RESUMO
Voltage-gated calcium channels (CaV) conduct Ca2+ influx promoting neurotransmitters and hormone release. CaV are finely regulated by voltage-dependent and independent pathways either by G-protein-coupled receptors (GPCRs) or intramembrane lipids, respectively, in neurons and glands. Interestingly, pancreatic ß-cells are abundantly innervated by both sympathetic and parasympathetic neurons, while a variety of high-voltage activated (HVA) Ca2+ channels are present in these cells. Thus, autonomic system seems to exert a tonic inhibition on HVA Ca2+ channels throughout GPCRs, constitutively preventing hormone secretion. Therefore, this work aimed to investigate noradrenergic and cholinergic inhibition of HVA Ca2+ channels in pancreatic ß-cells. Experiments were conducted in pancreatic ß-cells of rat by using patch-clamping methods, immunocytochemistry, pharmacological probes, and biochemical reagents. A voltage-clamp protocol with a strong depolarizing prepulse was used to unmask tonic inhibition. Herein, we consistently find a basal tonic inhibition of HVA Ca2+ channels according to a GPCRs regulation. Facilitation ratio is enhanced by noradrenaline (NA) according to a voltage-dependent regulation and a membrane-delimited mechanism, while no facilitation changes are observed with carbachol or phosphatidylinositol 4,5-bisphosphate (PIP2). Furthermore, carbachol or intramembrane lipids, such as PIP2, do not change facilitation ratio according to a voltage-independent regulation. Together, HVA Ca2+ channels of pancreatic ß-cells are constitutively inhibited by GPCRs, suggesting a natural brake preventing cells from exhaustive insulin secretion.NEW & NOTEWORTHY Our results support the hypothesis that GPCRs tonically inhibit HVA Ca2+ channels in pancreatic ß-cells. A voltage-clamp protocol with a strong depolarizing prepulse was used to unmask voltage-dependent inhibition of Ca2+ channels. The novelty of these results strengthens the critical role of Gßγ's in Ca2+ channel regulation, highlighting kinetic slowing and increased facilitation ratio. Together, HVA Ca2+ channels of pancreatic ß-cells are constitutively inhibited by GPCRs underlying fine-tuning modulation of insulin secretion.
Assuntos
Canais de Cálcio , Proteínas de Ligação ao GTP , Ratos , Animais , Canais de Cálcio/metabolismo , Carbacol , Proteínas de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Hormônios , Lipídeos , Cálcio/metabolismoRESUMO
Receptors are proteins coded by DNA, some of which have already been crystalized, thus allowing the details of their structure at the atomic level and some aspects of their function to be known. This review focuses on the most diverse and abundant family of receptors, G protein-coupled receptors. This family of receptors recognizes and mediates the action of several endogenous ligands (hormones, neurotransmitters, growth factors and local hormones) and also intervenes in the pathogenesis of various diseases, which is why they are targeted by approximately 30 to 40% of medications that are used in daily clinical practice and of various illegal drugs as well. X-ray crystallography is one of the essential tools that has allowed to observe the structure of these receptors in the amino acids that participate in this interaction, which allows to know the binding site of the endogenous ligand and of synthetic molecules that act on them to modulate their action. Molecular modeling or "docking" is also a computational bioinformatics tool that supports research on receptor-ligand binding, which allows the design and development of increasingly specific drugs. These developments have brought along significant changes in fundamental pharmacodynamic concepts.
Los receptores son proteínas codificadas por el ADN, algunos de los cuales ya han sido cristalizados, lo que permite conocer los detalles de su estructura a nivel atómico y algunos aspectos de su función. Esta revisión se enfoca en los más diversos y abundantes, los receptores acoplados a la proteína G. Esta familia de receptores reconoce y media la acción de varios ligandos endógenos (hormonas, neurotransmisores, factores de crecimiento y hormonas locales) y también interviene en la patogenia de diversas enfermedades, por lo que son el blanco terapéutico de aproximadamente 30 a 40 % de los medicamentos que se emplean en la práctica clínica cotidiana y de diversas drogas ilegales. La cristalografía de rayos X es una de las herramientas clave que ha permitido observar la estructura de estos receptores en los aminoácidos que participan en esta interacción, lo que posibilita conocer el sitio de unión del ligando endógeno y de moléculas sintéticas que actúan sobre ellos para modular su acción. El modelado molecular es también una herramienta bioinformática computacional que apoya la investigación sobre la unión receptor-ligando, que hace posible el diseño y desarrollo de fármacos cada vez más específicos. A estos desarrollos se suman importantes cambios en los conceptos farmacodinámicos fundamentales.
Assuntos
Aminoácidos , Receptores Acoplados a Proteínas G , Hormônios , Humanos , Ligantes , Modelos MolecularesRESUMO
Resumen Los receptores son proteínas codificadas por el ADN, algunos de los cuales ya han sido cristalizados, lo que permite conocer los detalles de su estructura a nivel atómico y algunos aspectos de su función. Esta revisión se enfoca en los más diversos y abundantes, los receptores acoplados a la proteína G. Esta familia de receptores reconoce y media la acción de varios ligandos endógenos (hormonas, neurotransmisores, factores de crecimiento y hormonas locales) y también interviene en la patogenia de diversas enfermedades, por lo que son el blanco terapéutico de aproximadamente 30 a 40 % de los medicamentos que se emplean en la práctica clínica cotidiana y de diversas drogas ilegales. La cristalografía de rayos X es una de las herramientas clave que ha permitido observar la estructura de estos receptores en los aminoácidos que participan en esta interacción, lo que posibilita conocer el sitio de unión del ligando endógeno y de moléculas sintéticas que actúan sobre ellos para modular su acción. El modelado molecular es también una herramienta bioinformática computacional que apoya la investigación sobre la unión receptor-ligando, que hace posible el diseño y desarrollo de fármacos cada vez más específicos. A estos desarrollos se suman importantes cambios en los conceptos farmacodinámicos fundamentales.
Abstract Receptors are proteins coded by DNA, some of which have already been crystalized, thus allowing the details of their structure at the atomic level and some aspects of their function to be known. This review focuses on the most diverse and abundant family of receptors, G protein-coupled receptors. This family of receptors recognizes and mediates the action of several endogenous ligands (hormones, neurotransmitters, growth factors and local hormones) and also intervenes in the pathogenesis of various diseases, which is why they are targeted by approximately 30 to 40% of medications that are used in daily clinical practice and of various illegal drugs as well. X-ray crystallography is one of the essential tools that has allowed to observe the structure of these receptors in the amino acids that participate in this interaction, which allows to know the binding site of the endogenous ligand and of synthetic molecules that act on them to modulate their action. Molecular modeling or "docking" is also a computational bioinformatics tool that supports research on receptor-ligand binding, which allows the design and development of increasingly specific drugs. These developments have brought along significant changes in fundamental pharmacodynamic concepts.
RESUMO
Fusarium oxysporum f. sp. lycopersici is an important plant pathogen that has been used to understand the virulence mechanisms that soil inhabiting fungi exhibit during the infection process. In F. oxysporum many of the virulence factors are secreted, and the secretion process requires the formation of vesicles. Arf family members, represented by Arf (ADP- Ribosylation Factor), Arl (Arf-like), and Sar (Secretion-associated and Ras-related) proteins, are involved in the vesicle creation process. In this study we identified the Arf family members in F. oxysporum f. sp. lycopersici, which includes seven putative proteins: Arf1, Arf3, Arl1 through Arl3, Arl8B, and Sar1. Quantification of the mRNA levels of each arf encoding gene revealed that the highest expression corresponds to arf1 in all tested conditions. The phylogenetic analysis revealed that no other Arf1 paralogue, such as Arf2 from yeast, is present in F. oxysporum f. sp. lycopersici. The essential function suggested of Arf1 in F. oxysporum f. sp. lycopersici was corroborated experimentally when, after several attempts, it was impossible to obtain a knockout mutant in arf1. Moreover, arl3 mRNA levels increased significantly when plant tissue was added as a sole carbon source, suggesting that the product of these genes could play pivotal roles during plant infection, the corresponding mutant ∆arl3 was less virulent compared to the wild-type strain. These results describe the role of arl3 as a critical regulator of the virulence in F. oxysporum f. sp. lycopersici and stablish a framework for the arf family members to be studied in deeper details in this phytopathogen.
Assuntos
Fusarium , Solanum lycopersicum , Fusarium/genética , Filogenia , Doenças das Plantas , Virulência/genéticaRESUMO
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
Reelin is a large extracellular matrix protein with relevant roles in mammalian central nervous system including neurogenesis, neuronal polarization and migration during development; and synaptic plasticity with its implications in learning and memory, in the adult. Dysfunctions in reelin signaling are associated with brain lamination defects such as lissencephaly, but also with neuropsychiatric diseases like autism, schizophrenia and depression as well with neurodegeneration. Reelin signaling involves a core pathway that activates upon reelin binding to its receptors, particularly ApoER2 (apolipoprotein E receptor 2)/LRP8 (low-density lipoprotein receptor-related protein 8) and very low-density lipoprotein receptor, followed by Src/Fyn-mediated phosphorylation of the adaptor protein Dab1 (Disabled-1). Phosphorylated Dab1 (pDab1) is a hub in the signaling cascade, from which several other downstream pathways diverge reflecting the different roles of reelin. Many of these pathways affect the dynamics of the actin and microtubular cytoskeleton, as well as membrane trafficking through the regulation of the activity of small GTPases, including the Rho and Rap families and molecules involved in cell polarity. The complexity of reelin functions is reflected by the fact that, even now, the precise mode of action of this signaling cascade in vivo at the cellular and molecular levels remains unclear. This review addresses and discusses in detail the participation of reelin in the processes underlying neurogenesis, neuronal migration in the cerebral cortex and the hippocampus; and the polarization, differentiation and maturation processes that neurons experiment in order to be functional in the adult brain. In vivo and in vitro evidence is presented in order to facilitate a better understanding of this fascinating system.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Diferenciação Celular , Membrana Celular/metabolismo , Movimento Celular , Citoesqueleto/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/fisiologia , Serina Endopeptidases/metabolismo , Animais , Transporte Biológico , Humanos , Proteína ReelinaRESUMO
It is well accepted that ethanol is able to produce major health and economic problems associated to its abuse. Because of its intoxicating and addictive properties, it is necessary to analyze its effect in the central nervous system. However, we are only now learning about the mechanisms controlling the modification of important membrane proteins such as ligand-activated ion channels by ethanol. Furthermore, only recently are these effects being correlated to behavioral changes. Current studies show that the glycine receptor (GlyR) is a susceptible target for low concentrations of ethanol (5-40mM). GlyRs are relevant for the effects of ethanol because they are found in the spinal cord and brain stem where they primarily express the α1 subunit. More recently, the presence of GlyRs was described in higher regions, such as the hippocampus and nucleus accumbens, with a prevalence of α2/α3 subunits. Here, we review data on the following aspects of ethanol effects on GlyRs: (1) direct interaction of ethanol with amino acids in the extracellular or transmembrane domains, and indirect mechanisms through the activation of signal transduction pathways; (2) analysis of α2 and α3 subunits having different sensitivities to ethanol which allows the identification of structural requirements for ethanol modulation present in the intracellular domain and C-terminal region; (3) Genetically modified knock-in mice for α1 GlyRs that have an impaired interaction with G protein and demonstrate reduced ethanol sensitivity without changes in glycinergic transmission; and (4) GlyRs as potential therapeutic targets.
Assuntos
Comportamento/efeitos dos fármacos , Etanol/farmacologia , Receptores de Glicina/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Anestésicos Gerais/farmacologia , Animais , Comportamento/fisiologia , Etanol/toxicidade , Humanos , Camundongos , Camundongos Mutantes , Modelos Neurológicos , Receptores de Glicina/química , Receptores de Glicina/genética , Receptores de Glicina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
Throughout evolution, organisms have created numerous mechanisms to sense and respond to their environment. One such highly conserved mechanism involves regulation by heterotrimeric G-protein complex comprised of alpha (Gα), beta (Gß) and gamma (Gγ) subunits. In plants, these proteins play important roles in signal transduction pathways related to growth and development including response to biotic and abiotic stresses and consequently affect yield. In this work, we have identified and characterized the complete heterotrimeric G-protein repertoire in the Capsicum annuum (Capsicum) genome which consists of one Gα, one Gß and three Gγ genes. We have also identified one RGS gene in the Capsicum genome that acts as a regulator of the G-protein signaling. Biochemical activities of the proteins were confirmed by assessing the GTP-binding and GTPase activity of the recombinant Gα protein and its regulation by the GTPase acceleration activity of the RGS protein. Interaction between different subunits was established using yeast- and plant-based analyses. Gene and protein expression profiles of specific G-protein components revealed interesting spatial and temporal regulation patterns, especially during root development and during fruit development and maturation. This research thus details the characterization of the first heterotrimeric G-protein family from a domesticated, commercially important vegetable crop.
Assuntos
Transdução de Sinais , Solanaceae/genética , Capsicum/genética , Capsicum/crescimento & desenvolvimento , Capsicum/metabolismo , Frutas/genética , Frutas/crescimento & desenvolvimento , Frutas/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Especificidade de Órgãos , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Mapeamento de Interação de Proteínas , Proteínas RGS/genética , Proteínas RGS/metabolismoRESUMO
En este estudio se investigaron los sitios probables de la acción inhibitoria de prolactina (Prl) sobre la esteroidogénesis ovárica inducida por la hormona folículo estimulante (FSH). Para esta finalidad se estudió la capacidad de cultivos primarios de células de la granulosa de la rata de sintetizar estradiol y AMPc bajo la estimulación con FSH o de activadores de la vía dependiente de AMPc en presencia de Prl humana. La participación de otros sistemas de transducción de señal como los dependientes de PKC y proteínas Gi en los mecanismos de acción inhibitoria de la Prl fue también investigada utilizando inhibidores de estos sistemas como la calfostina C y la toxina pertusis. Los resultados demostraron la habilidad de la Prl de alterar la esteroidogénesis previa y posterior a la generación de AMPc, muy probablemente por mecanismos que involucran la activación de la subunidad catalítica de la adenilato ciclasa, así como a través de interactuar con sistemas de transducción de señal dependientes de PKC y proteínas sensibles a la toxina pertusis. Nuestros resultados sugieren un mecanismo de interacción entre receptores acoplados a proteínas G con aquéllos acoplados a cinasas de tirosinas mediado muy probablemente por vías de señalización dependientes de proteínas Gi.
We studied the sites of prolactin inhibition upon FSH induced ovarian steroidogenesis and the ability of prolactin (Prl) to inhibit the synthesis of estradiol and cAMP accumulation under the stimulation of FSH or cAMP dependent activators. The participation of other signal pathways such as PKC and Gi proteins on the inhibitory actions of Prl was also investigated using calfostine C andpertusis toxin as inhibitors. Results showed a dose dependent prolactin decrease in FSH-induced estradiol and cAMP production prior and after the generation of the cyclic nucleotide by a mechanism involving the catalytic subunit of adenyl cyclase and/or through activation of PKC or by the interaction with pertusin toxin sensitive G proteins. Our results suggest a mechanism by which G protein coupled receptors are linked with those coupled with tyrosine kinase through the involvement of a Gi protein mediated mechanism.