RESUMO
De novo mutations in GNAO1, the gene encoding the major neuronal G protein Gαo, cause a spectrum of pediatric encephalopathies with seizures, motor dysfunction, and developmental delay. Of the >80 distinct missense pathogenic variants, many appear to uniformly destabilize the guanine nucleotide handling of the mutant protein, speeding up GTP uptake and deactivating GTP hydrolysis. Zinc supplementation emerges as a promising treatment option for this disease, as Zn2+ ions reactivate the GTP hydrolysis on the mutant Gαo and restore cellular interactions for some of the mutants studied earlier. The molecular etiology of GNAO1 encephalopathies needs further elucidation as a prerequisite for the development of efficient therapeutic approaches. In this work, we combine clinical and medical genetics analysis of a novel GNAO1 mutation with an in-depth molecular dissection of the resultant protein variant. We identify two unrelated patients from Norway and France with a previously unknown mutation in GNAO1, c.509C>G that results in the production of the Pro170Arg mutant Gαo, leading to severe developmental and epileptic encephalopathy. Molecular investigations of Pro170Arg identify this mutant as a unique representative of the pathogenic variants. Its 100-fold-accelerated GTP uptake is not accompanied by a loss in GTP hydrolysis; Zn2+ ions induce a previously unseen effect on the mutant, forcing it to lose the bound GTP. Our work combining clinical and molecular analyses discovers a novel, biochemically distinct pathogenic missense variant of GNAO1 laying the ground for personalized treatment development.
Assuntos
Encefalopatias , Humanos , Criança , Mutação/genética , Proteínas de Ligação ao GTP/metabolismo , Íons/metabolismo , Guanosina Trifosfato , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismoRESUMO
BACKGROUND: The GNAO1 gene, encoding the major neuronal G protein Gαo, is mutated in a subset of pediatric encephalopathies. Most such mutations consist of missense variants. METHODS: In this study, we present a precision medicine workflow combining next-generation sequencing (NGS) diagnostics, molecular etiology analysis, and personalized drug discovery. FINDINGS: We describe a patient carrying a de novo intronic mutation (NM_020988.3:c.724-8G>A), leading to epilepsy-negative encephalopathy with motor dysfunction from the second decade. Our data show that this mutation creates a novel splice acceptor site that in turn causes an in-frame insertion of two amino acid residues, Pro-Gln, within the regulatory switch III region of Gαo. This insertion misconfigures the switch III loop and creates novel interactions with the catalytic switch II region, resulting in increased GTP uptake, defective GTP hydrolysis, and aberrant interactions with effector proteins. In contrast, intracellular localization, Gßγ interactions, and G protein-coupled receptor (GPCR) coupling of the Gαo[insPQ] mutant protein remain unchanged. CONCLUSIONS: This in-depth analysis characterizes the heterozygous c.724-8G>A mutation as partially dominant negative, providing clues to the molecular etiology of this specific pathology. Further, this analysis allows us to establish and validate a high-throughput screening platform aiming at identifying molecules that could correct the aberrant biochemical functions of the mutant Gαo. FUNDING: This work was supported by the Joint Seed Money Funding scheme between the University of Geneva and the Hebrew University of Jerusalem.
Assuntos
Proteínas de Ligação ao GTP , Ensaios de Triagem em Larga Escala , Humanos , Criança , Avaliação Pré-Clínica de Medicamentos , Mutação/genética , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismoRESUMO
Dominant GNAO1 mutations cause an emerging group of childhood-onset neurological disorders characterized by developmental delay, intellectual disability, movement disorders, drug-resistant seizures and neurological deterioration. GNAO1 encodes the α-subunit of an inhibitory GTP/GDP-binding protein regulating ion channel activity and neurotransmitter release. The pathogenic mechanisms underlying GNAO1-related disorders remain largely elusive and there are no effective therapies. Here, we assessed the functional impact of two disease-causing variants associated with distinct clinical features, c.139A > G (p.S47G) and c.662C > A (p.A221D), using Caenorhabditis elegans as a model organism. The c.139A > G change was introduced into the orthologous position of the C. elegans gene via CRISPR/Cas9, whereas a knock-in strain carrying the p.A221D variant was already available. Like null mutants, homozygous knock-in animals showed increased egg laying and were hypersensitive to aldicarb, an inhibitor of acetylcholinesterase, suggesting excessive neurotransmitter release by different classes of motor neurons. Automated analysis of C. elegans locomotion indicated that goa-1 mutants move faster than control animals, with more frequent body bends and a higher reversal rate and display uncoordinated locomotion. Phenotypic profiling of heterozygous animals revealed a strong hypomorphic effect of both variants, with a partial dominant-negative activity for the p.A221D allele. Finally, caffeine was shown to rescue aberrant motor function in C. elegans harboring the goa-1 variants; this effect is mainly exerted through adenosine receptor antagonism. Overall, our findings establish a suitable platform for drug discovery, which may assist in accelerating the development of new therapies for this devastating condition, and highlight the potential role of caffeine in controlling GNAO1-related dyskinesia.
Assuntos
Proteínas de Caenorhabditis elegans , Discinesias , Acetilcolinesterase/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cafeína/farmacologia , Avaliação Pré-Clínica de Medicamentos , Discinesias/tratamento farmacológico , Discinesias/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/farmacologia , Proteínas de Ligação ao GTP/genética , Mutação , Neurotransmissores/metabolismoRESUMO
Heterotrimeric G proteins are crucial molecular switches that maintain a large number of physiological processes in cells. The signal is encoded into surface alterations of the Gα subunit that carries GTP in its active state and GDP in its inactive state. The ability of the Gα subunit to hydrolyze GTP is essential for signal termination. Regulator of G protein signaling (RGS) proteins accelerates this process. A key player in this catalyzed reaction is an arginine residue, Arg178 in Gαi1, which is already an intrinsic part of the catalytic center in Gα in contrast to small GTPases, at which the corresponding GTPase-activating protein (GAP) provides the arginine "finger." We applied time-resolved FTIR spectroscopy in combination with isotopic labeling and site-directed mutagenesis to reveal the molecular mechanism, especially of the role of Arg178 in the intrinsic Gαi1 mechanism and the RGS4-catalyzed mechanism. Complementary biomolecular simulations (molecular mechanics with molecular dynamics and coupled quantum mechanics/molecular mechanics) were performed. Our findings show that Arg178 is bound to γ-GTP for the intrinsic Gαi1 mechanism and pushed toward a bidentate α-γ-GTP coordination for the Gαi1·RGS4 mechanism. This movement induces a charge shift toward ß-GTP, increases the planarity of γ-GTP, and thereby catalyzes the hydrolysis.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/química , Arginina/química , Domínio Catalítico , Estabilidade Enzimática , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Hidrólise , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Neurofibromina 1/química , Neurofibromina 1/metabolismo , Proteínas RGS/química , Proteínas RGS/genética , Proteínas RGS/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
The existence of innate predator aversion evoked by predator-derived chemostimuli called kairomones offers a strong selective advantage for potential prey animals. However, it is unclear how chemically diverse kairomones can elicit similar avoidance behaviors. Using a combination of behavioral analyses and single-cell Ca(2+) imaging in wild-type and gene-targeted mice, we show that innate predator-evoked avoidance is driven by parallel, non-redundant processing of volatile and nonvolatile kairomones through the activation of multiple olfactory subsystems including the Grueneberg ganglion, the vomeronasal organ, and chemosensory neurons within the main olfactory epithelium. Perturbation of chemosensory responses in specific subsystems through disruption of genes encoding key sensory transduction proteins (Cnga3, Gnao1) or by surgical axotomy abolished avoidance behaviors and/or cellular Ca(2+) responses to different predator odors. Stimulation of these different subsystems resulted in the activation of widely distributed target regions in the olfactory bulb, as assessed by c-Fos expression. However, in each case, this c-Fos increase was observed within the same subnuclei of the medial amygdala and ventromedial hypothalamus, regions implicated in fear, anxiety, and defensive behaviors. Thus, the mammalian olfactory system has evolved multiple, parallel mechanisms for kairomone detection that converge in the brain to facilitate a common behavioral response. Our findings provide significant insights into the genetic substrates and circuit logic of predator-driven innate aversion and may serve as a valuable model for studying instinctive fear and human emotional and panic disorders.
Assuntos
Aprendizagem da Esquiva/fisiologia , Hipotálamo/fisiologia , Odorantes , Bulbo Olfatório/fisiologia , Animais , Comportamento Animal/fisiologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Gânglios/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Fenetilaminas , Feromônios , Comportamento Predatório , Proteínas Proto-Oncogênicas c-fos/metabolismo , Transdução de Sinais , Órgão Vomeronasal/fisiologiaRESUMO
OBJECTIVE: To determine whether Tai Chi Chuan or ballroom dancing promotes better performance with respect to postural balance, gait, and postural transfer among elderly people. METHODS: We evaluated 76 elderly individuals who were divided into two groups: the Tai Chi Chuan Group and the Dance Group. The subjects were tested using the NeuroCom Balance Master¯ force platform system with the following protocols: static balance tests (the Modified Clinical Tests of Sensory Interaction on Balance and Unilateral Stance) and dynamic balance tests (the Walk Across Test and Sit-to-stand Transfer Test). RESULTS: In the Modified Clinical Test of Sensory Interaction on Balance, the Tai Chi Chuan Group presented a lower sway velocity on a firm surface with open and closed eyes, as well as on a foam surface with closed eyes. In the Modified Clinical Test of Sensory Interaction on Unilateral Stance, the Tai Chi Chuan Group presented a lower sway velocity with open eyes, whereas the Dance Group presented a lower sway velocity with closed eyes. In the Walk Across Test, the Tai Chi Chuan Group presented faster walking speeds than those of the Dance Group. In the Sit-to-stand Transfer Test, the Tai Chi Chuan Group presented shorter transfer times from the sitting to the standing position, with less sway in the final standing position. CONCLUSION: The elderly individuals who practiced Tai Chi Chuan had better bilateral balance with eyes open on both types of surfaces compared with the Dance Group. The Dance Group had better unilateral postural balance with eyes closed. The Tai Chi Chuan Group had faster walking speeds, shorter transfer times, and better postural balance in the final standing position during the Sit-to-stand Test. .
Assuntos
/metabolismo , AMP Cíclico/metabolismo , Dictyostelium/enzimologia , Dictyostelium/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Proteínas de Protozoários/metabolismo , /genética , Dictyostelium/crescimento & desenvolvimento , Dictyostelium/metabolismo , Regulação para Baixo/efeitos dos fármacos , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Ácido Fólico/farmacologia , /deficiência , /genética , /metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/deficiência , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Mutação , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas de Protozoários/genética , Transdução de Sinais , Esporos de Protozoários/enzimologia , Esporos de Protozoários/genética , Complexo Vitamínico B/farmacologiaRESUMO
In ciliated mammalian cells, the precise migration of the primary cilium at the apical surface of the cells, also referred to as translational polarity, defines planar cell polarity (PCP) in very early stages. Recent research has revealed a co-dependence between planar polarization of some cell types and cilium positioning at the surface of cells. This important role of the primary cilium in mammalian cells is in contrast with its absence from Drosophila melanogaster PCP establishment. Here, we show that deletion of GTP-binding protein alpha-i subunit 3 (Gαi3) and mammalian Partner of inscuteable (mPins) disrupts the migration of the kinocilium at the surface of cochlear hair cells and affects hair bundle orientation and shape. Inhibition of G-protein function in vitro leads to kinocilium migration defects, PCP phenotype and abnormal hair bundle morphology. We show that Gαi3/mPins are expressed in an apical and distal asymmetrical domain, which is opposite and complementary to an aPKC/Par-3/Par-6b expression domain, and non-overlapping with the core PCP protein Vangl2. Thus G-protein-dependent signalling controls the migration of the cilium cell autonomously, whereas core PCP signalling controls long-range tissue PCP.
Assuntos
Cílios/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Externas/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Proteínas de Ciclo Celular , Movimento Celular , Polaridade Celular , Forma Celular , Cílios/genética , Citoesqueleto/genética , Citoesqueleto/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica , Células Ciliadas Auditivas Internas/citologia , Células Ciliadas Auditivas Externas/citologia , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteína Quinase C/genética , Proteína Quinase C/metabolismoRESUMO
Pasteurella multocida is the causative agent of a number of epizootic and zoonotic diseases. Its major virulence factor associated with atrophic rhinitis in animals and dermonecrosis in bite wounds is P. multocida toxin (PMT). PMT stimulates signal transduction pathways downstream of heterotrimeric G proteins, leading to effects such as mitogenicity, blockade of apoptosis, or inhibition of osteoblast differentiation. On the basis of Gα(i2), it was demonstrated that the toxin deamidates an essential glutamine residue of the Gα(i2) subunit, leading to constitutive activation of the G protein. Here, we studied the specificity of PMT for its G-protein targets by mass spectrometric analyses and by utilizing a monoclonal antibody, which recognizes specifically G proteins deamidated by PMT. The studies revealed deamidation of 3 of 4 families of heterotrimeric G proteins (Gα(q/11), Gα(i1,2,3), and Gα(12/13) of mouse or human origin) by PMT but not by a catalytic inactive toxin mutant. With the use of G-protein fragments and chimeras of responsive or unresponsive G proteins, the structural basis for the discrimination of heterotrimeric G proteins was studied. Our results elucidate substrate specificity of PMT on the molecular level and provide evidence for the underlying structural reasons of substrate discrimination.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidade , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Pasteurella multocida/metabolismo , Pasteurella multocida/patogenicidade , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Sequência de Bases , Sítios de Ligação , Células Cultivadas , DNA Complementar/genética , Subunidades alfa de Proteínas de Ligação ao GTP/deficiência , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Glutamina/química , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Pasteurella multocida/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais/efeitos dos fármacos , Especificidade por SubstratoRESUMO
Melanocortin-4 receptor (MC4R)-induced anorexigenic signaling in the hypothalamus controls body weight and energy homeostasis. So far, MC4R-induced signaling has been exclusively attributed to its coupling to G(s) proteins. In line with this monogamous G protein coupling profile, most MC4R mutants isolated from obese individuals showed a reduced ability to activate G(s). However, some mutants displayed enhanced G(s) coupling, suggesting that signaling pathways independent of G(s) may be involved in MC4R-mediated anorexigenic signaling. Here we report that the G(s) signaling-deficient MC4R-D90N mutant activates G proteins in a pertussis toxin-sensitive manner, indicating that this mutant is able to selectively interact with G(i/o) proteins. Analyzing a hypothalamic cell line (GT1-7 cells), we observed activation of pertussis toxin-sensitive G proteins by the wild-type MC4R as well, reflecting multiple coupling of the MC4R to G(s) and G(i/o) proteins in an endogenous cell system. Surprisingly, the agouti-related protein, which has been classified as a MC4R antagonist, selectively activates G(i/o) signaling in GT1-7 cells. Thus, the agouti-related protein antagonizes melanocortin-dependent G(s) activation not only by competitive antagonism but additionally by initiating G(i/o) protein-induced signaling as a biased agonist.
Assuntos
Proteína Relacionada com Agouti/metabolismo , Toxina Pertussis/farmacologia , Receptor Tipo 4 de Melanocortina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Relacionada com Agouti/genética , Animais , Linhagem Celular , AMP Cíclico/metabolismo , Ensaio de Imunoadsorção Enzimática , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Hipotálamo/citologia , Mutação , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ligação Proteica/efeitos dos fármacos , Ensaio Radioligante , Receptor Tipo 4 de Melanocortina/genética , Transfecção , alfa-MSH/metabolismo , alfa-MSH/farmacologiaRESUMO
Obesity results from an imbalance between food intake and energy expenditure, two vital functions that are tightly controlled by specialized neurons of the hypothalamus. The complex mechanisms that integrate these two functions are only beginning to be deciphered. The objective of this study was to determine the effect of two thermogenesis-inducing conditions, i.e., ingestion of a high-fat (HF) diet and exposure to cold environment, on the expression of 1,176 genes in the hypothalamus of Wistar rats. Hypothalamic gene expression was evaluated using a cDNA macroarray approach. mRNA and protein expressions were determined by reverse-transcription PCR (RT-PCR) and immunoblot. Cold exposure led to an increased expression of 43 genes and to a reduced expression of four genes. HF diet promoted an increased expression of 90 genes and a reduced expression of 78 genes. Only two genes (N-methyl-D-aspartate (NMDA) receptor 2B and guanosine triphosphate (GTP)-binding protein G-alpha-i1) were similarly affected by both thermogenesis-inducing conditions, undergoing an increment of expression. RT-PCR and immunoblot evaluations confirmed the modulation of NMDA receptor 2B and GTP-binding protein G-alpha-i1, only. This corresponds to 0.93% of all the responsive genes and 0.17% of the analyzed genes. These results indicate that distinct environmental thermogenic stimuli can modulate predominantly distinct profiles of genes reinforcing the complexity and multiplicity of the hypothalamic mechanisms that regulate energy conservation and expenditure.
Assuntos
Temperatura Baixa , Gorduras na Dieta/farmacologia , Hipotálamo/efeitos dos fármacos , Termogênese/efeitos dos fármacos , Termogênese/genética , Animais , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Metabolismo Energético/fisiologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Hipotálamo/metabolismo , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Termogênese/fisiologiaRESUMO
We report on a novel reconstitution method for G-protein-coupled receptors (GPCRs) that yields detergent-free, single, tubular membranes in porous anodic aluminum oxide (AAO) filters at concentrations sufficient for structural studies by solid-state NMR. The tubular membranes line the inner surface of pores that traverse the filters, permitting easy removal of detergents during sample preparation as well as delivery of ligands for functional studies. Reconstitution of bovine rhodopsin into AAO filters did not interfere with rhodopsin function. Photoactivation of rhodopsin in AAO pores, monitored by UV-vis spectrophotometry, was indistinguishable from rhodopsin in unsupported unilamellar liposomes. The rhodopsin in AAO pores is G-protein binding competent as shown by a [35S]GTPgammaS binding assay. The lipid-rhodopsin interaction was investigated by 2H NMR on sn-1- or sn-2-chain perdeuterated 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phospholine as a matrix lipid. Rhodopsin incorporation increased mosaic spread of bilayer orientations and contributed to spectral density of motions with correlation times in the range of nano- to microseconds, detected as a significant reduction in spin-spin relaxation times. The change in lipid chain order parameters due to interaction with rhodopsin was insignificant.
Assuntos
Bicamadas Lipídicas/química , Nanopartículas/química , Rodopsina/química , Óxido de Alumínio/química , Óxido de Alumínio/metabolismo , Animais , Bovinos , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/fisiologia , Filtração/instrumentação , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Ligantes , Luz , Bicamadas Lipídicas/metabolismo , Espectroscopia de Ressonância Magnética , Micelas , Fosfatidilcolinas/química , Porosidade , Ligação Proteica/genética , Proteolipídeos/química , Proteolipídeos/genética , Proteolipídeos/metabolismo , Rodopsina/genética , Rodopsina/fisiologia , Espalhamento de Radiação , Espectrofotometria UltravioletaRESUMO
Gbetagamma-activated inwardly rectifying K(+) (GIRK) channels have distinct gating properties when activated by receptors coupled specifically to Galpha(o) versus Galpha(i) subunit isoforms, with Galpha(o)-coupled currents having approximately 3-fold faster agonist-evoked activation kinetics. To identify the molecular determinants in Galpha subunits mediating these kinetic differences, chimeras were constructed using pertussis toxin (PTX)-insensitive Galpha(oA) and Galpha(i2) mutant subunits (Galpha(oA(C351G)) and Galpha(i2(C352G))) and examined in PTX-treated Xenopus oocytes expressing muscarinic m2 receptors and Kir3.1/3.2a channels. These experiments revealed that the alpha-helical N-terminal region (amino acids 1-161) and the switch regions of Galpha(i2) (amino acids 162-262) both partially contribute to slowing the GIRK activation time course when compared with the Galpha(oA(C351G))-coupled response. When present together, they fully reproduce Galpha(i2(C352G))-coupled GIRK kinetics. The Galpha(i2) C-terminal region (amino acids 263-355) had no significant effect on GIRK kinetics. Complementary responses were observed with chimeras substituting the Galpha(o) switch regions into the Galpha(i2(C352G)) subunit, which partially accelerated the GIRK activation rate. The Galpha(oA)/Galpha(i2) chimera results led us to examine an interaction between the alpha-helical domain and the Ras-like domain previously implicated in mediating a 4-fold slower in vitro basal GDP release rate in Galpha(i1) compared with Galpha(o). Mutations disrupting the interdomain contact in Galpha(i2(C352G)) at either the alphaD-alphaE loop (R145A) or the switch III loop (L233Q/A236H/E240T/M241T), significantly accelerated the GIRK activation kinetics consistent with the Galpha(i2) interdomain interface regulating receptor-catalyzed GDP release rates in vivo. We propose that differences in Galpha(i) versus Galpha(o)-coupled GIRK activation kinetics are due to intrinsic differences in receptor-catalyzed GDP release that rate-limit Gbetagamma production and is attributed to heterogeneity in Galpha(i) and Galpha(o) interdomain contacts.
Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Guanosina Difosfato/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Animais , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/genética , Modelos Moleculares , Dados de Sequência Molecular , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Toxina Pertussis/farmacologia , Canais de Potássio/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Xenopus laevisRESUMO
Guanosine triphosphate binding protein (G protein)-coupled receptors (GPCRs) are a large class of pharmaceutical drug targets. With the increasing popularity of functional assays for high throughput screening, there arises an increasing need for robust second messenger assays that reflect GPCR activation and are readily amenable for miniaturization. GPCRs that upon agonist stimulation modulate adenylyl cyclase activity, and, consequently, cellular cyclic adenosine monophosphate (cAMP) levels, via the G protein Gs or Gi, form a subset of therapeutic targets. While there are several cAMP assays currently available, most are not scalable for miniaturization into the 1536-well format employed for automated high throughput screening of large chemical libraries. Here, we describe a cAMP assay based on the enzyme fragmentation complementation (EFC) of beta-galactosidase. In this assay, recombinant cells expressing Gs- or Gi-coupled receptors exhibit robust and reproducible pharmacology for agonists and antagonists, as measured by cAMP levels. Furthermore, the EFC cAMP assay offers sufficient sensitivity to be used with cells expressing endogenous GPCRs. We demonstrate the miniaturization of this assay into a 1536-well format with comparable sensitivity and plate statistics to those of the 384-well assay for both Gs- and Gi-coupled receptors, and its suitability for miniaturized high throughput screening.
Assuntos
AMP Cíclico/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/efeitos dos fármacos , Subunidades alfa Gs de Proteínas de Ligação ao GTP/efeitos dos fármacos , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Animais , Células CHO , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Cricetinae , AMP Cíclico/análise , Fragmentação do DNA , Avaliação Pré-Clínica de Medicamentos , Ensaio de Imunoadsorção Enzimática , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Teste de Complementação Genética , Nanotecnologia , Receptores Acoplados a Proteínas G/genéticaRESUMO
The serine-threonine kinase Akt has been established as an important signaling intermediate in regulating cell survival, cell cycle progression, as well as agonist-induced platelet activation. Stimulation of platelets with various agonists including thrombin results in Akt activation. As thrombin can stimulate multiple G protein signaling pathways, we investigated the mechanism of thrombin-induced activation of Akt. Stimulation of platelets with a PAR1-activating peptide (SFLLRN), PAR4-activating peptide (AYPGKF), and thrombin resulted in Thr308 and Ser473 phosphorylation of Akt, which results in its activation. This phosphorylation and activation of Akt were dramatically inhibited in the presence of AR-C69931MX, a P2Y12 receptor-selective antagonist, or GF 109203X, a protein kinase C inhibitor, but Akt phosphorylation was restored by supplemental Gi or Gz signaling. Unlike wild-type mouse platelets, platelets from Galphaq-deficient mice failed to trigger Akt phosphorylation by thrombin and AYPGKF, whereas Akt phosphorylation was not affected by these agonists in platelets from mice that lack P2Y1 receptor. However, ADP caused Akt phosphorylation in Galphaq- and P2Y1-deficient platelets, which was completely blocked by AR-C69931MX. In contrast, ADP failed to cause Akt phosphorylation in platelets from mice treated with clopidogrel, and thrombin and AYPGKF induced minimal phosphorylation of Akt, which was not affected by AR-C69931MX in these platelets. These data demonstrate that Gi, but not Gq or G12/13, signaling pathways are required for activation of Akt in platelets, and Gi signaling pathways, stimulated by secreted ADP, play an essential role in the activation of Akt in platelets.
Assuntos
Plaquetas/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/sangue , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/sangue , Ticlopidina/análogos & derivados , Difosfato de Adenosina/farmacologia , Animais , Plaquetas/efeitos dos fármacos , Clopidogrel , Ativação Enzimática/efeitos dos fármacos , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/deficiência , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Humanos , Técnicas In Vitro , Camundongos , Camundongos Knockout , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Fosforilação , Proteína Quinase C/sangue , Proteínas Proto-Oncogênicas c-akt , Receptores Purinérgicos P2/sangue , Receptores Purinérgicos P2/deficiência , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2Y1 , Transdução de Sinais , Trombina/farmacologia , Ticlopidina/farmacologiaRESUMO
We have examined platelet functional responses and characterized a novel signaling defect in the platelets of a patient suffering from a chronic bleeding disorder. Platelet aggregation responses stimulated by weak agonists such as adenosine diphosphate (ADP) and adrenaline were severely impaired. In comparison, both aggregation and dense granule secretion were normal following activation with high doses of collagen, thrombin, or phorbol-12 myristate-13 acetate (PMA). ADP, thrombin, or thromboxane A2 (TxA2) signaling through their respective Gq-coupled receptors was normal as assessed by measuring either mobilization of intracellular calcium, diacylglycerol (DAG) generation, or pleckstrin phosphorylation. In comparison, Gi-mediated signaling induced by either thrombin, ADP, or adrenaline, examined by suppression of forskolin-stimulated rise in cyclic AMP (cAMP) was impaired, indicating dysfunctional Galphai signaling. Immunoblot analysis of platelet membranes with specific antiserum against different Galpha subunits indicated normal levels of Galphai2,Galphai3,Galphaz, and Galphaq in patient platelets. However, the Galphai1level was reduced to 25% of that found in normal platelets. Analysis of platelet cDNA and gDNA revealed no abnormality in either the Galphai1 or Galphai2 gene sequences. Our studies implicate the minor expressed Galphai subtype Galphai1 as having an important role in regulating signaling pathways associated with the activation of alphaIIbbeta3 and subsequent platelet aggregation by weak agonists.
Assuntos
Transtornos Plaquetários/etiologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Transdução de Sinais , Difosfato de Adenosina/farmacologia , Adulto , Ácido Araquidônico/sangue , Transtornos Plaquetários/genética , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Cálcio/sangue , Radioisótopos de Carbono , Membrana Celular/química , Colforsina/farmacologia , Colágeno/farmacologia , AMP Cíclico/sangue , DNA Complementar/química , Diglicerídeos/sangue , Epinefrina/farmacologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/sangue , Humanos , Masculino , Monócitos/química , Radioisótopos de Fósforo , Agregação Plaquetária/efeitos dos fármacos , Análise de Sequência de DNA , Serotonina/sangue , Trombina/farmacologia , Tromboxano B2/sangue , TrítioRESUMO
Compared with cloned, human (h)D(2) receptors (pK(i) = 6.9), the antiparkinsonian agent piribedil showed comparable affinity for halpha(2A)- (7.1) and halpha(2C)- (7.2) adrenoceptors (ARs), whereas its affinity for halpha(2B)-ARs was less marked (6.5). At halpha(2A)- and halpha(2C)-ARs, piribedil antagonized induction of [(35)S]guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) binding by norepinephrine (NE) with pK(b) values of 6.5 and 6.9, respectively. Furthermore, Schild analysis of the actions of piribedil at halpha(2A)-ARs indicated competitive antagonism, yielding a pA(2) of 6.5. At a porcine alpha(2A)-AR-Gi1alpha-Cys351C (wild-type) fusion protein, piribedil competitively abolished (pA(2) = 6.5) GTPase activity induced by epinephrine. However, at a alpha(2A)-AR-Gi1alpha-Cys351I (mutant) fusion protein of amplified sensitivity, although still acting as a competitive antagonist (pA(2) = 6.2) of epinephrine, piribedil itself manifested weak partial agonist properties. Similarly, piribedil weakly induced mitogen-activated protein kinase phosphorylation via wild-type halpha(2A)-ARs, although attenuating its phosphorylation by NE. As demonstrated by functional [(35)S]GTPgammaS autoradiography in rats, piribedil antagonized activation by NE of alpha(2)-ARs in cortex, amygdala, and septum. Antagonist properties were also expressed in a dose-dependent enhancement of the firing rate of adrenergic neurons in locus ceruleus (0.125-4.0 mg/kg i.v.). Furthermore, piribedil (2.5-4.0 mg/kg s.c.) accelerated hippocampal NE synthesis, elevated dialysis levels of NE in hippocampus and frontal cortex, and blocked hypnotic-sedative properties of the alpha(2)-AR agonist xylazine. Finally, piribedil showed only modest affinity for rat alpha(1)-ARs (5.9) and weakly antagonized NE-induced activation of phospholipase C via halpha(1A)-ARs (pK(b) = 5.6). In conclusion, piribedil displays essentially antagonist properties at cloned, human and cerebral, rat alpha(2)-ARs. Blockade of alpha(2)-ARs may, thus, contribute to its clinical antiparkinsonian profile.
Assuntos
Antagonistas de Receptores Adrenérgicos alfa 2 , Antiparkinsonianos/farmacologia , Piribedil/farmacologia , Antagonistas de Receptores Adrenérgicos alfa 1 , Agonistas alfa-Adrenérgicos/farmacologia , Animais , Ligação Competitiva/efeitos dos fármacos , Células CHO , Cricetinae , Relação Dose-Resposta a Droga , Lobo Frontal/efeitos dos fármacos , Lobo Frontal/metabolismo , GTP Fosfo-Hidrolases/antagonistas & inibidores , GTP Fosfo-Hidrolases/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacocinética , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Locus Cerúleo/citologia , Locus Cerúleo/efeitos dos fármacos , Locus Cerúleo/metabolismo , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Norepinefrina/metabolismo , Norepinefrina/farmacologia , Fosfatidilinositóis/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores Adrenérgicos alfa 2/genética , Receptores Adrenérgicos alfa 2/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Serotonina/metabolismoRESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) has been shown previously to be regulated by inhibitory G proteins. In the present study, we demonstrate inhibition of CFTR by alphaG(i2) and alphaG(i1), but not alphaG(0), in Xenopus oocytes. We further examined whether regulators of G protein signaling (RGS) proteins interfere with alphaG(i)-dependent inhibition of CFTR. Activation of CFTR by IBMX and forskolin was attenuated in the presence of alphaG(i2), indicating inhibition of CFTR by alphaG(i2) in Xenopus oocytes. Coexpression of the proteins RGS3 and RGS7 together with CFTR and alphaG(i2) partially recovered activation by IBMX/forskolin. 14-3-3, a protein that is known to interfere with RGS proteins, counteracted the effects of RGS3. These data demonstrate the regulation of CFTR by alphaG(i) in Xenopus oocytes. Because RGS proteins interfere with the G protein-dependent regulation of CFTR, this may offer new potential pathways for pharmacological intervention in cystic fibrosis.