDNA library based on the differential sensitivity of anterior and posterior genes to retinoic acid, we isolated a novel Xenopus nuclear GTP-binding protein (XGB). XGB is expressed prominently in the optic primordia at the tailbud stage. The N-terminal region of XGB contains a set of GTP-binding protein motifs, and the C-terminal region contains two putative nuclear localization signals and two coiled regions. A GFP-XGB fusion protein was expressed in the nucleus of NIH3T3 cells where it bound to subnuclear structures. Truncated C-terminal constructs of XGB containing both nuclear localization signal(s) and coiled region(s) suppressed eye formation, whereas neither the N-terminal construct nor constructs with a mutated GTP-binding protein motif affected eye formation. Expression of Pax6 and Rx1 genes, which are crucial for eye development, was reduced in embryos overexpressing the C-terminal constructs of XGB. Suppression of Pax6 and Rx1 at earlier developmental stages as well as perturbation of eye formation at later stages was counteracted by co-expression of wild-type XGB. We conclude that XGB plays a role in the formation of optic primordia through activation of at least two eye field transcription factors. [ABSTRACT FROM AUTHOR]

The translocation of tRNA and mRNA through the ribosome is promoted by elongation factor G (EF-G), a GTPase that hydrolyzes GTP during the reaction. Recently, it was reported that, in contrast to previous observations, the affinity of EF-G was much weaker for GTP than for GDP and that ribosome-catalyzed GDP-GTP exchange would be required for translocation [Zavialov AV, Hauryliuk VV, Ehrenberg M (2005) J Biol 4:9]. We have reinvestigated GTP/GDP binding and show that EF-G binds GTP and GDP with affinities in the 20 to 40 μM range (37°C), in accordance with earlier reports. Furthermore, GDP exchange, which is extremely rapid on unbound EF-G, is retarded, rather than accelerated, on the ribosome, which, therefore, is not a nucleotide-exchange factor for EF-G. The EF- GGDPNP complex, which is very labile, is stabilized 30,000-fold by binding to the ribosome. These findings, together with earlier kinetic results, reveal that EF-G enters the pretranslocation ribosome in the GTP-bound form and indicate that, upon ribosome- complex formation, the nucleotide-binding pocket of EF-G is closed, presumably in conjunction with GTPase activation. GTP hydrolysis is required for rapid tRNA-mRNA movement, and Pi release induces further rearrangements of both EF-G and the ribosome that are required for EF-G turnover. [ABSTRACT FROM AUTHOR]

The RAC/ROP family of small GTPases are central regulators of important cellular processes in plants. AtRAC2/ROP7 is an ancient member of the RAC/ROP gene family in Arabidopsis thaliana whose functions are generally unknown. In order to study the spatial expression pattern of the AtRAC2/ROP7 gene, transgenic plants expressing GUS or GFP under the control of the AtRAC2/ROP7 promoter were analysed. Functional analysis of AtRAC2/ROP7 was done using transgenic plants overexpressing wild-type and constitutively activated AtRAC2/ROP7 (Val15Gly), and an AtRAC2/ROP7T-DNA insertion mutant. The AtRAC2/ROP7 promoter directs a highly specific xylem-specific expression in the root, hypocotyl, stem, and leaves. The expression is developmentally limited to the late stages of xylem differentiation, and coincides with the formation of secondary cell walls. Leaf epidermal cells of transgenic plants overexpressing constitutively active AtRAC2/ROP7 exhibited highly impaired lobe formation, suggesting that AtRAC2/ROP7 is able to regulate polar cell expansion. Finally, GFP-AtRAC2/ROP7 fusion proteins were localized to the plasma membrane. The results indicate a role for AtRAC2/ROP7 in the development of secondary cell walls of xylem vessels. [ABSTRACT FROM PUBLISHER]

The full-length cDNA of the wheat ( Triticum aestivum L.) root hair defective 3 gene ( RHD3) has been cloned from the salt-tolerant hybrid wheat variety Shanrong No. 3 (Za3) using the mRNA differential display and 5’rapid amplification of cDNA ends (RACE) methods. Analysis of the amino acid sequence deduced from the wheat RHD3, gene shows that two conservative GTP-binding motifs, namely GXXXXGKS and DXXG, in eukaryotes also exist at the N-terminal of wheat RHD3. In addition, an 18 amino acid residue transmembrane domain, namely FYLAVMFVVFLVGKAIWV, exists at positions 701—718 of the C-terminal of the deduced protein of wheat RHD3 obtained, but this domain is absent in another three proteins aligned, including rice RHD3, Arabidopsis RHD3, and yeast homologue SEY1. Northern blot revealed that transcription of the wheat RHD3, gene is down-regulated in both the salt-tolerant line and in JN177 under saline stress. A possible stress-responsive mechanism for this gene is discussed. (Managing editor: Li-Hui ZHAO) [ABSTRACT FROM AUTHOR]

The Sec6/8 complex, also known as the exocyst complex, is an octameric protein complex that has been implicated in tethering of secretory vesicles to specific regions on the plasma membrane. Two subunits of the Sec6/8 complex, Exo84 and Sec5, have recently been shown to be effector targets for active Ral GTPases. However, the mechanism by which Ral proteins regulate the Sec6/8 activities remains unclear. Here, we present the crystal structure of the Ral-binding domain of Exo84 in complex with active RalA. The structure reveals that the Exo84 Ral-binding domain adopts a pleckstrin homology domain fold, and that RalA interacts with Exo84 via an extended interface that includes both switch regions. Key residues of Exo84 and RalA were found that determine the specificity of the complex interactions; these interactions were confirmed by mutagenesis binding studies. Structural and biochemical data show that Exo84 and Sec5 competitively bind to active RalA. Taken together, these results further strengthen the proposed role of RalA-regulated assembly of the Sec6/8 complex. [ABSTRACT FROM AUTHOR]

Abstract: The Ras-related human GTPase Rab4a is involved in the regulation of endocytosis through the sorting and recycling of early endosomes. Towards further insight, we have determined the three-dimensional crystal structure of human Rab4a in its GppNHp-bound state to 1.6Å resolution and in its GDP-bound state to 1.8Å resolution, respectively. Despite the similarity of the overall structure with other Rab proteins, Rab4a displays significant differences. The structures are discussed with respect to the recently determined structure of human Rab5a and its complex with the Rab5-binding domain of the bivalent effector Rabaptin-5. The Rab4 specific residue His39 modulates the nucleotide binding pocket giving rise to a reduced rate for nucleotide hydrolysis and exchange. In comparison to Rab5, Rab4a has a different GDP-bound conformation within switch 1 region and displays shifts in position and orientation of the hydrophobic triad. The observed differences at the S2–L3–S3 region represent a new example of structural plasticity among Rab proteins and may provide a structural basis to understand the differential binding of similar effector proteins. [Copyright &y& Elsevier]

Transglutaminase 2 (TG2) is a GTP-binding protein with transglutaminase activity. Despite advances in the characterization of TG2 functions and their impact on cellular processes, the role of TG2 in Human chronic myelogenous leukemia K562 cell line is still poorly understood. To understand the biological significance of TG2 during the differentiation of K562 cells, we established and characterized K562 cells that specifically express TG2. Non-transfected K562 cells showed the increase of membrane-bound-TG2 level after 3 days in the response to Hemin and all trans-retinoic acid (tRA), indicating that membrane recruitment of TG2 is occurred during the erythroid differentiation. However, membrane recruitment of TG2 in TG2-transfected cells revealed within earlier time period, compared with that in vector-transfected cells. The ability of membrane-bound-TG2 to be photoaffinity-labeled with [α-32P]GTP was also increased in TG2-transfected cells. TG2-transfected cells activated Akt phosphorylation and inactivated ERK1/2 phosphorylation, compared with vector-transfected cells. Furthermore, phosphorylation of CREB, one of the Akt substrates, was increased in TG2-transfected cells and this phenomenon was confirmed by RT-PCR analysis of several marker genes related with erythroid lineage in the absence of PI3K specific inhibitor, Wortmannin, indicating that PI3K/Akt signaling pathway also involved in the differentiation of the cell. Finally, as results of benzidine positive staining as well as hemoglobinization analysis, overexpression of TG2 revealed acceleration of the erythroid differentiation of K562 cells. Taken together, there was no increased TG2 expression level in the response of Hemin/tRA and delayed differentiation in vector transfected cells than in TG2-transfected cells, suggesting that suppression of TG2 expression may retard the erythroid differentiation of K562 cells. Therefore, our study may give a new insight for another aspect of the development of this disease. [Copyright &y& Elsevier]

Developmentally regulated GTP-binding proteins (DRGs) are a subclass of GTP-binding proteins that have been discovered recently. Here we report two zebrafish DRG cDNA clones closely related to human and mouse DRG genes. The two DRG sequences showed a high degree of similarity (55% identity, 72% similarity) at the amino acids level. Whole mount in situ hybridization revealed expression of zebrafish DRGs maternally, following the onset of zygotic transcription at the mid-blastula transition (MBT) and throughout embryonic. The expression of these two genes in different tissues follows a similar pattern, suggesting that they may serve a similar function. [ABSTRACT FROM AUTHOR]

Reactive oxygen intermediates (ROIs) such as hydrogen peroxide (H2O2) and the superoxide anion radical (O2·-) accumulate in many plants during attack by microbial pathogens. Despite a huge number of studies, the complete picture of the role of ROIs in the host–pathogen interaction is not yet fully understood. This situation is reflected by the controversially discussed question as to whether ROIs are key factors in the establishment and maintenance of either host cell inaccessibility or accessibility for fungal pathogens. On the one hand, ROIs have been implicated in signal transduction as well as in the execution of defence reactions such as cell wall strengthening and a rapid host cell death (hypersensitive reaction). On the other hand, ROIs accumulate in compatible interactions, and there are reports suggesting a function of ROIs in restricting the spread of leaf lesions and thus in suppressing cell death. Moreover, in situ analyses have demonstrated that different ROIs may trigger opposite effects in plants depending on their spatiotemporal distribution and subcellular concentrations. This demonstrates the need to determine the particular role of individual ROIs in distinct stages of pathogen development. The well-studied interaction of cereals with fungi from the genus Blumeria is an excellent model system in which signal transduction and defence reactions can be further elucidated in planta. This review article gives a synopsis of the role of ROI accumulation, with particular emphasis on the pathosystem Hordeum vulgare L.–Blumeria graminis. [ABSTRACT FROM AUTHOR]

The putative functions of NDP (nucleoside diaphosphate) kinases from various organisms focusing to fungi and plants are described. The biochemical reactions catalyzed by NDP kinase are as follows. (i) Phosphotransferring activity from mainly ATP to cognate NDPs generating nucleoside triphosphates (NTPs). (ii) Autophosphorylation activity from ATP and GTP. (iii) Protein kinase (phosphotransferring) activity phosphorylating such as myelin basic protein. NDP kinase could function to provide NTPs as a housekeeping enzyme. However, recent works proved possible functions of the NDP kinases in the processes of signal transduction in various organisms, as described below. By use of the extracts of the mycelia of a filamentous fungus Neurospora crassa blue-light irradiation could increase the phosphorylation of a 15-kDa protein, which was purified and identified to be NDP kinase (NDK-1). By use of the etiolated seedlings of Pisum sativum cv Alaska and Oryza sativa red-light irradiation of intact plants increased the phosphorylation of NDP kinase. However, successive irradiation by red–far-red reversed the reaction, indicating that phytochrome-mediated light signals are transduced to the phosphorylation of NDP kinase. NDP kinase localizing in mitochondria is encoded by nuclear genome and different from those localized in cytoplasm. NDP kinase in mitochondria formed a complex with succinyl CoA synthetase. In Spinicia oleraceae two different NDP kinases were detected in the chloroplast, and in Pisum sativum two forms of NDP kinase originated from single species of mRNA could be detected in the choloroplast. However, the function of NDP kinases in the choloroplast is not yet known. In Neurospora crassa a Pro72His mutation in NDP kinase ( ndk-1 Pro72His) deficient in the autophosphorylation and protein kinase activity resulted in lacking the light-induced polarity of perithecia. In wild-type directional light irradiation parallel to the solid medium resulted in the formation of the perithecial beak at the top of perithecia, which was designated as “light-induced polarity of perithecia.” In wild-type in darkness the beak was formed at random places on perithecia, and in ndk Pro72His mutant the perithecial beak was formed at random places even under directional light illumination. The introduction of genomic DNA and cDNA for ndk-1 demonstrated that the wild-type DNAs suppressed the mutant phenotype. With all these results except for the demonstration in Neurospora, most of the phenomena are elusive and should be solved in the molecular levels concerning with NDP kinases. [ABSTRACT FROM AUTHOR]

Salt stress results in a massive change in gene expression. An 837 bp cDNA designated ScRab was cloned from shoot cultures of the salt tolerant jojoba ( Simmondsia chinesis ). The cloned cDNA encodes a full length 200 amino acid long polypeptide that bears high homology to the Rab subfamily of small GTP binding proteins, particularly, the Rab5 subfamily. ScRab expression is reduced in shoots grown in the presence of salt compared to shoots from non-stressed cultures. His 6 -tagged ScRAB protein was expressed in E. coli , and purified to homogeneity. The purified protein bound radiolabelled GTP. The unlabelled guanine nucleotides GTP, GTPγS and GDP but not ATP, CTP or UTP competed with GTP binding. [ABSTRACT FROM AUTHOR]

In Saccharomyces cerevisiae, Rho1p plays an important role in cell wall integrity by regulating β-1,3-glucan synthase, Pkc1p and the actin cytoskeleton. To determine the physiological role of Rho1p in the dimorphic fungus Candida albicans, the major human fungal pathogen, we constructed mutants that conditionally express Rho1p from the glucose-repressible phosphoenolpyruvate carboxykinase promoter (pPCK1). We examined the growth of these cells in a range of conditions. Depletion of Rho1p from yeast cells resulted in cell death, lysis, and aggregation. The Rho1p conditional mutant was inviable on 10% serum indicating that Rho1p was also required for hyphal viability. Furthermore, in a mouse model of systemic candidiasis, strains dependent on pPCK1-driven RHO1 expression failed to colonise the kidneys and establish disease, suggesting that the level of glucose in serum was sufficient to repress the pPCK1 and that Rho1p-depleted strains were inviable within the host. Therefore, Rho1p is essential for the viability of C. albicans in vitro and in vivo. [ABSTRACT FROM AUTHOR]

1 Efaroxan induces membrane depolarization by interaction with the pore forming subunit of the ATP-sensitive potassium channel, Kir6.2. However, this effect is not responsible for its full secretory activity. 2 In this study we have used an anti-idiotypic approach to generate antibodies that recognize additional proteins that may be regulated by efaroxan in pancreatic β-cells. 3 Using these antisera in an expression cloning strategy we have identified a monomeric GTP-binding protein, Rhes. as a potential target for regulation by imidazoline ligands. Rhes is shown to be expressed in β-cells and its expression is regulated by efaroxan under conditions when a structurally related molecule. KU14R, is ineffective. 4 The results reveal that β-cells express Rhes and suggest that changes in the expression of this molecule may regulate the sensitivity of β-cells to imidazoline secretagogues. [ABSTRACT FROM AUTHOR]

Background: Nasal hyperresponsiveness is a common feature of allergic rhinitis, but the underlying mechanisms have yet to be elucidated. The effects of repeated antigen inhalation on the characteristics of histamine H[sub 1] receptors and expression levels of heterotrimeric guanosine 5′-triphosphate-binding proteins in nasal mucosa were investigated to understand the mechanisms of the pathogenesis of nasal hyperresponsiveness in allergic rhinitis. Methods: Male Hartley guinea pigs were sensitized by the inhalation of dinitrophenylated ovalbumin antigen (10 mg of protein/ml) and repeatedly challenged by inhaling aerosolized dinitrophenylated ovalbumin antigen for 3 weeks. Twenty-four hours after the last antigen inhalation, in vivo nasal responsiveness to histamine was measured. [[sup 3] H]Mepyramine binding assays and immunoblotting for α subunits of the G[sub q] protein were also performed using membrane preparations of isolated nasal mucosae. Results: The histamine-induced increase in intranasal pressure was significantly augmented after repeated antigen challenge, indicating that nasal hyperresponsiveness was achieved. In saturation binding studies, no significant change was observed in the density and antagonist affinity of H[sub 1] receptors in the hyperresponsive animals. On the other hand, the affinity of histamine for high-affinity agonist binding sites in the hyperresponsive group, measured by histamine competition binding studies, was much greater than that in control animals, and these results were affected by guanosine 5′-O-(3-thiotriphosphate) in both groups. Moreover, Gα[sub q] levels in nasal mucosal homogenates were significantly increased after repeated antigen challenge. Conclusions: Elevated G protein levels in nasal mucosa might induce an increased binding affinity of histamine to its receptors, resulting in an augmented nasal response to histamine, that is, nasal hyperresponsiveness, in guinea pigs.Copyright © 2002 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

GTP-binding proteins of the Rab family were cloned from human platelets using RT-PCR. Clones corresponding to two novel Rab proteins, Rab31 and Rab32, and to Rab11A, which had not been detected in platelets previously, were isolated. The coding sequence of Rab31 (GenBank accession no. U59877) corresponded to a 194 amino-acid protein of 21.6 kDa. The Rab32 sequence was extended to 1000 nucleotides including 630 nucleotides of coding sequence (GenBank accession no. U59878) but the 5′ coding sequence was only completed later by others (GenBank accession no. U71127). Human Rab32 cDNA encodes a 225 amino-acid protein of 25.0 kDa with the unusual GTP-binding sequence DIAGQE in place of DTAGQE. Northern blots for Rab31 and Rab32 identified 4.4 kb and 1.35 kb mRNA species, respectively, in some human tissues and in human erythroleukemia (HEL) cells. Rabbit polyclonal anti-peptide antibodies to Rab31, Rab32 and Rab11A detected platelet proteins of 22 kDa, 28 kDa and 26 kDa, respectively. Human platelets were highly enriched in Rab11A (0.85 µg·mg of platelet protein-1 ) and contained substantial amounts of Rab32 (0.11 µg·mg protein-1 ). Little Rab31 was present (0.005 µg·mg protein-1 ). All three Rab proteins were found in both granule and membrane fractions from platelets. In rat platelets, the 28-kDa Rab32 was replaced by a 52-kDa immunoreactive protein. Rab31 and Rab32, expressed as glutathione S -transferase (GST)-fusion proteins, did not bind [α-32 P]GTP on nitrocellulose blots but did bind [35 S]GTP[S] in a Mg2+ -dependent manner. Binding of [35 S]GTP[S] was optimal with 5 µm Mg2+ free and was markedly inhibited by higher Mg2+ concentrations in the case of GST–Rab31 but not GST–Rab32. Both proteins displayed low steady-state GTPase activities, which were not inhibited by mutations... [ABSTRACT FROM AUTHOR]

We studied cAMP-mediated exocytosis in rat pancreatic acinar cells. We monitored changes in the membrane capacitance (ΔC), which reflects the granule fusion/retrieval process, with whole-cell patch-clamp capacitance measurement. The rise in cellular cAMP, caused indirectly by receptor activation by vasoactive intestinal polypeptide or directly by dibutyryl cyclic AMP, was able to induce an increase in ΔC independently of cellular Ca2+. Using the latter stimulation, we estimated the magnitude of the response to internal GTPγS [guanosine 5′-(γ-thio)trisphosphate] and/or GDPβS [guanosine 5′-(β-thio)diphosphate]. The internal GTPγS and GDPβS amplified and depressed the response, respectively. Thus, the cellular cAMP alone can trigger granule insertion independently of cellular Ca2+ and it can be controlled by cellular GTP-binding proteins, presumably those belonging to the Rab family. [ABSTRACT FROM AUTHOR]

The Trichoderma reesei gene, rho3, encoding the functional homologue of the Saccharomyces cerevisiae small GTP-binding protein Rho3p was cloned as a suppressor of the secretion-deficient mutation sec15-1 in yeast. The encoded protein showed 61% amino acid identity to the Rho3 protein. Rescue of the growth defect of a RHO3 disruption strain by an expression vector carrying rho3 cDNA confirmed the functional homology with the S. cerevisiae RHO3 gene. In addition, overproduction of T. reesei RHOIII in this yeast strain appeared to improve the actin organization and chitin localization of the cells. Three putative mutant alleles of the T. reesei rho3 gene (rho3Gly20Val, rho3Thr25Asn, rho3Asp126Ala) were introduced into the wild-type yeast, in yeast with sec15 mutation and in yeast with Rho3p depletion. Cells expressing rho3Gly20Val displayed wild-type growth and those expressing rho3Thr25Asn and rho3Asp126Ala had a loss-of-function phenotype. [ABSTRACT FROM AUTHOR]

The interferon-γ-induced guanylate-binding protein 1 (GBP1) belongs to a special class of large GTP- binding proteins of 60–100 kDa with unique characteristics. Here we present the structure of human GBP1 in complex with the non-hydrolysable GTP analogue GppNHp. Basic features of guanine nucleotide binding, such as the P-loop orientation and the Mg2+ co-ordination, are analogous to those of Ras-related and heterotrimeric GTP-binding proteins. However, the glycosidic bond and thus the orientation of the guanine base and its interaction with the protein are very different. Furthermore, two unique regions around the base and the phosphate-binding areas, the guanine and the phosphate caps, respectively, give the nucleotide-binding site a unique appearance not found in the canonical GTP-binding proteins. The phosphate cap, which constitutes the region analogous to switch I, completely shields the phosphate-binding site from solvent such that a potential GTPase-activating protein cannot approach. This has consequences for the GTPase mechanism of hGBP1 and possibly of other large GTP-binding proteins. [ABSTRACT FROM AUTHOR]

Plant Rho-type GTPases (ROPs) are versatile molecular switches involved in a number of signal transduction pathways. Although it is well known that they are indirectly linked to protein kinases, our knowledge about their direct functional interaction with upstream or downstream protein kinases is scarce. It is reasonable to suppose that similarly to their animal counterparts, ROPs might also be regulated by phosphorylation. There is only, however, very limited experimental evidence to support this view. Here, we present the analysis of two potential phosphorylation sites of AtROP1 and two types of potential ROP-kinases. The S74 site of AtROP1 has been previously shown to potentially regulate AtROP1 activation dependent on its phosphorylation state. However, the kinase phosphorylating this evolutionarily conserved site could not be identified: we show here that despite of the appropriate phosphorylation site consensus sequences around S74 neither the selected AGC nor CPK kinases phosphorylate S74 of AtROP1 in vitro. However, we identified several phosphorylation sites other than S74 for the CPK17 and 34 kinases in AtROP1. One of these sites, S97, was tested for biological relevance. Although the mutation of S97 to alanine (which cannot be phosphorylated) or glutamic acid (which mimics phosphorylation) somewhat altered the protein interaction strength of AtROP1 in yeast cells, the mutant proteins did not modify pollen tube growth in an in vivo test. [ABSTRACT FROM AUTHOR]

Gi3, a member of the Gi family of heterotrimeric GTP-binding proteins, regulates vesicle trafficking along both the constitutive and regulated pathways. In mast cells, specialized secretory cells which secrete a variety of inflammatory mediators by regulated exocytosis, activation of Gi3 provides a sufficient signal for exocytosis [Aridor, M., Rajmilevich, G., Beaven, M. A. & Sagi-Eisenberg, R. (1993) Science 262, 1569-1572]. Such activation can be achieved in patch-clamped or streptolysin-O (SLO)-permeabilized mast cells by a combination of Ca2+ and nonhydrolyzable analogs of GTP. In contrast, Ca2+-activated exocytosis in intact cells is Gi3 independent. We show here that overexpression of a GTPase-deficient mutant (Gαi3Q204L), but not of the wild-type form of Gαi3, in rat basophilic leukemia cells (RBL-2H3), a tumor analog of mucosal mast cells, resulted in marked potentiation of exocytosis and release of arachidonic acid in intact cells activated by a Ca2+ ionophore alone or in combination with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. In contrast, exocytosis and arachidonic acid release stimulated by aggregation of the cell surface receptors for immunoglobulin E (IgE) were unaffected. These results strongly suggest that the intracellular receptor, responsible for the activation of Gi3, is a low-affinity Ca2+-binding protein that can only be activated during Ca2+ ionophore stimulation. Moreover, these results also suggest that the propagation of the Ca2+-activated and Gi3-mediated signaling pathway requires the blocking of Gi3 GTPase activity. Finally, our results indicate that release of arachidonic acid is at least one of the downstream effectors of Gi3. [ABSTRACT FROM AUTHOR]

To investigate the mechanisms of receptor-mediated regulation of heart muscle contraction, we developed a tension-recording system using β-escin-skinned single cardiac cells of rats and studied the effects of agonists on myofibrillar Ca2+ sensitivity and Ca2+ release from the sarcoplasmic reticulum (SR). In pCa/tension relations, 1 µM isoproterenol plus 100 µM guanosine 5′-triphosphate (GTP) decreased the myofibrillar Ca2+ sensitivity (pCa50, the [Ca2+] required for half-maximal tension, as an indicator of the sensitivity; from 6.07 to 5.92); this effect was blocked by 1 µM metoprolol or 1 mM guanosine 5′-O-(2-thiodiphosphate) (GDPβS). Phenylephrine (10 µM) plus 100 µM GTP increased the Ca2+ sensitivity (pCa50; from 6.12 to 6.28), and this effect was blocked by 1 µM phentolamine or 1 mM GDPβS. After Ca2+ loading into the SR, 10 µM phenylephrine plus 100 µM GTP in a low-ethylene- glycol-bis(β-aminoethylether)- N, N, N′, N′-tetraacetic acid (EGTA, 0.1 mM) relaxing solution induced oscillatory contractions that were attenuated by either 1 µM phentolamine or pre-treatment with 10 µM inositol 1,4,5-trisphosphate. Our results demonstrate that β1-adrenergic stimulation decreases myofibrillar Ca2+ sensitivity and that α1-adrenergic stimulation both increases the Ca2+ sensitivity and activates Ca2+ release from the agonist-sensitive SR through GTP-binding protein-related mechanisms. [ABSTRACT FROM AUTHOR]

1. Antidepressants have been used clinically for many years; however, the neurochemical mechanism for their therapeutic effect has not been clarified yet. Recent reports indicate that chronic antidepressant treatment directly affects the postsynaptic membrane to increase the coupling between the stimulatory GTP-binding (G) protein, Gs, and adenylyl cyclase. Tubulin, a cytoskeletal element, is involved in the stimulatory and inhibitory regulation of adenylyl cyclase in rat cerebral cortex via direct transfer of GTP to G proteins. In this study, we investigated whether the functional change of the adenylyl cyclase system caused by chronic antidepressant treatment involves an alteration of tubulin function in the regulation of adenylyl cyclase activity. 2. Male Sprague–Dawley rats were treated once daily with amitriptyline or saline by intraperitoneal injection (10 mg/kg) for 21 days, and their cerebral cortex membranes and GppNHp-liganded tubulin (tubulin-GppNHp) were prepared for what. 3. GppNHp-stimulated adenylyl cyclase activity in cortex membranes from amitriptyline-treated rats was significantly higher than that in control membranes. Furthermore, tubulin–GppNHp prepared from amitriptyline-treated rats was more potent than that from control rats in the stimulation of adenylyl cyclase activity in the cortex membranes of the controls. However, there was no significant difference in manganese-stimulated adenylyl cyclase activity between control and amitriptyline-treated rats. 4. The present results suggest that chronic antidepressant treatment enhances not only the coupling between Gs and the catalytic subunit of adenylyl cyclase but also tubulin interaction with Gs in the cerebral cortex of the rat. [ABSTRACT FROM AUTHOR]

The interaction of various hormones and regucalcin on (Ca−Mg)-ATPase activity in rat liver plasma membranes was investigated. The presence of epinephrine (10-10 M), and insulin (10-10 M) in the reaction mixture produced a significant increase in (Ca−Mg)-ATPase activity, while the enzyme activity was decreased significantly by calcitonin, (3×10-3×10 M). These hormonal effects, except for calcitonin, were clearly inhibited by the presence of vanadate (10 M) which can inhibit the Ca-dependent phosphorylation of enzyme. Meanwhile, regucalcin (0.25 and 0.50 μM), isolated from rat liver cytosol, elevated significantly (Ca−Mg)-ATPase activity in the plasma membranes, although this elevation was not inhibited by vanadate (10 M). the epinephrine (10 M) or phenylephrine (10 M)-induced increase in (Ca−Mg)-ATPase activity was disappeared in the presence of regucalcin; in this case the effect of regucalcin was also weakened. However, the inhibitory effect of calcitonin (3×10 M) was not weakened by the presence of regucalcin (0.5 μM). Moreover, GTP (10 and 10 M)-induced increase in (Ca−Mg)-ATPase activity was not seen in the presence of regucalcin (0.25 μM). The present finding suggests that the activating mechanism of regucalcin on (Ca−Mg)-ATPase is not involved on GTP-binding protein which modulates the receptor-mediated hormonal effect in rat liver plasma membranes. [ABSTRACT FROM AUTHOR]

Three G proteins from human brain membranes were purified to near homogeneity by conventional techniques including preparative electrophoresis. These G proteins were characterized by their ability to bind GTP, GDP and GTP analogs. Two of these proteins have molecular weights of 50,000 (G) and 36,000 (G), as determined on SDS-gels. G was ADP-ribosylated by pertussis toxin. Thus, G could represent a Gα subunit, whereas G could be Gα or Gα. G was phosphorylated by cAMP dependent protein kinase and protein kinase C. G was phosphorylated by a protein kinase independent of calcium and phospholipid, a proteolytic product of protein kinase C, analogous to protein kinase M. Phosphorylation of G by this protein kinase induced a dramatic decrease in its GTPase activity. The third G protein, of molecular weight 22,000 probably belongs to the group of monomeric G proteins possessing functional similarities with ras gene products. The regulation of G proteins involving calcium-dependent and independent pathways is delineated. [ABSTRACT FROM AUTHOR]

In order to examine the status of cell signal transduction in depression, we measured in the same subjects both the platelet guanine nucleotide binding protein (G protein) levels and G protein function in drug-free depressed patients. G protein levels were measured thanks to the method of Western immunoblot of three G protein subunits, i.e. Gαi2; Gαq and Gβ. The G protein function was assessed by the method of GTPγS binding through platelet α2-adrenoceptor stimulation. The results showed an increase in immunoreactivity of the three G proteins in major depressed patients. The study of GTPγS binding on platelet membrane showed a significant decrease in drug-free depressed patients with respect to age- and gender-matched controls. These data suggest that uncoupling of α2-adrenoceptor from G proteins, whose levels were increased, may be found in depression. [ABSTRACT FROM AUTHOR]