RESUMEN
Highly homologous members of the Gαi family, Gαi1-3, have distinct tissue distributions and physiological functions, yet their biochemical and functional properties are very similar. We recently identified PDZ-RhoGEF (PRG) as a novel Gαi1 effector that is poorly activated by Gαi2. In a proteomic proximity labeling screen we observed a strong preference for Gαi1 relative to Gαi2 with respect to engagement of a broad range of potential targets. We investigated the mechanistic basis for this selectivity using PRG as a representative target. Substitution of either the helical domain (HD) from Gαi1 into Gαi2 or substitution of a single amino acid, A230 in Gαi2 with the corresponding D in Gαi1, largely rescues PRG activation and interactions with other potential Gαi targets. Molecular dynamics simulations combined with Bayesian network models revealed that in the GTP bound state, separation at the HD-Ras-like domain (RLD) interface is more pronounced in Gαi2 than Gαi1. Mutation of A230 to D in Gαi2 stabilizes HD-RLD interactions via ionic interactions with R145 in the HD which in turn modify the conformation of Switch III. These data support a model where D229 in Gαi1 interacts with R144 and stabilizes a network of interactions between HD and RLD to promote protein target recognition. The corresponding A230 in Gαi2 is unable to stabilize this network leading to an overall lower efficacy with respect to target interactions. This study reveals distinct mechanistic properties that could underly differential biological and physiological consequences of activation of Gαi1 or Gαi2 by G protein-coupled receptors.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go , Transducción de Señal , Humanos , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Simulación de Dinámica Molecular , Subunidad alfa de la Proteína de Unión al GTP Gi2/metabolismo , Subunidad alfa de la Proteína de Unión al GTP Gi2/genética , Subunidad alfa de la Proteína de Unión al GTP Gi2/química , Células HEK293 , Dominios Proteicos , Estabilidad Proteica , Unión ProteicaRESUMEN
BACKGROUND AND AIMS: NASH is an advanced stage of liver disease accompanied by lipid accumulation, inflammation, and liver fibrosis. Guanine nucleotide-binding protein G(i) subunit alpha-2 (GNAI2) is a member of the "inhibitory" class of α-subunits, and recent studies showed that Gnai2 deficiency is known to cause reduced weight in mice. However, the role of GNAI2 in hepatocytes, particularly in the context of liver inflammation and lipid metabolism, remains to be elucidated. Herein, we aim to ascertain the function of GNAI2 in hepatocytes and its impact on the development of NASH. APPROACH AND RESULTS: Human liver tissues were obtained from NASH patients and healthy persons to evaluate the expression and clinical relevance of GNAI2. In addition, hepatocyte-specific Gnai2-deficient mice (Gnai2hep-/- ) were fed either a Western diet supplemented with fructose in drinking water (WDF) for 16 weeks or a methionine/choline-deficient diet (MCD) for 6 weeks to investigate the regulatory role and underlying mechanism of Gnai2 in NASH. GNAI2 was significantly up-regulated in liver tissues of patients with NASH. Following feeding with WDF or MCD diets, livers from Gnai2hep-/- mice had reduced steatohepatitis with suppression of markers of inflammation and an increase in lipophagy compared to Gnai2flox/flox mice. Toll-like receptor 4 signals through nuclear factor kappa B to trigger p65-dependent transcription of Gnai2. Intriguingly, immunoprecipitation, immunofluorescence, and mass spectrometry identified peroxiredoxin 1 (PRDX1) as a binding partner of GNAI2. Moreover, the function of PRDX1 in the suppression of TNF receptor-associated factor 6 ubiquitin-ligase activity and glycerophosphodiester phosphodiesterase domain-containing 5-related phosphatidylcholine metabolism was inhibited by GNAI2. Suppression of GNAI2 combined with overexpression of PRDX1 reversed the development of steatosis and fibrosis in vivo. CONCLUSIONS: GNAI2 is a major regulator that leads to the development of NASH. Thus, inhibition of GNAI2 could be an effective therapeutic target for the treatment of NASH.
Asunto(s)
Subunidad alfa de la Proteína de Unión al GTP Gi2/metabolismo , Enfermedad del Hígado Graso no Alcohólico/inmunología , Peroxirredoxinas/metabolismo , Adulto , Animales , Autofagia/inmunología , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Femenino , Subunidad alfa de la Proteína de Unión al GTP Gi2/genética , Hepatocitos , Humanos , Hígado/inmunología , Hígado/patología , Masculino , Ratones , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Unión Proteica/inmunología , Transducción de Señal/inmunología , Adulto JovenRESUMEN
Plant microRNAs have shown the capacity to regulate mammalian systems. The potential bioactivity of miR171vr, an isoform of the plant miR171, on human embryonic kidney 293 (HEK293) cells was investigated. Bioinformatics simulations revealed that human G protein subunit alpha 12 (GNA12) transcript could represent an excellent target for miR171vr. To confirm this prediction, in vitro experiments were performed using a synthetic microRNA designed on miR171vr sequence. MiR-treated cells showed a significant decrease of GNA12 mRNA and protein levels, confirming the putative cross-kingdom interaction. In addition, miR171vr determined the modulation of GNA12 downstream signaling factors, including mTOR, as expected. Finally, the effect of the plant miRNA on HEK293 cell growth and its stability in presence of several stressors, such as those miming digestive processes and procedures for preparing food, were evaluated. All this preliminary evidence would suggest that miR171vr, introduced by diet or as supplement in gene therapies, could potentially influence human gene expression, especially for treating disorders where GNA12 is over-expressed (i.e. oral cancer, breast and prostate adenocarcinoma) or mTOR kinase is down-regulated (e.g. obesity, type 2 diabetes, neurodegeneration).
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Subunidad alfa de la Proteína de Unión al GTP Gi2/genética , MicroARNs/genética , Mapas de Interacción de Proteínas/genética , Serina-Treonina Quinasas TOR/genética , Proliferación Celular/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/terapia , Regulación Neoplásica de la Expresión Génica/genética , Células HEK293 , Humanos , MicroARNs/farmacología , Neoplasias/genética , Neoplasias/terapia , Obesidad/genética , Obesidad/terapia , Transducción de Señal/genéticaRESUMEN
Stress-related alterations in brain-derived neurotrophic factor (BDNF) expression, a neurotrophin that plays a key role in synaptic plasticity, are believed to contribute to the pathophysiology of depression. Here, we show that in a chronic mild stress (CMS) model of depression the Gαi1 and Gαi3 subunits of heterotrimeric G proteins are down-regulated in the hippocampus, a key limbic structure associated with major depressive disorder. We provide evidence that Gαi1 and Gαi3 (Gαi1/3) are required for the activation of TrkB downstream signaling pathways. In mouse embryonic fibroblasts (MEFs) and CNS neurons, Gαi1/3 knockdown inhibited BDNF-induced tropomyosin-related kinase B (TrkB) endocytosis, adaptor protein activation, and Akt-mTORC1 and Erk-MAPK signaling. Functional studies show that Gαi1 and Gαi3 knockdown decreases the number of dendrites and dendritic spines in hippocampal neurons. In vivo, hippocampal Gαi1/3 knockdown after bilateral microinjection of lentiviral constructs containing Gαi1 and Gαi3 shRNA elicited depressive behaviors. Critically, exogenous expression of Gαi3 in the hippocampus reversed depressive behaviors in CMS mice. Similar results were observed in Gαi1/Gαi3 double-knockout mice, which exhibited severe depressive behaviors. These results demonstrate that heterotrimeric Gαi1 and Gαi3 proteins are essential for TrkB signaling and that disruption of Gαi1 or Gαi3 function could contribute to depressive behaviors.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Depresión/metabolismo , Subunidad alfa de la Proteína de Unión al GTP Gi2/biosíntesis , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/biosíntesis , Hipocampo/metabolismo , Animales , Dendritas/metabolismo , Dendritas/patología , Espinas Dendríticas/metabolismo , Espinas Dendríticas/patología , Depresión/patología , Trastorno Depresivo Mayor/metabolismo , Trastorno Depresivo Mayor/patología , Regulación hacia Abajo , Femenino , Subunidad alfa de la Proteína de Unión al GTP Gi2/genética , Subunidad alfa de la Proteína de Unión al GTP Gi2/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Ratones , Ratones Noqueados , Neuronas/metabolismo , Neuronas/patología , Transducción de Señal/efectos de los fármacos , Estrés Fisiológico/fisiologíaRESUMEN
Hepatic ischemia-reperfusion (I/R) injury is a major challenge in liver resection and transplantation surgeries. Previous studies have revealed that guanine nucleotide-binding protein G(i)α2 (GNAI2) was involved in the progression of myocardial and cerebral I/R injury, but the role and function of GNAI2 in hepatic I/R have not been elucidated. The hepatocyte-specific GNAI2 knockout (GNAI2hep-/-) mice were generated and subjected to hepatic I/R injury. Primary hepatocytes isolated from GNAI2hep-/- and GNAI2flox/flox mice were cultured and challenged to hypoxia-reoxygenation insult. The specific function of GNAI2 in I/R-triggered hepatic injury and the underlying molecular mechanism were explored by various phenotypic analyses and molecular biology methods. In this study, we demonstrated that hepatic GNAI2 expression was significantly increased in liver transplantation patients and wild-type mice after hepatic I/R. Interestingly, hepatocyte-specific GNAI2 deficiency attenuated I/R-induced liver damage, inflammation cytokine expression, macrophage/neutrophil infiltration, and hepatocyte apoptosis in vivo and in vitro. Mechanistically, up-regulation of GNAI2 phosphorylates mixed-lineage protein kinase 3 (MLK3) through direct binding, which exacerbated hepatic I/R damage via MAPK and NF-κB pathway activation. Furthermore, blocking MLK3 signaling reversed GNAI2-mediated hepatic I/R injury. Our study firstly identifies GNAI2 as a promising target for prevention of hepatic I/R-induced injury and related liver diseases.-Sun, Q., He, Q., Xu, J., Liu, Q., Lu, Y., Zhang, Z., Xu, X., Sun, B. Guanine nucleotide-binding protein G(i)α2 aggravates hepatic ischemia-reperfusion injury in mice by regulating MLK3 signaling.
Asunto(s)
Subunidad alfa de la Proteína de Unión al GTP Gi2/metabolismo , Hepatopatías/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Daño por Reperfusión/fisiopatología , Animales , Apoptosis , Células Cultivadas , Regulación hacia Abajo , Subunidad alfa de la Proteína de Unión al GTP Gi2/genética , Regulación de la Expresión Génica , Hepatocitos/metabolismo , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Transducción de Señal , Proteina Quinasa Quinasa Quinasa 11 Activada por MitógenoRESUMEN
Salt sensitivity of blood pressure (BP) increases hypertension risk and associated adverse cardiovascular outcomes. At present, there are no validated rapid tests or diagnostic markers to identify salt sensitivity of BP in clinical practice. Based on our prior animal studies that report a role for brain Gαi2 proteins in the salt sensitivity of BP and evidence that GNAI2 single nucleotide polymorphisms (SNPs) associate with hypertension risk, we investigated the hypothesis that GNAI2 SNPs associate with salt sensitivity of BP in humans. Our data provide the first evidence that a GNAI2 SNP ( rs10510755 ) positively associates with salt sensitivity of BP in the Genetic Epidemiology of Salt Sensitivity data set (continuous phenotype P = 0.049, case-control phenotype P = 0.039; n = 968), independently of subject sex or age. These observations suggest that genotyping at GNAI2 may be a useful biomarker in identifying individuals at risk for developing salt-sensitive BP and related complications or in identifying salt sensitivity within the hypertensive population.
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Presión Sanguínea/genética , Subunidad alfa de la Proteína de Unión al GTP Gi2/genética , Polimorfismo de Nucleótido Simple/genética , Cloruro de Sodio Dietético/efectos adversos , Adulto , Femenino , Humanos , Masculino , Epidemiología MolecularRESUMEN
The heterotrimeric G-protein Go with its splice variants, Go1α and Go2α, seems to be involved in the regulation of motor function but isoform-specific effects are still unclear. We found that Go1α-/- knockouts performed worse on the rota-rod than Go2α-/- and wild-type (WT) mice. In Go1+2α-/- mice motor function was partially recovered. Furthermore, Go1+2α-/- mice showed an increased spontaneous motor activity. Compared to wild types or Go2α-/- mice, Go1+2α-/- mice developed increased behavioural sensitization following repetitive cocaine treatment, but failed to develop conditioned place preference. Analysis of dopamine concentration and expression of D1 and D2 receptors unravelled splice-variant-specific imbalances in the striatal dopaminergic system: In Go1α-/- mice dopamine concentration and vesicular monoamine uptake were increased compared to wild types. The expression of the D2 receptor was higher in Go1α-/- compared to wild type littermates, but unchanged in Go2α-/- mice. Deletion of both Go1α and Go2α re-established both dopamine and D2 receptor levels comparable to those in the wild-type. Cocaine treatment had no effect on the ratio of D1 receptor to D2 receptor in Go1+2α-/- mutants, but decreased this ratio in Go2α-/- mice. Finally, we observed that the deletion of Go1α led to a threefold higher striatal expression of Go2α. Taken together our data suggest that a balance in the expression of Go1α and Go2α sustains normal motor function. Deletion of either splice variant results in divergent behavioural and molecular alterations in the striatal dopaminergic system. Deletion of both splice variants partially restores the behavioural and molecular changes. Open Data: Materials are available on https://cos.io/our-services/open-science-badges/ https://osf.io/93n6m/.
Asunto(s)
Cuerpo Estriado/metabolismo , Dopamina/metabolismo , Subunidad alfa de la Proteína de Unión al GTP Gi2/metabolismo , Actividad Motora/genética , Animales , Animales Recién Nacidos , Monoaminas Biogénicas/metabolismo , Cocaína/administración & dosificación , Condicionamiento Operante/fisiología , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/ultraestructura , Inhibidores de Captación de Dopamina/administración & dosificación , Subunidad alfa de la Proteína de Unión al GTP Gi2/genética , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Masculino , Ratones , Ratones Transgénicos , Monoaminooxidasa/metabolismo , Actividad Motora/fisiología , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismo , Sinapsis/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/metabolismoRESUMEN
BACKGROUND/AIMS: From invertebrates to mammals, Gαi proteins act together with their common binding partner Gpsm2 to govern cell polarization and planar organization in virtually any polarized cell. Recently, we demonstrated that Gαi3-deficiency in pre-hearing murine cochleae pointed to a role of Gαi3 for asymmetric migration of the kinocilium as well as the orientation and shape of the stereociliary ("hair") bundle, a requirement for the progression of mature hearing. We found that the lack of Gαi3 impairs stereociliary elongation and hair bundle shape in high-frequency cochlear regions, linked to elevated hearing thresholds for high-frequency sound. How these morphological defects translate into hearing phenotypes is not clear. METHODS: Here, we studied global and conditional Gnai3 and Gnai2 mouse mutants deficient for either one or both Gαi proteins. Comparative analyses of global versus Foxg1-driven conditional mutants that mainly delete in the inner ear and telencephalon in combination with functional tests were applied to dissect essential and redundant functions of different Gαi isoforms and to assign specific defects to outer or inner hair cells, the auditory nerve, satellite cells or central auditory neurons. RESULTS: Here we report that lack of Gαi3 but not of the ubiquitously expressed Gαi2 elevates hearing threshold, accompanied by impaired hair bundle elongation and shape in high-frequency cochlear regions. During the crucial reprogramming of the immature inner hair cell (IHC) synapse into a functional sensory synapse of the mature IHC deficiency for Gαi2 or Gαi3 had no impact. In contrast, double-deficiency for Gαi2 and Gαi3 isoforms results in abnormalities along the entire tonotopic axis including profound deafness associated with stereocilia defects. In these mice, postnatal IHC synapse maturation is also impaired. In addition, the analysis of conditional versus global Gαi3-deficient mice revealed that the amplitude of ABR wave IV was disproportionally elevated in comparison to ABR wave I indicating that Gαi3 is selectively involved in generation of neural gain during auditory processing. CONCLUSION: We propose a so far unrecognized complexity of isoform-specific and overlapping Gαi protein functions particular during final differentiation processes.
Asunto(s)
Proteínas Portadoras/metabolismo , Factores de Transcripción Forkhead/metabolismo , Subunidad alfa de la Proteína de Unión al GTP Gi2/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Audición/fisiología , Proteínas del Tejido Nervioso/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas de Ciclo Celular , Factores de Transcripción Forkhead/genética , Subunidad alfa de la Proteína de Unión al GTP Gi2/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Células Ciliadas Auditivas Internas/citología , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genéticaRESUMEN
Chemokines are required for leukocyte recruitment and appropriate host defense and act through G protein-coupled receptors (GPCRs), which induce downstream signaling leading to integrin activation. Although the α and ß subunits of the GPCRs are the first intracellular molecules that transduce signals after ligand binding and are therefore indispensable for downstream signaling, relatively little is known about their contribution to lymphocyte function-associated antigen 1 (LFA-1) activation and leukocyte recruitment. We used knockout mice and short hairpin RNA to knock down guanine nucleotide binding protein (GNB) isoforms (GNB1, GNB2, GNB4, and GNB5) in HL60 cells and primary murine hematopoietic cells. Neutrophil function was assessed by using intravital microscopy, flow chamber assays, and chemotaxis and biochemistry studies. We unexpectedly discovered that all expressed GNB isoforms are required for LFA-1 activation. Their downregulation led to a significant impairment of LFA-1 activation, which was demonstrated in vitro and in vivo. Furthermore, we showed that GPCR activation leads to Ras-related C3 botulinum toxin substrate 1 (Rac1)-dependent activation of both phospholipase C ß2 (Plcß2) and Plcß3. They act nonredundantly to produce inositol triphosphate-mediated intracellular Ca(2+) flux and LFA-1 activation that support chemokine-induced arrest in vivo. In a complex inflammatory disease model, Plcß2-, Plcß3-, or Rac1-deficient mice were protected from lipopolysaccharide-induced lung injury. Taken together, we demonstrated that all Gnb isoforms are required for chemokine-induced downstream signaling, and Rac1, Plcß2, and Plcß3 are critically involved in integrin activation and leukocyte arrest.
Asunto(s)
Puntos de Control del Ciclo Celular , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Neutrófilos/metabolismo , Fosfolipasa C beta/metabolismo , Transducción de Señal , Proteína de Unión al GTP rac1/metabolismo , Animales , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Calcio/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Línea Celular , Quimiocinas/farmacología , Quimiotaxis/efectos de los fármacos , Quimiotaxis/genética , Quimiotaxis/inmunología , Modelos Animales de Enfermedad , Regulación hacia Abajo , Subunidad alfa de la Proteína de Unión al GTP Gi2/genética , Subunidad alfa de la Proteína de Unión al GTP Gi2/metabolismo , Subunidades beta de la Proteína de Unión al GTP/genética , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Lipopolisacáridos/efectos adversos , Ratones , Modelos Biológicos , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Fosfolipasa C beta/genética , Neumonía/inducido químicamente , Neumonía/genética , Neumonía/inmunología , Neumonía/metabolismo , Unión Proteica , Isoformas de Proteínas , ARN Interferente Pequeño/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteína de Unión al GTP rac1/genéticaRESUMEN
Leukocyte recruitment to inflammation sites progresses in a multistep cascade. Chemokines regulate multiple steps of the cascade, including arrest, transmigration, and chemotaxis. The most important chemokine receptor in mouse neutrophils is CXCR2, which couples through Gαi2- and Gαi3-containing heterotrimeric G proteins. Neutrophils arrest in response to CXCR2 stimulation. This is defective in Gαi2-deficient neutrophils. In this study, we show that Gαi3-deficient neutrophils showed reduced transmigration but normal arrest in mice. We also tested Gαi2- or Gαi3-deficient neutrophils in a CXCL1 gradient generated by a microfluidic device. Gαi3-, but not Gαi2-, deficient neutrophils showed significantly reduced migration and directionality. This was confirmed in a model of sterile inflammation in vivo. Gαi2-, but not Gαi3-, deficient neutrophils showed decreased Ca(2+) flux in response to CXCR2 stimulation. Conversely, Gαi3-, but not Gαi2-, deficient neutrophils exhibited reduced AKT phosphorylation upon CXCR2 stimulation. We conclude that Gαi2 controls arrest and Gαi3 controls transmigration and chemotaxis in response to chemokine stimulation of neutrophils.
Asunto(s)
Subunidad alfa de la Proteína de Unión al GTP Gi2/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Neutrófilos/inmunología , Animales , Señalización del Calcio/genética , Movimiento Celular/genética , Células Cultivadas , Quimiocina CXCL1/metabolismo , Quimiotaxis/genética , Subunidad alfa de la Proteína de Unión al GTP Gi2/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Unión Proteica , Receptores de Interleucina-8B/metabolismo , Migración Transendotelial y Transepitelial/genéticaRESUMEN
Canine mast cell tumours (CMCT) are one of the most common skin tumours in dogs with a major impact on canine health. Certain breeds have a higher risk of developing mast cell tumours, suggesting that underlying predisposing germ-line genetic factors play a role in the development of this disease. The genetic risk factors are largely unknown, although somatic mutations in the oncogene C-KIT have been detected in a proportion of CMCT, making CMCT a comparative model for mastocytosis in humans where C-KIT mutations are frequent. We have performed a genome wide association study in golden retrievers from two continents and identified separate regions in the genome associated with risk of CMCT in the two populations. Sequence capture of associated regions and subsequent fine mapping in a larger cohort of dogs identified a SNP associated with development of CMCT in the GNAI2 gene (p = 2.2x10-16), introducing an alternative splice form of this gene resulting in a truncated protein. In addition, disease associated haplotypes harbouring the hyaluronidase genes HYAL1, HYAL2 and HYAL3 on cfa20 and HYAL4, SPAM1 and HYALP1 on cfa14 were identified as separate risk factors in European and US golden retrievers, respectively, suggesting that turnover of hyaluronan plays an important role in the development of CMCT.
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Enfermedades de los Perros/genética , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Mutación de Línea Germinal , Mastocitoma/veterinaria , Empalme Alternativo , Animales , Perros , Subunidad alfa de la Proteína de Unión al GTP Gi2/genética , Mastocitoma/genética , Polimorfismo de Nucleótido SimpleRESUMEN
Chemokines engage B lymphocyte surface receptors, triggering heterotrimeric G protein Gαi subunit guanine nucleotide exchange. RGS proteins limit the duration that Gαi subunits remain GTP bound, and the loss of an individual RGS protein typically enhances chemokine receptor signaling. In this study, we show that B cells carrying a Gαi2 (G184S/G184S) mutation that disables all RGS protein/Gαi2 interactions exhibit an unexpectedly severe reduction in chemokine receptor signaling. The Gαi2 (G184S/G184S) B cells have markedly elevated basal calcium levels, but poor chemokine-induced increases, enhanced nonspecific migration, but extremely poor chemotaxis. In striking contrast, the Gαi2 (G184S/G184S) B cells exhibited enhanced sensitivity to sphingosine 1-phosphate (S1P). S1P elicited heightened intracellular calcium responses and enhanced S1P-triggered cell migration. Mice with the Gαi2 (G184S/G184S) mutation displayed excessive numbers of germinal center-like structures; abnormal serum Ig profiles; and aberrant B lymphocyte trafficking. These findings establish an essential role for RGS proteins in B cell chemoattractant signaling and for the proper position of B lymphocytes in lymphoid organs.
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Subgrupos de Linfocitos B/metabolismo , Quimiotaxis de Leucocito/efectos de los fármacos , Subunidad alfa de la Proteína de Unión al GTP Gi2/metabolismo , Proteínas RGS/metabolismo , Bazo/metabolismo , Animales , Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/efectos de los fármacos , Subgrupos de Linfocitos B/inmunología , Sitios de Unión , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Calcio/inmunología , Calcio/metabolismo , Quimiocinas/farmacología , Femenino , Subunidad alfa de la Proteína de Unión al GTP Gi2/genética , Subunidad alfa de la Proteína de Unión al GTP Gi2/inmunología , Regulación de la Expresión Génica , Centro Germinal/citología , Centro Germinal/efectos de los fármacos , Centro Germinal/inmunología , Centro Germinal/metabolismo , Lisofosfolípidos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Cultivo Primario de Células , Unión Proteica , Proteínas RGS/genética , Proteínas RGS/inmunología , Transducción de Señal , Esfingosina/análogos & derivados , Esfingosina/farmacología , Bazo/citología , Bazo/efectos de los fármacos , Bazo/inmunologíaRESUMEN
P2Y receptors are G-protein-coupled receptors (GPCRs) for extracellular nucleotides. The platelet ADP-receptor which has been denominated P2Y12 receptor is an important target in pharmacotherapy. The receptor couples to Gαi2 mediating an inhibition of cyclic AMP accumulation and additional downstream events including the activation of phosphatidylinositol-3-kinase and Rap1b proteins. The nucleoside analogue ticagrelor and active metabolites of the thienopyridine compounds ticlopidine, clopidogrel and prasugrel block P2Y12 receptors and, thereby, inhibit ADP-induced platelet aggregation. These drugs are used for the prevention and therapy of cardiovascular events such as acute coronary syndromes or stroke. The recently published three-dimensional crystal structures of the human P2Y12 receptor in complex with agonists and antagonists will facilitate the development of novel therapeutic agents with reduced adverse effects. P2Y12 receptors are also expressed on vascular smooth muscle cells and may be involved in the pathophysiology of atherogenesis. P2Y12 receptors on microglial cells operate as sensors for adenine nucleotides released during brain injury. A recent study indicated the involvement of microglial P2Y12 receptors in the activity-dependent neuronal plasticity. Interestingly, there is evidence for changes in P2Y12 receptor expression in CNS pathologies including Alzheimer's diseases and multiple sclerosis. P2Y12 receptors may also be involved in systemic immune modulating responses and the susceptibility to develop bronchial asthma.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Asma/metabolismo , Aterosclerosis/metabolismo , Plaquetas/metabolismo , Lesiones Encefálicas/metabolismo , Músculo Liso Vascular/metabolismo , Agregación Plaquetaria , Receptores Purinérgicos P2Y12 , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Animales , Asma/genética , Asma/patología , Aterosclerosis/genética , Aterosclerosis/patología , Plaquetas/patología , Lesiones Encefálicas/genética , Lesiones Encefálicas/patología , Subunidad alfa de la Proteína de Unión al GTP Gi2/genética , Subunidad alfa de la Proteína de Unión al GTP Gi2/metabolismo , Humanos , Músculo Liso Vascular/patología , Dominios Proteicos , Receptores Purinérgicos P2Y12/química , Receptores Purinérgicos P2Y12/genética , Receptores Purinérgicos P2Y12/metabolismo , Relación Estructura-Actividad , Proteínas de Unión al GTP rap/genética , Proteínas de Unión al GTP rap/metabolismoRESUMEN
The planar cell polarity (PCP) pathway is responsible for polarizing and orienting cochlear hair cells during development through movement of a primary cilium, the kinocilium. GPSM2/LGN, a mitotic spindle-orienting protein associated with deafness in humans, is a PCP effector involved in kinocilium migration. Here, we link human and mouse truncating mutations in the GPSM2/LGN gene, both leading to hearing loss. The human variant, p.(Trp326*), was identified by targeted genomic enrichment of genes associated with deafness, followed by massively parallel sequencing. Lgn (ΔC) mice, with a targeted deletion truncating the C-terminal GoLoco motifs, are profoundly deaf and show misorientation of the hair bundle and severe malformations in stereocilia shape that deteriorates over time. Full-length protein levels are greatly reduced in mutant mice, with upregulated mRNA levels. The truncated Lgn (ΔC) allele is translated in vitro, suggesting that mutant mice may have partially functioning Lgn. Gαi and aPKC, known to function in the same pathway as Lgn, are dependent on Lgn for proper localization. The polarization of core PCP proteins is not affected in Lgn mutants; however, Lgn and Gαi are misoriented in a PCP mutant, supporting the role of Lgn as a PCP effector. The kinocilium, previously shown to be dependent on Lgn for robust localization, is essential for proper localization of Lgn, as well as Gαi and aPKC, suggesting that cilium function plays a role in positioning of apical proteins. Taken together, our data provide a mechanism for the loss of hearing found in human patients with GPSM2/LGN variants.
Asunto(s)
Proteínas Portadoras/genética , Subunidad alfa de la Proteína de Unión al GTP Gi2/genética , Células Ciliadas Auditivas/metabolismo , Pérdida Auditiva Sensorineural/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteína Quinasa C/genética , Alelos , Animales , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Movimiento Celular , Polaridad Celular , Cilios/genética , Cilios/metabolismo , Cilios/patología , Femenino , Subunidad alfa de la Proteína de Unión al GTP Gi2/metabolismo , Eliminación de Gen , Regulación de la Expresión Génica , Células Ciliadas Auditivas/patología , Pérdida Auditiva Sensorineural/metabolismo , Pérdida Auditiva Sensorineural/patología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Motivos de Nucleótidos , Linaje , Proteína Quinasa C/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
TPRC channels are Ca(2+)-permeable, nonselective cation channels that are activated by a wide variety of stimuli, including G protein-coupled receptors (GPCRs). TRPC4 is commonly assumed to be activated by Gq/phospholipase C-coupled receptors. However, the other molecular mechanisms by which Gα proteins regulate TRPC4 remain unclear. Here, we found that Gαi2 regulates TRPC4 activation by direct binding. To investigate this mechanism, we used whole patch clamp and fluorescence resonance energy transfer (FRET). We tagged an isoform of mTRPC4 and G protein with CFP and YFP, respectively, and transiently transfected cells with the FRET pair. The FRET efficiency between TRPC4ß-CFP and the constitutively active mutant form of Gαi2 was nearly 15% and was greater than that observed with wild-type Gαi2 (nearly 5%). Gßγ and the TRPC4 channel showed a FRET efficiency lower than 6%. In HEK293 cells transfected with the M2 muscarinic receptor, the application of carbachol increased the FRET efficiency between TRPC4ß-CFP and Gαi2(WT)-YFP from 4.7 ± 0.4% (n = 7) to 12.6 ± 1.4% (n = 7). We also found that the TRPC4 channel directly interacts with Gαi2, but not with Gαq, when the channel is open. We analyzed the calcium levels in HEK293 cells expressing the channels and Gαi2 or Gαq using the calcium indicator YC6.1 (Yellow Cameleon 6.1). In response to the muscarinic agonist carbachol, M2-, Gαi2-, and TRPC4-expressing cells showed a prolonged Ca(2+) influx compared with cells expressing only M2. Together, these data suggest that Gαi2 activates the TRPC4 channel by direct binding, which then induces Ca(2+) entry.
Asunto(s)
Señalización del Calcio/genética , Calcio/metabolismo , Subunidad alfa de la Proteína de Unión al GTP Gi2/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Canales Catiónicos TRPC/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Señalización del Calcio/efectos de los fármacos , Proteínas de Unión al Calcio/química , Carbacol/farmacología , Agonistas Colinérgicos/farmacología , Transferencia Resonante de Energía de Fluorescencia , Subunidad alfa de la Proteína de Unión al GTP Gi2/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Canales Catiónicos TRPC/metabolismoRESUMEN
Group II activator of G-protein signaling (AGS) proteins contain one or more G-protein regulatory motifs (GPR), which serve as docking sites for GαiGDP independent of Gßγ and stabilize the GDP-bound conformation of Gαi, acting as guanine nucleotide dissociation inhibitors. The GαGPR interaction is regulated by seven-transmembrane-spanning (7TM) receptors in the intact cell as determined by bioluminescence resonance energy transfer (BRET). It is hypothesized that a 7TM receptor directly couples to the GαGPR complex in a manner analogous to receptor coupling to the Gαßγ heterotrimer. As an initial approach to test this hypothesis, we used BRET to examine 7TM receptor-mediated regulation of GαGPR in the intact cell when Gαi2 yellow fluorescent protein (YFP) was tethered to the carboxyl terminus of the α2A adrenergic receptor (α2AAR-Gαi2YFP). AGS3- and AGS4-Renilla luciferase (Rluc) exhibited robust BRET with the tethered GαiYFP, and this interaction was regulated by receptor activation localizing the regulation to the receptor microenvironment. Agonist regulation of the receptor-Gαi-GPR complex was also confirmed by coimmunoprecipitation and cell fractionation. The tethered Gαi2 was rendered pertussis toxin-insensitive by a C352I mutation, and receptor coupling to endogenous Gαi/oßγ was subsequently eliminated by cell treatment with pertussis toxin (PT). Basal and agonist-induced regulation of α2AAR-Gαi2YFP(C352I):AGS3Rluc and α2AAR-Gαi2YFP(C352I):AGS4Rluc BRET was not altered by PT treatment or Gßγ antagonists. Thus, the localized regulation of GαGPR by receptor activation appears independent of endogenous Gαi/oßγ, suggesting that GαiAGS3 and GαiAGS4 directly sense agonist-induced conformational changes in the receptor, as is the case for 7TM receptor coupling to the Gαßγ heterotrimer. The direct coupling of a receptor to the GαiGPR complex provides an unexpected platform for signal propagation with broad implications.
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Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas de Unión al GTP/metabolismo , Toxina del Pertussis/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Animales , Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Subunidad alfa de la Proteína de Unión al GTP Gi2/química , Subunidad alfa de la Proteína de Unión al GTP Gi2/genética , Subunidad alfa de la Proteína de Unión al GTP Gi2/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Proteínas de Unión al GTP/química , Células HEK293 , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Mutación , Conformación Proteica/efectos de los fármacos , Ratas , Receptores Acoplados a Proteínas G/químicaRESUMEN
The increase of reactive oxygen species in infracted heart significantly reduces the survival of donor mesenchymal stem cells, thereby attenuating the therapeutic efficacy for myocardial infarction. In our previous study, we demonstrated that lysophosphatidic acid (LPA) protects bone marrow-derived mesenchymal stem cells (BMSCs) against hypoxia and serum deprivation-induced apoptosis. However, whether LPA protects BMSCs from H2O2-induced apoptosis was not examined. In this study, we report that H2O2 induces rat BMSC apoptosis whereas LPA pre-treatment effectively protects BMSCs from H2O2-induced apoptosis. LPA protection of BMSC from the induced apoptosis is mediated mostly through LPA3 receptor. Furthermore, we found that membrane G protein Gi2 and Gi3 are involved in LPA-elicited anti-apoptotic effects through activation of ERK1/2- and PI3 K-pathways. Additionally, H2O2 increases levels of type II of light chain 3B (LC3B II), an autophagy marker, and H2O2-induced autophagy thus protected BMSCs from apoptosis. LPA further increases the expression of LC3B II in the presence of H2O2. In contrast, autophagy flux inhibitor bafilomycin A1 has no effect on LPA's protection of BMSC from H2O2-induced apoptosis. Taken together, our data suggest that LPA rescues H2O2-induced apoptosis mainly by interacting with Gi-coupled LPA3, resulting activation of the ERK1/2- and PI3 K/AKT-pathways and inhibition caspase-3 cleavage, and LPA protection of BMSCs against the apoptosis is independent of it induced autophagy.
Asunto(s)
Apoptosis/efectos de los fármacos , Subunidad alfa de la Proteína de Unión al GTP Gi2/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Peróxido de Hidrógeno/antagonistas & inhibidores , Lisofosfolípidos/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Caspasa 3/genética , Caspasa 3/metabolismo , Subunidad alfa de la Proteína de Unión al GTP Gi2/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Peróxido de Hidrógeno/farmacología , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Cultivo Primario de Células , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores del Ácido Lisofosfatídico/genética , Receptores del Ácido Lisofosfatídico/metabolismoRESUMEN
129/SvEv mice with a loss-of-function mutation in the heterotrimeric G protein α-subunit gene Gnai3 have fusions of ribs and lumbar vertebrae, indicating a requirement for Gα(i) (the "inhibitory" class of α-subunits) in somite derivatives. Mice with mutations of Gnai1 or Gnai2 have neither defect, but loss of both Gnai3 and one of the other two genes increases the number and severity of rib fusions without affecting the lumbar fusions. No myotome defects are observed in Gnai3/Gnai1 double-mutant embryos, and crosses with a conditional allele of Gnai2 indicate that Gα(i) is specifically required in cartilage precursors. Penetrance and expressivity of the rib fusion phenotype is altered in mice with a mixed C57BL/6 × 129/SvEv genetic background. These phenotypes reveal a previously unknown role for G protein-coupled signaling pathways in development of the axial skeleton.
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Desarrollo Óseo , Huesos/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Mamíferos/crecimiento & desarrollo , Transducción de Señal , Alelos , Animales , Subunidad alfa de la Proteína de Unión al GTP Gi2/genética , Subunidad alfa de la Proteína de Unión al GTP Gi2/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/deficiencia , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Vértebras Lumbares/anomalías , Vértebras Lumbares/crecimiento & desarrollo , Ratones , Ratones de la Cepa 129 , Ratones Mutantes , Mutación/genética , Fenotipo , Costillas/anomalías , Costillas/crecimiento & desarrollo , Fusión Vertebral , Esternón/anomalías , Esternón/crecimiento & desarrolloRESUMEN
Bordetella pertussis toxin (PTx), also known as islet-activating protein, induces insulin secretion by ADP-ribosylation of inhibitory G proteins. PTx-induced insulin secretion may result either from inactivation of Gα(o) proteins or from combined inactivation of Gα(o), Gα(i1), Gα(i2), and Gα(i3) isoforms. However, the specific role of Gα(i2) in pancreatic ß-cells still remains unknown. In global (Gα(i2)(-/-)) and ß-cell-specific (Gα(i2)(ßcko)) gene-targeted Gα(i2) mouse models, we studied glucose homeostasis and islet functions. Insulin secretion experiments and intracellular Ca²âº measurements were used to characterize Gα(i2) function in vitro. Gα(i2)(-/-) and Gα(i2)(ßcko) mice showed an unexpected metabolic phenotype, i.e., significantly lower plasma insulin levels upon intraperitoneal glucose challenge in Gα(i2)(-/-) and Gα(i2)(ßcko) mice, whereas plasma glucose concentrations were unchanged in Gα(i2)(-/-) but significantly increased in Gα(i2)(ßcko) mice. These findings indicate a novel albeit unexpected role for Gα(i2) in the expression, turnover, and/or release of insulin from islets. Detection of insulin secretion in isolated islets did not show differences in response to high (16 mM) glucose concentrations between control and ß-cell-specific Gα(i2)-deficient mice. In contrast, the two- to threefold increase in insulin secretion evoked by L-arginine or L-ornithine (in the presence of 16 mM glucose) was significantly reduced in islets lacking Gα(i2). In accord with a reduced level of insulin secretion, intracellular calcium concentrations induced by the agonistic amino acid L-arginine did not reach control levels in ß-cells. The presented analysis of gene-targeted mice provides novel insights in the role of ß-cell Gα(i2) showing that amino acid-induced insulin-release depends on Gα(i2).
Asunto(s)
Arginina/metabolismo , Subunidad alfa de la Proteína de Unión al GTP Gi2/agonistas , Hiperglucemia/prevención & control , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Ornitina/metabolismo , Regulación hacia Arriba , Animales , Glucemia/análisis , Señalización del Calcio , Cruzamientos Genéticos , Regulación hacia Abajo , Técnica del Anticuerpo Fluorescente , Subunidad alfa de la Proteína de Unión al GTP Gi2/genética , Subunidad alfa de la Proteína de Unión al GTP Gi2/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/agonistas , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Hiperglucemia/sangre , Hiperglucemia/metabolismo , Hipoglucemia/sangre , Hipoglucemia/metabolismo , Hipoglucemia/prevención & control , Insulina/sangre , Secreción de Insulina , Islotes Pancreáticos/citología , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Ornitina/sangre , Organismos Libres de Patógenos Específicos , Técnicas de Cultivo de TejidosRESUMEN
Others and we have characterized several Gßγ-dependent effectors in smooth muscle, including G protein-coupled receptor kinase 2 (GRK2), PLCß3, and phosphatidylinositol (PI) 3-kinase-γ, and have identified various signaling targets downstream of PI 3-kinase-γ, including cSrc, integrin-linked kinase, and Rac1-Cdc42/p21-activated kinase/p38 MAP kinase. This study identified a novel mechanism whereby Gßγ acting via PI 3-kinase-γ and cSrc exerts an inhibitory influence on Gαi activity. The Gi2-coupled δ-opioid receptor agonist d-penicillamine (2,5)-enkephalin (DPDPE) activated cSrc, stimulated tyrosine phosphorylation of Gαi2, and induced regulator of G protein signaling 12 (RGS12) association; all three events were blocked by PI 3-kinase (LY294002) and cSrc (PP2) inhibitors and by expression of the COOH-terminal sequence of GRK2-(495-689), a Gßγ-scavenging peptide. Inhibition of forskolin-stimulated cAMP and muscle relaxation by DPDPE was augmented by PP2, LY294002, and a selective PI 3-kinase-γ inhibitor, AS-605420. Expression of tyrosine-deficient (Y69F, Y231F, or Y321F) Gαi2 mutant or knockdown of RGS12 blocked Gαi2 phosphorylation and Gαi2-RGS12 association and caused greater inhibition of cAMP. Parallel studies using somatostatin, cyclopentyl adenosine, or ACh to activate, respectively, Gi1-coupled somatostatin (sstr3) receptors, and Gi3-coupled adenosine A1 or muscarinic m2 receptors elicited cSrc activation, Gαi1 or Gαi3 phosphorylation, Gαi1-RGS12 or Gαi3-RGS12 association, and inhibition of cAMP. Inhibition of cAMP and muscle relaxation was greatly increased by AS-605240 and PP2. The results demonstrate that Gßγ-dependent tyrosine phosphorylation of Gαi1/2/3 by cSrc facilitated recruitment of RGS12, a Gαi-specific RGS protein with a unique phosphotyrosine-binding domain, resulting in rapid deactivation of Gαi and facilitation of smooth muscle relaxation.