RESUMO
Mucor circinelloides, a dimorphic opportunistic pathogen, expresses three heterotrimeric G-protein beta subunits (Gpb1, Gpb2 and Gpb3). The Gpb1-encoding gene is up-regulated during mycelial growth compared with that in the spore or yeast stage. gpb1 deletion mutation analysis revealed its relevance for an adequate development during the dimorphic transition and for hyphal growth under low oxygen concentrations. Infection assays in mice indicated a phenotype with considerably reduced virulence and tissue invasiveness in the deletion mutants (Δgpb1) and decreased host inflammatory response. This finding could be attributed to the reduced filamentous growth in animal tissues compared with that of the wild-type strain. Mutation in a regulatory subunit of cAMP-dependent protein kinase A (PKA) subunit (PkaR1) resulted in similar phenotypes to Δgpb1. The defects exhibited by the Δgpb1 strain were genetically suppressed by pkaR1 overexpression, indicating that the PKA pathway is controlled by Gpb1 in M. circinelloides. Moreover, during growth under low oxygen levels, cAMP levels were much higher in the Δgpb1 than in the wild-type strain, but similar to those in the ΔpkaR1 strain. These findings reveal that M. circinelloides possesses a signal transduction pathway through which the Gpb1 heterotrimeric G subunit and PkaR1 control mycelial growth in response to low oxygen levels.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Fúngicas/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Mucor/crescimento & desenvolvimento , AMP Cíclico/metabolismo , Proteínas Fúngicas/genética , Subunidades beta da Proteína de Ligação ao GTP/genética , Genes Fúngicos , Hifas/crescimento & desenvolvimento , Mucor/metabolismo , Mucor/patogenicidade , Mutação , Micélio/crescimento & desenvolvimento , Oxigênio/análise , Transdução de Sinais , Virulência/genéticaRESUMO
Wnt ligands play crucial roles in the development and regulation of synapse structure and function. Specifically, Wnt-5a acts as a secreted growth factor that regulates dendritic spine formation in rodent hippocampal neurons, resulting in postsynaptic development that promotes the clustering of the PSD-95 (postsynaptic density protein 95). Here, we focused on the early events occurring after the interaction between Wnt-5a and its Frizzled receptor at the neuronal cell surface. Additionally, we studied the role of heterotrimeric G proteins in Wnt-5a-dependent synaptic development. We report that FZD9 (Frizzled9), a Wnt receptor related to Williams syndrome, is localized in the postsynaptic region, where it interacts with Wnt-5a. Functionally, FZD9 is required for the Wnt-5a-mediated increase in dendritic spine density. FZD9 forms a precoupled complex with Gαo under basal conditions that dissociates after Wnt-5a stimulation. Accordingly, we found that G protein inhibition abrogates the Wnt-5a-dependent pathway in hippocampal neurons. In particular, the activation of Gαo appears to be a key factor controlling the Wnt-5a-induced dendritic spine density. In addition, we found that Gßγ is required for the Wnt-5a-mediated increase in cytosolic calcium levels and spinogenesis. Our findings reveal that FZD9 and heterotrimeric G proteins regulate Wnt-5a signaling and dendritic spines in cultured hippocampal neurons.
Assuntos
Espinhas Dendríticas/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Hipocampo/metabolismo , Receptores de Neurotransmissores/metabolismo , Transdução de Sinais/fisiologia , Proteína Wnt-5a/metabolismo , Animais , Linhagem Celular Transformada , Espinhas Dendríticas/genética , Receptores Frizzled , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Hipocampo/citologia , Camundongos , Ratos , Ratos Sprague-Dawley , Receptores de Neurotransmissores/genética , Proteína Wnt-5a/genéticaRESUMO
Diverse G protein-coupled receptors depend on Gßγ heterodimers to promote cell polarization and survival via direct activation of PI3Kγ and potentially other effectors. These events involve full activation of AKT via its phosphorylation at Ser473, suggesting that mTORC2, the kinase that phosphorylates AKT at Ser473, is activated downstream of Gßγ. Thus, we tested the hypothesis that Gßγ directly contributes to mTOR signaling. Here, we demonstrate that endogenous mTOR interacts with Gßγ. Cell stimulation with serum modulates Gßγ interaction with mTOR. The carboxyl terminal region of mTOR, expressed as a GST-fusion protein, including the serine/threonine kinase domain, binds Gßγ heterodimers containing different Gß subunits, except Gß4. Both, mTORC1 and mTORC2 complexes interact with Gß1γ2 which promotes phosphorylation of their respective substrates, p70S6K and AKT. In addition, chronic treatment with rapamycin, a condition known to interfere with assembly of mTORC2, reduces the interaction between Gßγ and mTOR and the phosphorylation of AKT; whereas overexpression of Gαi interfered with the effect of Gßγ as promoter of p70S6K and AKT phosphorylation. Altogether, our results suggest that Gßγ positively regulates mTOR signaling via direct interactions and provide further support to emerging strategies based on the therapeutical potential of inhibiting different Gßγ signaling interfaces.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Western Blotting , Ativação Enzimática/efeitos dos fármacos , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Células HEK293 , Humanos , Imunoprecipitação , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
TASK-2 (K2P5.1) is a background K(+) channel opened by extra- or intracellular alkalinisation that plays a role in renal bicarbonate handling, central chemoreception and cell volume regulation. Here, we present results that suggest that TASK-2 is also modulated by Gßγ subunits of heterotrimeric G protein. TASK-2 was strongly inhibited when GTP-γ-S was used as a replacement for intracellular GTP. No inhibition was present using GDP-ß-S instead. Purified Gßγ introduced intracellularly also inhibited TASK-2 independently of whether GTP or GDP-ß-S was present. The effects of GTP-γ-S and Gßγ subunits were abolished by neutralisation of TASK-2 C terminus double lysine residues K257-K258 or K296-K297. Use of membrane yeast two hybrid (MYTH) experiments and immunoprecipitation assays using tagged proteins gave evidence for a physical interaction between Gß1 and Gß2 subunits and TASK-2, in agreement with expression of these subunits in proximal tubule cells. Co-immunoprecipitation was impeded by mutating C terminus K257-K258 (but not K296-K297) to alanines. Gating by extra- or intracellular pH was unaltered in GTP-γ-S-insensitive TASK-2-K257A-K258A mutant. Shrinking TASK-2-expressing cells in hypertonic solution decreased the current to 36 % of its initial value. The same manoeuvre had a significantly diminished effect on TASK-2-K257A-K258A- or TASK-2-K296-K297-expressing cells, or in cells containing intracellular GDP-ß-S. Our data are compatible with the concept that TASK-2 channels are modulated by Gßγ subunits of heterotrimeric G protein. We propose that this modulation is a novel way in which TASK-2 can be tuned to its physiological functions.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Sequência de Aminoácidos , Animais , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacologia , Células HEK293 , Proteínas Heterotriméricas de Ligação ao GTP/genética , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos , Tionucleotídeos/farmacologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
We isolated two transcription factor genes, heterotrimeric G protein beta 2 subunit (Gß2) and ArcA1, from pepper (Capsicum annuum). The complete coding sequences were amplified using reversed transcriptase PCR based on conserved sequence information of Solanum lycopersicum and several other plant species. Nucleotide sequence analysis of these two genes revealed that the pepper Gß2 gene encodes a protein of 376 amino acids that belongs to the WD40 superfamily. Tissue expression analysis indicated that this gene is highly expressed in the pericarp, moderately expressed in stem, flower, placenta, and leaves, and weakly expressed in seed. There was no expression in the roots. The ArcA1 gene encodes a protein of 331 amino acids that also belongs to the WD40 superfamily. Tissue expression analysis indicated that the pepper ArcA1 gene is moderately expressed in the pericarp and weakly expressed in seed. There was no expression in root, stem, flower, placenta, or leaves.
Assuntos
Capsicum/genética , Subunidades beta da Proteína de Ligação ao GTP/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Sequência de Bases , Capsicum/metabolismo , Clonagem Molecular , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Expressão Gênica , Dados de Sequência Molecular , Fases de Leitura Aberta , Especificidade de Órgãos/genética , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Conformação Proteica , Alinhamento de SequênciaRESUMO
Reflex syncope is defined by a self-terminating transient loss of consciousness associated with an exaggerated response of the vagal reflexes upon orthostatic challenges. A hereditary component has previously been suggested. We hypothesized that variations in genes encoding proteins mediating the vagal signaling in the heart may be involved in reflex syncope pathogenesis. We systematically resequenced the entire coding regions and flanking intron sequences in 5 genes in the cardiac post-synaptic parasympathetic signaling pathway [muscarinic acetylcholine receptor M2 (CHRM2); G-protein beta-1 subunit (GNB1); G-protein gamma-2 subunit (GNG2); potassium inwardly rectifying channel, subfamily J, member 3 (KCNJ3); and potassium inwardly rectifying channel, subfamily J, member 5 (KCNJ5)] in 74 patients with well-characterized reflex syncope of either cardioinhibitory [Vasovagal Syncope International Study (VASIS-IIB), N = 38] or vasodepressor (VASIS-III, N = 36) type. We identified 2 novel genetic variants (CHRM2 c.1114C>G and GNG2 c.87+34G>A) and several known variants (GNB1: c.267+14G>A, c.267+19C>T, and c.738C>T; KCNJ3: c.119A>G, c.591C>T, c.1038T>C, and c.1494T>C; KCNJ5: c. 171T>C, c.810T>G, c.834T>C, c.844C>G, c.938+7C>T, and c.938-10G>A). The minor allele frequency of the KCNJ5 c.938+7C>T variant was significantly lower in patients than in the control group (0.014 versus 0.089, P = 0.001), and the frequency of heterozygosity and homozygosity was lower in cardioinhibitory patients compared to controls. Genetic variations in genes responsible for the vagal signaling in the heart, including CHRM2, GNB1, GNG2, KCNJ3, and KCNJ5, are not major contributors to the pathogenesis of reflex syncope of vasodepressor or cardioinhibitory types.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Subunidades beta da Proteína de Ligação ao GTP/genética , Polimorfismo de Nucleotídeo Único , Receptor Muscarínico M2/genética , Síncope Vasovagal/genética , Adulto , Estudos de Casos e Controles , Feminino , Frequência do Gene , Estudos de Associação Genética , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Gßγ interaction with GlyR is an important determinant in ethanol potentiation of this channel. RESULTS: A small peptide, RQH(C7), can inhibit ethanol potentiation of GlyR currents. CONCLUSION: Results with RQH(C7) indicate that ethanol mediated potentiation of GlyR is in part by Gßγ activation. SIGNIFICANCE: Molecular interaction between Gßγ and GlyR could be used as a target for pharmacological modification of ethanol effects. Previous studies indicate that ethanol can modulate glycine receptors (GlyR), in part, through Gßγ interaction with basic residues in the intracellular loop. In this study, we show that a seven-amino acid peptide (RQH(C7)), which has the primary structure of a motif in the large intracellular loop of GlyR (GlyR-IL), was able to inhibit the ethanol-elicited potentiation of this channel from 47 ± 2 to 16 ± 4%, without interfering with the effect of Gßγ on GIRK (G protein activated inwardly rectifying potassium channel) activation. RQH(C7) displayed a concentration-dependent effect on ethanol action in evoked and synaptic currents. A fragment of GlyR-IL without the basic amino acids did not interact with Gßγ or inhibit ethanol potentiation of GlyR. In silico analysis using docking and molecular dynamics allowed to identify a region of ~350Å(2) involving aspartic acids 186, 228, and 246 in Gßγ where we propose that RQH(C7) binds and exerts its blocking action on the effect of ethanol in GlyR.
Assuntos
Etanol/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Peptídeos/metabolismo , Receptores de Glicina/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Células HEK293 , Humanos , Cinética , Peptídeos/química , Ligação Proteica , Receptores de Glicina/química , Receptores de Glicina/genéticaRESUMO
The Kluyveromyces lactis heterotrimeric G protein is a canonical Galphabetagamma complex; however, in contrast to Saccharomyces cerevisiae, where the Ggamma subunit is essential for mating, disruption of the KlGgamma gene yielded cells with almost intact mating capacity. Expression of a nonfarnesylated Ggamma, which behaves as a dominant-negative in S. cerevisiae, did not affect mating in wild-type and DeltaGgamma cells of K. lactis. In contrast to the moderate sterility shown by the single DeltaKlGalpha, the double DeltaKlGalpha DeltaKlGgamma mutant displayed full sterility. A partial sterile phenotype of the DeltaKlGgamma mutant was obtained in conditions where the KlGbeta subunit interacted defectively with the Galpha subunit. The addition of a CCAAX motif to the C-end of KlGbeta, partially suppressed the lack of both KlGalpha and KlGgamma subunits. In cells lacking KlGgamma, the KlGbeta subunit cofractionated with KlGalpha in the plasma membrane, but in the DeltaKlGalpha DeltaKlGgamma strain was located in the cytosol. When the KlGbeta-KlGalpha interaction was affected in the DeltaKlGgamma mutant, most KlGbeta fractionated to the cytosol. In contrast to the generic model of G-protein function, the Gbeta subunit of K. lactis has the capacity to attach to the membrane and to activate mating effectors in absence of the Ggamma subunit.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Kluyveromyces/fisiologia , Feromônios/metabolismo , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Técnicas do Sistema de Duplo-HíbridoRESUMO
Xenopus laevis oocyte maturation is induced by the steroid hormone progesterone through a nongenomic mechanism that implicates the inhibition of the effector system adenylyl cyclase (AC). Recently, it has been shown that the G protein betagamma heterodimer is involved in oocyte maturation arrest. Since AC is the proposed target for Gbetagamma action, we considered of importance to identify and characterize the Gbetagamma regulated AC isoform(s) that are expressed in the Xenopus oocyte. Through biochemical studies, we found that stage VI plasma membrane oocyte AC activity showed attributes of an AC2 isoform. Furthermore, exogenous Gbetagamma was capable to activate oocyte AC only in the presence of the activated form of Galphas (Galphas-GTPgammaS), which is in agreement with the Ggammabeta conditional activation reported for the mammalian AC2 and AC4 isotypes. In order to study the functional role of AC in oocyte maturation we cloned from a Xenopus oocyte cDNA library a gene encoding an AC with high identity to AC7 (xAC7). Based on this sequence, we constructed a minigene encoding the AC-Gbetagamma interacting region (xAC7pep) to block, within the oocyte, this interaction. We found that microinjection of the xAC7pep potentiated progesterone-induced maturation, as did the AC2 minigene. From these results we can conclude that a Gbetagamma-activated AC is playing an important role in Xenopus oocyte meiotic arrest in a Galphas-GTP dependent manner.