RESUMEN
Chronic stress is one of the main risk factors of bone loss. While the neurons and neural circuits of the ventromedial hypothalamus (VMH) mediate bone loss induced by chronic stress, the detailed intrinsic mechanisms within the VMH nucleus still need to be explored. Astrocytes in brain regions play important roles in the regulation of metabolism and anxiety-like behavior through interactions with surrounding neurons. However, whether astrocytes in the VMH affect neuronal activity and therefore regulate chronic stress-induced anxiety and bone loss remain elusive. In this study, we found that VMH astrocytes were activated during chronic stress-induced anxiety and bone loss. Pharmacogenetic activation of the Gi and Gq pathways in VMH astrocytes reduced and increased the levels of anxiety and bone loss, respectively. Furthermore, activation of VMH astrocytes by optogenetics induced depolarization in neighboring steroidogenic factor-1 (SF-1) neurons, which was diminished by administration of N-methyl-D-aspartic acid (NMDA) receptor blocker but not by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor blocker. These results suggest that there may be a functional "glial-neuron microcircuit" in VMH nuclei that mediates anxiety and bone loss induced by chronic stress. This study not only advances our understanding of glial cell function but also provides a potential intervention target for chronic stress-induced anxiety and bone loss therapy.
Asunto(s)
Trastornos de Ansiedad/fisiopatología , Astrocitos/fisiología , Resorción Ósea/fisiopatología , Estrés Psicológico/complicaciones , Núcleo Hipotalámico Ventromedial/patología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Densidad Ósea , Resorción Ósea/etiología , Resorción Ósea/prevención & control , Enfermedad Crónica , Clozapina/farmacología , Clozapina/uso terapéutico , Prueba de Laberinto Elevado , Emociones , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/agonistas , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/fisiología , Genes Reporteros , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/fisiología , Prueba de Campo Abierto , Optogenética , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Proteínas Proto-Oncogénicas c-fos/genética , Distribución Aleatoria , Receptores de N-Metil-D-Aspartato/fisiología , Núcleo Hipotalámico Ventromedial/fisiopatologíaRESUMEN
Pimavanserin is claimed as the first antipsychotic drug that shows selectivity for serotonin 5-HT2 receptors (5-HT2Rs) and lacks of affinity for dopamine D2 receptors (D2Rs). Cell-based functional assays suggest that pimavanserin and antipsychotics with D2R/5-HT2R affinity could act as inverse agonists of 5-HT2ARs. However, there is not evidence of such pharmacological profile in native brain tissue. 5-HT2ARs are able to engage both canonical Gαq/11- and non-canonical Gαi1-proteins. In the present study, the response to pimavanserin of the 5-HT2AR coupling to Gαq/11- and Gαi1-proteins was measured in membranes of postmortem human prefrontal cortex by antibody-capture [35S]GTPγS binding scintillation proximity assays. Pimavanserin promoted a concentration-dependant inhibition of the 5-HT2AR coupling to Gαi1-proteins whereas the response of Gαq/11-proteins was unaltered, suggesting inverse agonism and neutral antagonism properties, respectively. The inhibition was abolished in the presence of the selective 5-HT2AR antagonist MDL-11,939 and was absent in brain cortex of 5-HT2AR knock-out mice when compared to respective 5-HT2AR wild-type animals. In conclusion, the results demonstrate the existence of constitutive 5-HT2AR activity in human brain for the signalling pathway mediated by Gαi1-proteins. Pimavanserin demonstrates 5-HT2AR functional selectivity and exhibits inverse agonist profile towards Gαi1-proteins, which is considered the effector pathway promoting hallucinogenic responses. In contrast, pimavanserin behaves as neutral antagonist on the 5-HT2AR coupling to the canonical Gαq/11-protein pathway. The results strengthen the relevance of inverse agonism as potential mechanism of antipsychotic activity. Moreover, the existence of functional selectivity of 5-HT2ARs for different Gα-proteins could contribute to better design of 5-HT2AR-related antipsychotic drugs.
Asunto(s)
Corteza Cerebral/efectos de los fármacos , Agonismo Inverso de Drogas , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/antagonistas & inhibidores , Piperidinas/farmacología , Agonistas del Receptor de Serotonina 5-HT2/farmacología , Antagonistas del Receptor de Serotonina 5-HT2/farmacología , Urea/análogos & derivados , Adulto , Anciano , Animales , Corteza Cerebral/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/agonistas , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Receptor de Serotonina 5-HT2A/metabolismo , Urea/farmacologíaRESUMEN
In mouse ileal myocytes, muscarinic receptor-mediated cationic current (mIcat) occurs mainly through synergism of M2 and M3 subtypes involving Gi/o-type GTP-binding proteins and phospholipase C (PLC). We have further studied the M2/M3 synergistic pathway. Carbachol-induced mIcat was markedly depressed by YM-254890, a Gq/11 protein inhibitor. However, the mIcat was unaffected by heparin, calphostin C, or chelerythrine, suggesting that mIcat activation does not involve signaling molecules downstream of phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown. M2-knockout (KO) mice displayed a reduced mIcat (~10% of wild-type mIcat) because of the lack of M2-Gi/o signaling. The impaired mIcat was insensitive to neuropeptide Y possessing a Gi/o-stimulating activity. M3-KO mice also displayed a reduced mIcat (~6% of wild-type mIcat) because of the lack of M3-Gq/11 signaling, and the mIcat was insensitive to prostaglandin F2α possessing a Gq/11-stimulating activity. These results suggest the importance of Gq/11/PLC-hydrolyzed PIP2 breakdown itself in mIcat activation and also support the idea that the M2/M3 synergistic pathway represents a signaling complex consisting of M2-Gi/o and M3-Gq/11-PLC systems in which both G proteins are special for this pathway but not general in receptor coupling.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Mucosa Intestinal/metabolismo , Miocitos del Músculo Liso/metabolismo , Receptor Muscarínico M2/metabolismo , Receptor Muscarínico M3/metabolismo , Animales , Agonistas Colinérgicos/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/agonistas , Cobayas , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Péptidos Cíclicos/farmacología , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M3/agonistasRESUMEN
G protein-coupled receptors (GPCRs) play key roles in intercellular signaling in the brain. Their effects on cellular function have been largely studied in neurons, but their functional consequences on astrocytes are less known. Using both endogenous and chemogenetic approaches with DREADDs, we have investigated the effects of Gq and Gi/o GPCR activation on astroglial Ca2+ -based activity, gliotransmitter release, and the functional consequences on neuronal electrical activity. We found that while Gq GPCR activation led to cellular activation in both neurons and astrocytes, Gi/o GPCR activation led to cellular inhibition in neurons and cellular activation in astrocytes. Astroglial activation by either Gq or Gi/o protein-mediated signaling stimulated gliotransmitter release, which increased neuronal excitability. Additionally, activation of Gq and Gi/o DREADDs in vivo increased astrocyte Ca2+ activity and modified neuronal network electrical activity. Present results reveal additional complexity of the signaling consequences of excitatory and inhibitory neurotransmitters in astroglia-neuron network operation and brain function.
Asunto(s)
Astrocitos/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Inhibición Neural/fisiología , Neuroglía/metabolismo , Neuronas/metabolismo , Animales , Astrocitos/efectos de los fármacos , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/agonistas , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Antagonistas Muscarínicos/farmacología , Inhibición Neural/efectos de los fármacos , Neuroglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Técnicas de Cultivo de Órganos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
The short neuropeptide F (sNPF) neuropeptides, closely related to vertebrate neuropeptide Y (NPY), have been suggested to exert pleiotropic effects on many physiological processes in insects. In the silkworm (Bombyx mori) two orphan G protein-coupled receptors, Bombyx neuropeptide G protein-coupled receptor (BNGR) A10 and A11, have been identified as cognate receptors for sNPFs, but other sNPF receptors and their signaling mechanisms in B. mori remain unknown. Here, we cloned the full-length cDNA of the orphan receptor BNGR-A7 from the brain of B. mori larvae and identified it as a receptor for Bombyx sNPFs. Further characterization of signaling and internalization indicated that BNGR-A7, -A10, and -A11 are activated by direct interaction with synthetic Bombyx sNPF-1 and -3 peptides. This activation inhibited forskolin or adipokinetic hormone-induced adenylyl cyclase activity and intracellular Ca2+ mobilization via a Gi/o-dependent pathway. Upon activation by sNPFs, BNGR-A7, -A10, and -A11 evoked ERK1/2 phosphorylation and underwent internalization. On the basis of these findings, we designated the receptors BNGR-A7, -A10, and -A11 as Bommo-sNPFR-1, -2, and -3, respectively. Moreover, the results obtained with quantitative RT-PCR analysis revealed that the three Bombyx sNPF receptor subtypes exhibit differential spatial and temporal expression patterns, suggesting possible roles of sNPF signaling in the regulation of a wide range of biological processes. Our findings provide the first in-depth information on sNPF signaling for further elucidation of the roles of the Bombyx sNPF/sNPFR system in the regulation of physiological activities.
Asunto(s)
Bombyx/metabolismo , Señalización del Calcio , Regulación hacia Abajo , Proteínas de Insectos/agonistas , Neuropéptidos/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores de Neuropéptido/agonistas , Animales , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/agonistas , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/antagonistas & inhibidores , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Proteínas de Insectos/química , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Sistema de Señalización de MAP Quinasas , Neuropéptidos/química , Neuropéptidos/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosforilación , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropéptido/genética , Receptores de Neuropéptido/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Células Sf9 , SpodopteraRESUMEN
Low testosterone levels are strongly related to obesity in males. The balance between the classically M1 and alternatively M2 polarized macrophages also plays a critical role in obesity. It is not clear whether testosterone regulates macrophage polarization and then affects adipocyte differentiation. In this report, we demonstrate that testosterone strengthens interleukin (IL) -4-induced M2 polarization and inhibits lipopolysaccharide (LPS)-induced M1 polarization, but has no direct effect on adipocyte differentiation. Cellular signaling studies indicate that testosterone regulates macrophage polarization through the inhibitory regulative G-protein (Gαi) mainly, rather than via androgen receptors, and phosphorylation of Akt. Moreover, testosterone inhibits pre-adipocyte differentiation induced by M1 macrophage medium. Lowering of serum testosterone in mice by injecting a luteinizing hormone receptor (LHR) peptide increases epididymal white adipose tissue. Testosterone supplementation reverses this effect. Therefore, our findings indicate that testosterone inhibits pre-adipocyte differentiation by switching macrophages to M2 polarization through the Gαi and Akt signaling pathways.
Asunto(s)
Adipocitos Blancos/efectos de los fármacos , Adipogénesis/efectos de los fármacos , Adiposidad/efectos de los fármacos , Terapia de Reemplazo de Hormonas , Macrófagos/efectos de los fármacos , Obesidad/prevención & control , Testosterona/uso terapéutico , Células 3T3-L1 , Adipocitos Blancos/inmunología , Adipocitos Blancos/metabolismo , Adipocitos Blancos/patología , Animales , Polaridad Celular/efectos de los fármacos , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/agonistas , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/antagonistas & inhibidores , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/inmunología , Obesidad/metabolismo , Obesidad/patología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células RAW 264.7 , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Organismos Libres de Patógenos Específicos , Testosterona/sangre , Testosterona/farmacologíaRESUMEN
G protein-coupled receptor 30 (GPR30), also called G protein-coupled estrogen receptor 1 (GPER1), is thought to play important roles in breast cancer and cardiometabolic regulation, but many questions remain about ligand activation, effector coupling, and subcellular localization. We showed recently that GPR30 interacts through the C-terminal type I PDZ motif with SAP97 and protein kinase A (PKA)-anchoring protein (AKAP) 5, which anchor the receptor in the plasma membrane and mediate an apparently constitutive decrease in cAMP production independently of Gi/o Here, we show that GPR30 also constitutively increases ERK1/2 activity. Removing the receptor PDZ motif or knocking down specifically AKAP5 inhibited the increase, showing that this increase also requires the PDZ interaction. However, the increase was inhibited by pertussis toxin as well as by wortmannin but not by AG1478, indicating that Gi/o and phosphoinositide 3-kinase (PI3K) mediate the increase independently of epidermal growth factor receptor transactivation. FK506 and okadaic acid also inhibited the increase, implying that a protein phosphatase is involved. The proposed GPR30 agonist G-1 also increased ERK1/2 activity, but this increase was only observed at a level of receptor expression below that required for the constitutive increase. Furthermore, deleting the PDZ motif did not inhibit the G-1-stimulated increase. Based on these results, we propose that GPR30 increases ERK1/2 activity via two Gi/o-mediated mechanisms, a PDZ-dependent, apparently constitutive mechanism and a PDZ-independent G-1-stimulated mechanism.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/agonistas , Sistema de Señalización de MAP Quinasas , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Modelos Moleculares , Fosfatidilinositol 3-Quinasa/metabolismo , Receptores de Estrógenos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Anclaje a la Quinasa A/antagonistas & inhibidores , Proteínas de Anclaje a la Quinasa A/genética , Proteínas de Anclaje a la Quinasa A/metabolismo , Sustitución de Aminoácidos , Ciclopentanos/farmacología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/química , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/química , Proteína Quinasa 3 Activada por Mitógenos/genética , Mutación , Dominios PDZ , Fosfatidilinositol 3-Quinasa/química , Fosfatidilinositol 3-Quinasa/genética , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Quinolinas/farmacología , Interferencia de ARN , Ensayo de Unión Radioligante , Receptores de Estrógenos/química , Receptores de Estrógenos/genética , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Gαq inhibitor UBO-QIC (FR900359) is becoming an important pharmacological tool, but its selectivity against other G proteins and their subunits, especially ßγ, has not been well characterized. We examined UBO-QIC's effect on diverse signaling pathways mediated via various G protein-coupled receptors (GPCRs) and G protein subunits by comparison with known Gαi inhibitor pertussis toxin. As expected, UBO-QIC inhibited Gαq signaling in all assay systems examined. However, other non-Gαq-events, e.g. Gßγ-mediated intracellular calcium release and inositol phosphate production, following activation of Gi-coupled A1 adenosine and M2 muscarinic acetylcholine receptors, were also blocked by low concentrations of UBO-QIC, indicating that its effect is not limited to Gαq. Thus, UBO-QIC also inhibits Gßγ-mediated signaling similarly to pertussis toxin, although UBO-QIC does not affect Gαi-mediated inhibition or Gαs-mediated stimulation of adenylyl cyclase activity. However, the blockade by UBO-QIC of GPCR signaling, such as carbachol- or adenosine-mediated calcium or inositol phosphate increases, does not always indicate inhibition of Gαq-mediated events, as the ßγ subunits released from Gi proteins following the activation of Gi-coupled receptors, e.g. M2 and A1Rs, may produce similar signaling events. Furthermore, UBO-QIC completely inhibited Akt signaling, but only partially blocked ERK1/2 activity stimulated by the Gq-coupled P2Y1R. Thus, we have revealed new aspects of the pharmacological interactions of UBO-QIC.
Asunto(s)
Depsipéptidos/farmacología , Inhibidores Enzimáticos/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/antagonistas & inhibidores , Modelos Biológicos , Inhibidores de Agregación Plaquetaria/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Animales , Células CHO , Cricetulus , 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 , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/antagonistas & inhibidores , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/química , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Subunidades beta de la Proteína de Unión al GTP/agonistas , Subunidades beta de la Proteína de Unión al GTP/antagonistas & inhibidores , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/agonistas , Subunidades gamma de la Proteína de Unión al GTP/antagonistas & inhibidores , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Toxina del Pertussis/farmacología , Proteínas Proto-Oncogénicas c-akt/agonistas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor de Adenosina A1/química , Receptor de Adenosina A1/genética , Receptor de Adenosina A1/metabolismo , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/antagonistas & inhibidores , Receptor Muscarínico M2/genética , Receptor Muscarínico M2/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
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
Short lipidated peptide sequences derived from various intracellular loop regions of G protein-coupled receptors (GPCRs) are named pepducins and act as allosteric modulators of a number of GPCRs. Recently, a pepducin selectively targeting the C-X-C chemokine receptor type 4 (CXCR4) was found to be an allosteric agonist, active in both cell-based assays and in vivo. However, the precise mechanism of action of this class of ligands remains poorly understood. In particular, given the diversity of signaling effectors that can be engaged by a given receptor, it is not clear whether pepducins can show biased signaling leading to functional selectivity. To explore the ligand-biased potential of pepducins, we assessed the effect of the CXCR4 selective pepducin, ATI-2341, on the ability of the receptor to engage the inhibitory G proteins (Gi1, Gi2 and Gi3), G13, and ß-arrestins. Using bioluminescence resonance energy transfer-based biosensors, we found that, in contrast to the natural CXCR4 ligand, stromal cell-derived factor-1α, which promotes the engagement of the three Gi subtypes, G13 and the two ß-arrestins, ATI-2341 leads to the engagement of the Gi subtypes but not G13 or the ß-arrestins. Calculation of the transduction ratio for each pathway revealed a strong negative bias of ATI-2341 toward G13 and ß-arrestins, revealing functional selectivity for the Gi pathways. The negative bias toward ß-arrestins results from the reduced ability of the pepducin to promote GPCR kinase-mediated phosphorylation of the receptor. In addition to revealing ligand-biased signaling of pepducins, these findings shed some light on the mechanism of action of a unique class of allosteric regulators.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/agonistas , Células Madre Hematopoyéticas/metabolismo , Lipopéptidos/metabolismo , Receptores CXCR4/metabolismo , Transferencia de Energía por Resonancia de Bioluminiscencia , Western Blotting , Citometría de Flujo , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Células HEK293 , HumanosRESUMEN
Classically, opioids produce their effects by activating Gi-proteins that inhibit adenylate cyclase activity. Previous studies proposed that mu-opioid receptors can also stimulate adenylate cyclase due to an initial transient coupling to Gs-proteins. Treatment with ultra-low doses of the nonselective opioid antagonist (-)-naloxone or its inactive enantiomer (+)-naloxone blocks this excitatory effect and enhances Gi-coupling. Previously we reported that infusion of the mu-opioid receptor agonist [D-Ala2, N-Me-Phe4, Glycinol5]-Enkephalin (DAMGO) into the mu-opioid receptor expressing lateral parabrachial nucleus increases feeding. Pretreatment with (-)-naloxone blocks this effect. We used this parabrachial circuit as a model to assess cellular actions of ultra-low doses of (-)-naloxone and (+)-naloxone in modifying the effects of DAMGO. Our results showed that an ultra-low concentration of (-)-naloxone (0.001 nM) and several concentrations of (+)-naloxone (0.01-10 nM) enhanced DAMGO-stimulated guanosine-5'-0-(γ-thio)-triphosphate incorporation in parabrachial sections in vitro. Further, we analyzed the relevance of these effects in vivo. In the present study, we show that (+)-naloxone can potentiate DAMGO-induced feeding at doses at which (-)-naloxone was an antagonist. These results implicated (+)-naloxone as a novel tool for studying mu-opioid receptor functions and suggest that (+)-naloxone may have therapeutic value to enhance clinical actions of opiate drugs.
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Subunidades alfa de la Proteína de Unión al GTP Gi-Go/agonistas , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Neuronas/efectos de los fármacos , Puente/efectos de los fármacos , Receptores Opioides mu/agonistas , Transducción de Señal/efectos de los fármacos , Analgésicos Opioides/agonistas , Analgésicos Opioides/antagonistas & inhibidores , Analgésicos Opioides/farmacología , Animales , Conducta Animal/efectos de los fármacos , Sinergismo Farmacológico , Ingestión de Alimentos/efectos de los fármacos , Encefalina Ala(2)-MeFe(4)-Gli(5)/agonistas , Encefalina Ala(2)-MeFe(4)-Gli(5)/antagonistas & inhibidores , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Técnicas In Vitro , Masculino , Proteínas del Tejido Nervioso/agonistas , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Concentración Osmolar , Puente/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Opioides mu/metabolismo , EstereoisomerismoRESUMEN
BACKGROUND: Takotsubo cardiomyopathy is an acute heart failure syndrome characterized by myocardial hypocontractility from the mid left ventricle to the apex. It is precipitated by extreme stress and can be triggered by intravenous catecholamine administration, particularly epinephrine. Despite its grave presentation, Takotsubo cardiomyopathy is rapidly reversible, with generally good prognosis. We hypothesized that this represents switching of epinephrine signaling through the pleiotropic ß(2)-adrenergic receptor (ß(2)AR) from canonical stimulatory G-protein-activated cardiostimulant to inhibitory G-protein-activated cardiodepressant pathways. METHODS AND RESULTS: We describe an in vivo rat model in which a high intravenous epinephrine, but not norepinephrine, bolus produces the characteristic reversible apical depression of myocardial contraction coupled with basal hypercontractility. The effect is prevented via G(i) inactivation by pertussis toxin pretreatment. ß(2)AR number and functional responses were greater in isolated apical cardiomyocytes than in basal cardiomyocytes, which confirmed the higher apical sensitivity and response to circulating epinephrine. In vitro studies demonstrated high-dose epinephrine can induce direct cardiomyocyte cardiodepression and cardioprotection in a ß(2)AR-Gi-dependent manner. Preventing epinephrine-G(i) effects increased mortality in the Takotsubo model, whereas ß-blockers that activate ß(2)AR-G(i) exacerbated the epinephrine-dependent negative inotropic effects without further deaths. In contrast, levosimendan rescued the acute cardiac dysfunction without increased mortality. CONCLUSIONS: We suggest that biased agonism of epinephrine for ß(2)AR-G(s) at low concentrations and for G(i) at high concentrations underpins the acute apical cardiodepression observed in Takotsubo cardiomyopathy, with an apical-basal gradient in ß(2)ARs explaining the differential regional responses. We suggest this epinephrine-specific ß(2)AR-G(i) signaling may have evolved as a cardioprotective strategy to limit catecholamine-induced myocardial toxicity during acute stress.
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Modelos Animales de Enfermedad , Epinefrina/sangre , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Receptores Adrenérgicos beta 2/fisiología , Cardiomiopatía de Takotsubo/sangre , Animales , Antiarrítmicos/administración & dosificación , Antiarrítmicos/sangre , Células Cultivadas , Epinefrina/administración & dosificación , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/agonistas , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/sangre , Humanos , Masculino , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Flow cytometry enables comparative quantification, population analysis, and high-throughput screening of agonist-mediated G-protein-coupled receptor (GPCR) signaling in genetically engineered yeasts. By using flow cytometry, we found that transformation of yeast cells with a low plasmid number is critical both for the construction of large screening libraries and for stable signal transmission in cell ensembles. Based on these findings, we constructed an engineered yeast strain for the improved identification of signal promotion by Gα(i)-specific human GPCRs using flow cytometry.
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Inhibidores Enzimáticos/análisis , Citometría de Flujo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Saccharomyces cerevisiae/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Humanos , Plásmidos/metabolismo , Receptores de Somatostatina/genética , Receptores de Somatostatina/metabolismo , Transducción de SeñalRESUMEN
ER-to-Golgi protein transport involves transport vesicles of which formation is initiated by assembly of Sar1. The assembly of Sar1 is suppressed by protein kinase inhibitor H89, suggesting that ER-to-Golgi transport is regulated progressively by H89 sensitive kinase. ER-resident G(i2) protein suppresses vesicle formation with inhibition of Sar1 assembly. This study examined whether these promotion and suppression of vesicle transport share the same signal pathway, by examining the effects of G(i/o) protein activator mastoparan 7 (Mp-7) and H89 on Sar1 and Sec23 recruitment onto microsomes. In a cell-free system for Sar1 translocation assay, GTPγS addition induced the translocation of Sar1 onto microsomes. Mp-7 and H89 decreased the Sar1 translocation. Double treatment of Mp-7 and H89 strongly decreased Sar1 translocation. In single and double treatments, however, G(i/o) protein inactivator pertussis toxin (IAP) partially restored the suppressive effect of Mp-7, but had not any effect on H89-induced effect. Then, the assembly of Sec23 onto the microsome was also increased by the addition of GTPγS. Sec23 translocation was decreased by Mp-7 and/or H89 treatment and recovered by IAP pretreatment except for H89 single treatment, similarly to Sar1 translocation in each treatment. Inhibitory effects of H89 and Mp-7on ER-to-Golgi vesicle transport by H89 or Mp-7 were also confirmed in a cell culture system by BFA-dispersion and BFA-reconstruction experiments. These findings indicate that promotion and suppression of ER-to-Golgi vesicle transport are modulated through separate signal pathways.
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Vesículas Citoplasmáticas/metabolismo , Retículo Endoplásmico/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Aparato de Golgi/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas Quinasas/metabolismo , Animales , Brefeldino A/farmacología , Sistema Libre de Células , Células Cultivadas , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/agonistas , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/antagonistas & inhibidores , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Péptidos y Proteínas de Señalización Intercelular , Membranas Intracelulares/metabolismo , Isoquinolinas/farmacología , Hígado/citología , Masculino , Microsomas/metabolismo , Péptidos/farmacología , Toxina del Pertussis/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Transporte de Proteínas , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Sulfonamidas/farmacología , Proteínas de Transporte Vesicular/metabolismoRESUMEN
The chemokine receptor CXCR3 is a G-protein-coupled receptor that signals through the Gα(i) class of heterotrimeric G-proteins. CXCR3 is highly expressed on activated T cells and has been proposed to be a therapeutic target in autoimmune disease. CXCR3 is activated by the chemokines CXCL9, CXCL10 and CXCL11. CXCR3 signaling properties in response to CXCL10, CXCL11 and the synthetic agonist VUF10661 have previously been evaluated using conventional endpoint assays. In the present study, label-free impedance measurements were used to characterize holistic responses of CXCR3-expressing cells to stimulation with chemokines and VUF10661 in real time and to compare these responses with both G-protein and non-G-protein (ß-arrestin2) mediated responses. Differences in response kinetics were apparent between the chemokines and VUF10661. Moreover, CXCR3-independent effects could be distinguished from CXCR3-specific responses with the use of the selective CXCR3 antagonist NBI-74330 and the Gα(i) inhibitor pertussis toxin. By comparing the various responses, we observed that CXCL9 is a biased CXCR3 agonist, stimulating solely G-protein-dependent pathways. Moreover, CXCR3-mediated changes in cellular impedance correlated with G-protein signaling, but not ß-arrestin2 recruitment.
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Impedancia Eléctrica , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/agonistas , Isoquinolinas/farmacología , Receptores CXCR3/agonistas , Acetamidas/farmacología , Quimiocina CXCL10/farmacología , Quimiocina CXCL11/farmacología , Quimiocina CXCL9/farmacología , Células HEK293 , Humanos , Ligandos , Redes y Vías Metabólicas , Toxina del Pertussis/farmacología , Pirimidinas/farmacología , Receptores CXCR3/antagonistas & inhibidoresRESUMEN
Pharmacological characterization of adenosine A(1) and A(2A) receptors in human brain caudate nucleus membranes led to non-cooperative binding of radiolabelled ligands. In human caudate nucleus but not in cortex, the agonist binding to A(1) receptors was modulated by the agonist binding to A(2A) receptors indicating a functional negative cross-talk. Accordingly, the A(1) receptor-activation-mediated G(i)-dependent guanosine 5'-o-(3-[(35)S]thio-triphosphate) binding was modulated by agonist binding to A(2A) receptors. A(2A) receptors occupation led to a decrease in the potency of A(1) receptor agonists. These results indicate that A(1) but not A(2A) receptors activation, likely occurring at low adenosine concentrations, engages a G(i)-mediated signaling; however, when both receptors are occupied by adenosine, there is an A(2A) receptor-mediated impairment of G(i)-operated transducing units. These findings are relevant to get insight into the complex relationships derived from co-expression of multiple neurotransmitter/neuromodulator receptors subtypes that individually are coupled to different G proteins. A further finding was the demonstration that the A(2A) receptor agonist, CGS 21680, at high concentrations able to significantly bind to the A(1) receptor, behaved as a partial agonist of the later receptor. This fact might be taken into account when characterizing CGS 21680 actions in human cells expressing A(1) receptors when the compound is used at micromolar concentrations.
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Núcleo Caudado/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Complejos Multiproteicos/metabolismo , Receptor Cross-Talk/fisiología , Receptor de Adenosina A1/metabolismo , Receptor de Adenosina A2A/metabolismo , Agonistas del Receptor de Adenosina A1 , Agonistas del Receptor de Adenosina A2 , Unión Competitiva/fisiología , Núcleo Caudado/efectos de los fármacos , Membrana Celular/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/agonistas , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Humanos , Complejos Multiproteicos/agonistas , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiologíaRESUMEN
In the present study, we evaluated the effects of the synthetic cannabinoid receptor agonist (R)-(+)-[2,3-Dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN55,212-2) and the active component of Cannabis delta-9-tetrahydrocannabinol (triangle up(9)-THC) on Na(+),K(+)-ATPase activity in synaptosomal mice brain preparation. Additionally, the potential exogenous cannabinoids and endogenous opioid peptides interaction as well as the role of G(i/o) proteins in mediating Na(+),K(+)-ATPase activation were also explored. The ouabain-sensitive Na(+),K(+)-ATPase activity was measured in whole-brain pure intact synaptosomes (obtained by Percoll gradient method) of female CF-1 mice and was calculated as the difference between the total and the ouabain (1 mM)-insensitive Na(+),K(+)-ATPase activities. Incubation in vitro of the synaptosomes with WIN55,212-2 (0.1 pM-10 microM) or triangle up(9)-THC (0.1 pM-0.1 microM), in a concentration-dependent manner, stimulated ouabain-sensitive Na(+),K(+)-ATPase activity. WIN55,212-2 was less potent but more efficacious than triangle up(9)-THC. N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM-251) (10 nM), a CB(1) cannabinoid receptor selective antagonist, had not effect per se but antagonized the enhancement of Na(+),K(+)-ATPase activity induced by both, WIN55,212-2 and triangle up(9)-THC. AM-251 produced a significant reduction in the E(max) of cannabinoid-induced increase in Na(+),K(+)-ATPase activity, but did not significantly modify their EC(50). On the other hand, co-incubation with naloxone (1 microM), an opioid receptor antagonist, did not significantly modify the effect of WIN55,212-2 and completely failed to modify the effect of triangle up(9)-THC on synaptosomal Na(+),K(+)-ATPase. Finally, pre-incubation with 0.5 microg of pertussis toxin (G(i/o) protein blocker) completely abolished the enhancement of ouabain-sensitive Na(+),K(+)-ATPase activity induced by WIN55,212-2. A lower dose, 0.25 microg, decreased the E(max) of WIN55,212-2 by 70% but did not significantly affect its EC(50). These results suggest that WIN55212-2 and triangle up(9)-THC indirectly enhance Na(+),K(+)-ATPase activity in the brain by activating cannabinoid CB(1) receptors in a naloxone-insensitive manner. In addition, the effect of WIN55,212-2 on neuronal Na(+),K(+)-ATPase is apparently due to activation of G(i/o) proteins.
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Benzoxazinas/farmacología , Encéfalo/efectos de los fármacos , Dronabinol/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/agonistas , Morfolinas/farmacología , Naftalenos/farmacología , Receptor Cannabinoide CB1/fisiología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Encéfalo/enzimología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Inhibidores Enzimáticos/farmacología , Femenino , Técnicas In Vitro , Ratones , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Ouabaína/farmacología , Toxina del Pertussis/administración & dosificación , Toxina del Pertussis/farmacología , Piperidinas/farmacología , Pirazoles/farmacología , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/antagonistas & inhibidores , Sinaptosomas/efectos de los fármacos , Sinaptosomas/enzimologíaRESUMEN
Simple photoreceptors, namely photoresponsive neurons, designated as A-P-1, Es-1, Ip-2 and Ip-1, exist in the sea slug Onchidium ganglion. Previous works has shown that, of these, Ip-2 and Ip-1 respond to light with a hyperpolarizing receptor potential, caused by the opening of light-dependent, cGMP-gated K+ channels, whereas A-P-1 and Es-1 are depolarized by light, owing to the closing of the same K+ channels. The present study of Ip-2 or Ip-1 cells was undertaken to identify the G-proteins that couple light to the activation of guanylate cyclase (GC), thereby leading to the opening of K+ channels and the consequent hyperpolarizing photocurrents. The specific channel blocker, 4-aminopyridine (4-AP), and a GC inhibitor, LY-83583, both suppressed this hyperpolarizing photocurrent. N-ethylmaleimide and GDP-beta-S also inhibited this photocurrent, consistent with the involvement of G-proteins. Mastoparan an activator of both Go- and Gi-type G-proteins, induced an outward current. Furthermore, benzalkonium chloride (C(16)BAC), a selective activator of Go, dose-dependently generated an outward current similar to that induced by mastoparan. Both of these outward currents were susceptible to 4-AP, LY-83583 and N-ethylmaleimide. Taken together, these results suggest that phototransduction in Ip-2 or Ip-1 cells is triggered by a Go-type G-protein coupled to GC. Thus, this new cGMP cascade contrasts with the conventional phototransduction cGMP cascade mediated by the Gt-type G-protein coupled to phosphodiesterase, seen in the vertebrate photoreceptors and the above A-P-1 or Es-1 cells.
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GMP Cíclico/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Guanilato Ciclasa/metabolismo , Células Fotorreceptoras/fisiología , Visión Ocular/fisiología , Animales , Interacciones Farmacológicas , Inhibidores Enzimáticos/farmacología , Etilmaleimida/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/agonistas , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/antagonistas & inhibidores , Ganglios de Invertebrados/citología , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intercelular , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Moluscos , Técnicas de Placa-Clamp , Péptidos/farmacología , Estimulación Luminosa/métodos , Visión Ocular/efectos de los fármacos , Venenos de Avispas/farmacologíaRESUMEN
The concept of pharmacological efficacy has been much discussed recently with significant interest both in inverse agonists and in protean agonists (i.e., compounds with functional selectivity for different effector responses). Although first proposed in the mid-1990s, the pharmacological and therapeutic importance of these concepts is now receiving wider support. Two articles in recent issues of Molecular Pharmacology, Lane et al. (p. 1349, current issue) and Galandrin and Bouvier (Mol Pharmacol 70:1575-1584, 2006), provide new mechanistic information on functionally selective ligands at the pharmacologically important D2 dopamine receptor and the beta(1) and beta(2) adrenergic receptors. Each article bridges a gap between recent biophysical studies showing distinct receptor conformations produced by different ligands and the increasing number of reports of discordant outputs by a single ligand to two effector readouts. The Lane et al. study clearly demonstrates G protein-specific actions of D(2) dopamine receptor ligands. These range from equivalent responses for Galpha(o) and Galpha(i) activation by norapomorphine and 7-hydroxy-2-dipropylaminotetralin to S-(-)-3-(3-hydroxyphenyl)-N-propylpiperidine, which is an agonist for Galpha(o) activation and an inverse agonist at Galpha(i1) and Galpha(i2). Likewise, Galandrin and Bouvier describe a two-dimensional Cartesian efficacy approach in which propranolol is an agonist for extracellular signal-regulated kinase activation, probably through beta-arrestin, while functioning as an inverse agonist for adenylyl cyclase activation. Thus, these two important articles further solidify the concepts of functional selectivity and protean agonism and begin to define the first postreceptor step in actions of protean agonist ligands.
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Receptores de Dopamina D2/agonistas , Animales , Subunidad alfa de la Proteína de Unión al GTP Gi2/antagonistas & inhibidores , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/agonistas , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/antagonistas & inhibidores , Humanos , Piperidinas/farmacología , RatasRESUMEN
G protein-coupled receptors transmit extracellular signals into the cells by activating heterotrimeric G proteins, a process that is often followed by receptor desensitization. Monitoring such a process in real time and in living cells will help better understand how G protein activation occurs. Energy transfer-based approaches [fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET)] were recently shown to be powerful methods to monitor the G protein-coupled receptors (GPCRs)-G protein association in living cells. Here, we used a BRET technique to monitor the coupling between the protease-activated receptor 1 (PAR1) and Galpha(i1) protein. A specific constitutive BRET signal can be measured between nonactivated PAR1 and the Galpha(i1) protein expressed at a physiological level. This signal is insensitive to pertussis toxin (PTX) and probably reflects the preassembly of these two proteins. The BRET signal rapidly increases upon receptor activation in a PTX-sensitive manner. The BRET signal then returns to the basal level after few minutes. The desensitization of the BRET signal is concomitant with beta-arrestin-1 recruitment to the receptor, consistent with the known rapid desensitization of PARs. The agonist-induced BRET increase was dependent on the insertion site of fluorophores in proteins. Taken together, our results show that BRET between GPCRs and Galpha proteins can be used to monitor the receptor activation in real time and in living cells. Our data also revealed that PAR1 can be part of a preassembled complex with Galpha(i1) protein, resulting either from a direct interaction between these partners or from their colocalization in specific microdomains, and that receptor activation probably results in rearrangements within such complexes.