Assuntos
Proteínas de Ligação ao GTP , Guanosina Trifosfato , Transdução de Sinais , Transdução de Sinais/genética , Humanos , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/genética , Ligação Proteica , AnimaisRESUMO
G protein-coupled receptors (GPCRs) are mainly regulated by GPCR kinase (GRK) phosphorylation and subsequent ß-arrestin recruitment. The ubiquitously expressed GRKs are classified into cytosolic GRK2/3 and membrane-tethered GRK5/6 subfamilies. GRK2/3 interact with activated G protein ßγ-subunits to translocate to the membrane. Yet, this need was not linked as a factor for bias, influencing the effectiveness of ß-arrestin-biased agonist creation. Using multiple approaches such as GRK2/3 mutants unable to interact with Gßγ, membrane-tethered GRKs and G protein inhibitors in GRK2/3/5/6 knockout cells, we show that G protein activation will precede GRK2/3-mediated ß-arrestin2 recruitment to activated receptors. This was independent of the source of free Gßγ and observable for Gs-, Gi- and Gq-coupled GPCRs. Thus, ß-arrestin interaction for GRK2/3-regulated receptors is inseparably connected with G protein activation. We outline a theoretical framework of how GRK dependence on free Gßγ can determine a GPCR's potential for biased agonism. Due to this inherent cellular mechanism for GRK2/3 recruitment and receptor phosphorylation, we anticipate generation of ß-arrestin-biased ligands to be mechanistically challenging for the subgroup of GPCRs exclusively regulated by GRK2/3, but achievable for GRK5/6-regulated receptors, that do not demand liberated Gßγ. Accordingly, GRK specificity of any GPCR is foundational for developing arrestin-biased ligands.
Assuntos
Quinases de Receptores Acoplados a Proteína G , Subunidades beta da Proteína de Ligação ao GTP , Subunidades gama da Proteína de Ligação ao GTP , Humanos , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/genética , Células HEK293 , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/genética , Quinases de Receptores Acoplados a Proteína G/metabolismo , Quinases de Receptores Acoplados a Proteína G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Fosforilação , Animais , Transdução de SinaisRESUMO
Pulmonary arterial hypertension (PAH) is a life-threatening disease with limited survival. Herein, we propose the pharmacological inhibition of Gq proteins as a novel concept to counteract pulmonary vasoconstriction and proliferation/migration of pulmonary artery smooth muscle cells (PASMCs) in PAH. We demonstrate that the specific pan-Gq inhibitor FR900359 (FR) induced a strong vasorelaxation in large and small pulmonary arteries in mouse, pig, and human subjects ex vivo. Vasorelaxation by FR proved at least as potent as the currently used triple therapy. We also provide in vivo evidence that local pulmonary application of FR prevented right ventricular systolic pressure increase in healthy mice as well as in mice suffering from hypoxia (Hx)-induced pulmonary hypertension (PH). In addition, we demonstrate that chronic application of FR prevented and also reversed Sugen (Su)Hx-induced PH in mice. We also demonstrate that Gq inhibition reduces proliferation and migration of PASMCs in vitro. Thus, our work illustrates a dominant role of Gq proteins for pulmonary vasoconstriction as well as remodeling and proposes direct Gq inhibition as a powerful pharmacological strategy in PH.
Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Hipertensão Pulmonar , Artéria Pulmonar , Animais , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/antagonistas & inibidores , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/fisiopatologia , Humanos , Camundongos , Artéria Pulmonar/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Suínos , Vasodilatação/efeitos dos fármacos , Masculino , Proliferação de Células/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Camundongos Endogâmicos C57BL , DepsipeptídeosRESUMO
A conserved intracellular allosteric binding site (IABS) was recently identified at several G protein-coupled receptors (GPCRs). This target site allows the binding of allosteric modulators and enables a new mode of GPCR inhibition. Herein, we report the development of a NanoBRET-based assay platform based on the fluorescent ligand LT221 (5), to detect intracellular binding to CCR6 and CXCR1, two chemokine receptors that have been pursued as promising drug targets in inflammation and immuno-oncology. Our assay platform enables cell-free as well as cellular NanoBRET-based binding studies in a nonisotopic and straightforward manner. By combining this screening platform with a previously reported CXCR2 assay, we investigated CXCR1/CXCR2/CCR6 selectivity profiles for both known and novel squaramide analogues derived from navarixin, a known intracellular CXCR1/CXCR2 antagonist and phaseâ II clinical candidate for the treatment of pulmonary diseases. By means of these studies we identified compound 10, a previously reported tert-butyl analogue of navarixin, as a low nanomolar intracellular CCR6 antagonist. Further, our assay platform clearly indicated intracellular binding of the CCR6 antagonist PF-07054894, currently evaluated in phaseâ I clinical trials for the treatment of ulcerative colitis, thereby providing profound evidence for the existence and the pharmacological relevance of a druggable IABS at CCR6.
RESUMO
The marine sponge-derived fungus Stachylidium bicolor 293 K04 is a prolific producer of specialized metabolites, including certain cyclic tetrapeptides called endolides, which are characterized by the presence of the unusual amino acid N-methyl-3-(3-furyl)-alanine. This rare feature can be used as bait to detect new endolide-like analogs through customized fragment pattern searches of tandem mass spectrometry data using the Mass Spec Query Language (MassQL). Here, we integrate endolide-specific MassQL queries with molecular networking to obtain substructural information guiding the targeted isolation and structure elucidation of the new proline-containing endolides E (1) and F (2). We showed that endolide F (but not E) is a moderate antagonist of the arginine vasopressin V1A receptor, a member of the G protein-coupled receptor superfamily.
Assuntos
Peptídeos Cíclicos , Poríferos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Estrutura Molecular , Animais , Poríferos/química , Espectrometria de Massas em Tandem , Biologia MarinhaRESUMO
Recent pan-cancer genomic analyses have identified numerous oncogenic driver mutations that occur in a cell-type and tissue-specific distribution. For example, oncogenic mutations in Braf and Nras genes arise predominantly in melanocytic neoplasms of the epidermis, while oncogenic mutations in Gnaq/11 genes arise mostly in melanocytic lesions of the dermis or the uvea. The mechanisms promoting cell-type and tissue-specific oncogenic events currently remain poorly understood. Here, we report that Gnaq/11 hotspot mutations occur as early oncogenic drivers during the evolution of primary melanomas in Hgf-Cdk4 mice. Additional single base substitutions in the Trp53 gene and structural chromosomal aberrations favoring amplifications of the chromosomal region containing the Met receptor gene accumulate during serial tumor transplantation and in cell lines established in vitro. Mechanistically, we found that the GnaqQ209L mutation transactivates the Met receptor. Overexpression of oncogenic GnaqQ209L in the immortalized melanocyte cell line promoted in vivo growth that was enhanced by transgenic Hgf expression in the tumor microenvironment. This cross-signaling mechanism explains the selection of oncogenic Gnaq/11 in primary Hgf-Cdk4 melanomas and provides an example of how oncogenic driver mutations, intracellular signaling cascades, and microenvironmental cues cooperate to drive cancer development in a tissue-specific fashion.
Assuntos
Quinase 4 Dependente de Ciclina , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Fator de Crescimento de Hepatócito , Melanoma , Proteínas Proto-Oncogênicas c-met , Transdução de Sinais , Animais , Camundongos , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Ativação Transcricional , Mutação , Linhagem Celular Tumoral , Camundongos Transgênicos , Microambiente Tumoral/genéticaRESUMO
Melanocortin 4 receptor (MC4R) mutations are the most common cause of human monogenic obesity and are associated with hyperphagia and increased linear growth. While MC4R is known to activate Gsα/cAMP signaling, a substantial proportion of obesity-associated MC4R mutations do not affect MC4R/Gsα signaling. To further explore the role of specific MC4R signaling pathways in the regulation of energy balance, we examined the signaling properties of one such mutant, MC4R (F51L), as well as the metabolic consequences of MC4RF51L mutation in mice. The MC4RF51L mutation produced a specific defect in MC4R/Gq/11α signaling and led to obesity, hyperphagia, and increased linear growth in mice. The ability of a melanocortin agonist to acutely inhibit food intake when delivered to the paraventricular nucleus (PVN) was lost in MC4RF51L mice, as well as in WT mice in which a specific Gq/11α inhibitor was delivered to the PVN; this provided evidence that a Gsα-independent signaling pathway, namely Gq/11α, significantly contributes to the actions of MC4R on food intake and linear growth. These results suggest that a biased MC4R agonist that primarily activates Gq/11α may be a potential agent to treat obesity with limited untoward cardiovascular and other side effects.
Assuntos
Hiperfagia , Receptor Tipo 4 de Melanocortina , Humanos , Camundongos , Animais , Receptor Tipo 4 de Melanocortina/metabolismo , Hiperfagia/genética , Hiperfagia/metabolismo , Obesidade/metabolismo , Transdução de Sinais/fisiologia , MutaçãoRESUMO
Herein, we report the structure-based development of fluorescent ligands targeting the intracellular allosteric binding site (IABS) of CXC chemokine receptor 2 (CXCR2), a G protein-coupled receptor (GPCR) that has been pursued as a drug target in oncology and inflammation. Starting from the cocrystallized intracellular CXCR2 antagonist 00767013 (1), tetramethylrhodamine (TAMRA)-labeled CXCR2 ligands were designed, synthesized, and tested for their suitability as fluorescent reporters to probe binding to the IABS of CXCR2. By means of these studies, we developed Mz438 (9a) as a high-affinity and selective fluorescent CXCR2 ligand, enabling cell-free as well as cellular NanoBRET-based binding studies in a nonisotopic and high-throughput manner. Further, we show that 9a can be used as a tool to visualize intracellular target engagement for CXCR2 via fluorescence microscopy. Thus, our small-molecule-based fluorescent CXCR2 ligand 9a represents a promising tool for future studies of CXCR2 pharmacology.
Assuntos
Receptores Acoplados a Proteínas G , Receptores de Interleucina-8B , Sítio Alostérico , Ligantes , Ligação Proteica , Receptores Acoplados a Proteínas G/metabolismoRESUMO
The cyclic depsipeptide FR900359 (FR) is derived from the soil bacterium Chromobacterium vaccinii and known to bind Gq proteins of mammals and insects, thereby abolishing the signal transduction of their Gq protein-coupled receptors, a process that leads to severe physiological consequences. Due to their highly conserved structure, Gq family of proteins are a superior ecological target for FR producing organisms, resulting in a defense towards a broad range of harmful organisms. Here, we focus on the question whether bacteria like C. vaccinii are important factors in soil in that their secondary metabolites impair, e.g., plant harming organisms like nematodes. We prove that the Gq inhibitor FR is produced under soil-like conditions. Furthermore, FR inhibits heterologously expressed Gαq proteins of the nematodes Caenorhabditis elegans and Heterodera schachtii in the micromolar range. Additionally, in vivo experiments with C. elegans and the plant parasitic cyst nematode H. schachtii demonstrated that FR reduces locomotion of C. elegans and H. schachtii. Finally, egg-laying of C. elegans and hatching of juvenile stage 2 of H. schachtii from its cysts is inhibited by FR, suggesting that FR might reduce nematode dispersion and proliferation. This study supports the idea that C. vaccinii and its excreted metabolome in the soil might contribute to an ecological equilibrium, maintaining and establishing the successful growth of plants.
Assuntos
Depsipeptídeos , Nematoides , Animais , Solo , Caenorhabditis elegans , Depsipeptídeos/farmacologia , Bactérias , Transdução de Sinais , MamíferosRESUMO
AIMS/HYPOTHESIS: After birth, the neonatal islets gradually acquire glucose-responsive insulin secretion, a process that is subjected to maternal imprinting. Although NEFA are major components of breastmilk and insulin secretagogues, their role for functional maturation of neonatal beta cells is still unclear. NEFA are the endogenous ligands of fatty acid receptor 1 (FFA1, encoded by Ffar1 in mice), a Gq-coupled receptor with stimulatory effect on insulin secretion. This study investigates the role of FFA1 in neonatal beta cell function and in the adaptation of offspring beta cells to parental high-fat feeding. METHODS: Wild-type (WT) and Ffar1-/- mice were fed high-fat (HFD) or chow diet (CD) for 8 weeks before mating, and during gestation and lactation. Blood variables, pancreas weight and insulin content were assessed in 1-, 6-, 11- and 26-day old (P1-P26) offspring. Beta cell mass and proliferation were determined in P1-P26 pancreatic tissue sections. FFA1/Gq dependence of insulin secretion was evaluated in isolated islets and INS-1E cells using pharmacological inhibitors and siRNA strategy. Transcriptome analysis was conducted in isolated islets. RESULTS: Blood glucose levels were higher in CD-fed Ffar1-/- P6-offspring compared with CD-fed WT P6-offspring. Accordingly, glucose-stimulated insulin secretion (GSIS) and its potentiation by palmitate were impaired in CD Ffar1-/- P6-islets. In CD WT P6-islets, insulin secretion was stimulated four- to fivefold by glucose and five- and sixfold over GSIS by palmitate and exendin-4, respectively. Although parental HFD increased blood glucose in WT P6-offspring, it did not change insulin secretion from WT P6-islets. In contrast, parental HFD abolished glucose responsiveness (i.e. GSIS) in Ffar1-/- P6-islets. Inhibition of Gq by FR900359 or YM-254890 in WT P6-islets mimicked the effect of Ffar1 deletion, i.e. suppression of GSIS and of palmitate-augmented GSIS. The blockage of Gi/o by pertussis toxin (PTX) enhanced (100-fold) GSIS in WT P6-islets and rendered Ffar1-/- P6-islets glucose responsive, suggesting constitutive activation of Gi/o. In WT P6-islets, FR900359 cancelled 90% of PTX-mediated stimulation, while in Ffar1-/- P6-islets it completely abolished PTX-elevated GSIS. The secretory defect of Ffar1-/- P6-islets did not originate from insufficient beta cells, since beta cell mass increased with the offspring's age irrespective of genotype and diet. In spite of that, in the breastfed offspring (i.e. P1-P11) beta cell proliferation and pancreatic insulin content had a genotype- and diet-driven dynamic. Under CD, the highest proliferation rate was reached by the Ffar1-/- P6 offspring (3.95% vs 1.88% in WT P6), whose islets also showed increased mRNA levels of genes (e.g. Fos, Egr1, Jun) typically high in immature beta cells. Although parental HFD increased beta cell proliferation in both WT (4.48%) and Ffar1-/- (5.19%) P11 offspring, only the WT offspring significantly increased their pancreatic insulin content upon parental HFD (5.18 µg under CD to 16.93 µg under HFD). CONCLUSIONS/INTERPRETATION: FFA1 promotes glucose-responsive insulin secretion and functional maturation of newborn islets and is required for adaptive offspring insulin secretion in the face of metabolic challenge, such as parental HFD.
Assuntos
Células Secretoras de Insulina , Ilhotas Pancreáticas , Feminino , Camundongos , Animais , Glucose/farmacologia , Glucose/metabolismo , Secreção de Insulina , Glicemia/metabolismo , Animais Recém-Nascidos , Ilhotas Pancreáticas/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Palmitatos/metabolismoRESUMO
BACKGROUND: Purinergic P2Y1 and P2Y12 receptors (P2Y1-R and P2Y12-R) are G protein-coupled receptors (GPCR) activated by adenosine diphosphate (ADP) to mediate platelet activation, thereby playing a pivotal role in hemostasis and thrombosis. While P2Y12-R is the major target of antiplatelet drugs, no P2Y1-R antagonist has yet been developed for clinical use. However, accumulating data suggest that P2Y1-R inhibition would ensure efficient platelet inhibition with minimal effects on bleeding. In this context, an accurate characterization of P2Y1-R antagonists constitutes an important preliminary step. RESULTS: Here, we investigated the pharmacology of P2Y1-R signaling through Gq and ß-arrestin pathways in HEK293T cells and in mouse and human platelets using highly sensitive resonance energy transfer-based technologies (BRET/HTRF). We demonstrated that at basal state, in the absence of agonist ligand, P2Y1-R activates Gq protein signaling in HEK293T cells and in mouse and human platelets, indicating that P2Y1-R is constitutively active in physiological conditions. We showed that P2Y1-R also promotes constitutive recruitment of ß-arrestin 2 in HEK293T cells. Moreover, the P2Y1-R antagonists MRS2179, MRS2279 and MRS2500 abolished the receptor dependent-constitutive activation, thus behaving as inverse agonists. CONCLUSIONS: This study sheds new light on P2Y1-R pharmacology, highlighting for the first time the existence of a constitutively active P2Y1-R population in human platelets. Given the recent interest of P2Y12-R constitutive activity in patients with diabetes, this study suggests that modification of constitutive P2Y1-R signaling might be involved in pathological conditions, including bleeding syndrome or high susceptibility to thrombotic risk. Thus, targeting platelet P2Y1-R constitutive activation might be a promising and powerful strategy for future antiplatelet therapy.
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Agonismo Inverso de Drogas , Proteínas de Ligação ao GTP , Receptores Purinérgicos P2Y1 , Transdução de Sinais , beta-Arrestina 2 , Animais , Humanos , Camundongos , beta-Arrestina 2/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Receptores Purinérgicos P2Y1/metabolismo , PlaquetasRESUMO
Endosomes, long considered as sorting stations for downregulation and recycling of cell surface G protein-coupled receptors (GPCRs), are now well-established sites of signal transduction. Recent work from the groups of Jin Zhang and Roger Sunahara features endosomes as signaling hubs and physical platforms for noncanonical activation of ERK by GPCRs.
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Endossomos , Sistema de Sinalização das MAP Quinases , Receptores Acoplados a Proteínas G , Humanos , Membrana Celular/metabolismo , Endossomos/metabolismo , Transporte Proteico/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Carvedilol is among the most effective ß-blockers for improving survival after myocardial infarction. Yet the mechanisms by which carvedilol achieves this superior clinical profile are still unclear. Beyond blockade of ß1-adrenoceptors, arrestin-biased signalling via ß2-adrenoceptors is a molecular mechanism proposed to explain the survival benefits. Here, we offer an alternative mechanism to rationalize carvedilol's cellular signalling. Using primary and immortalized cells genome-edited by CRISPR/Cas9 to lack either G proteins or arrestins; and combining biological, biochemical, and signalling assays with molecular dynamics simulations, we demonstrate that G proteins drive all detectable carvedilol signalling through ß2ARs. Because a clear understanding of how drugs act is imperative to data interpretation in basic and clinical research, to the stratification of clinical trials or to the monitoring of drug effects on the target pathway, the mechanistic insight gained here provides a foundation for the rational development of signalling prototypes that target the ß-adrenoceptor system.
Assuntos
Antagonistas Adrenérgicos beta , Infarto do Miocárdio , Humanos , Carvedilol/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Receptores Adrenérgicos beta 2/genética , Infarto do Miocárdio/tratamento farmacológicoRESUMO
Oxytocin (OXT) and OXT receptor (OXTR)-mediated signaling control excitability, firing patterns, and plasticity of hippocampal CA2 pyramidal neurons, which are pivotal in generation of brain oscillations and social memory. Nonetheless, the ionic mechanisms underlying OXTR-induced effects in CA2 neurons are not fully understood. Using slice physiology in a reporter mouse line and interleaved current-clamp and voltage-clamp experiments, we systematically identified the ion channels modulated by OXT signaling in CA2 pyramidal cells (PYRs) in mice of both sexes and explored how changes in channel conductance support altered electrical activity. Activation of OXTRs inhibits an outward potassium current mediated by inward rectifier potassium channels (I Kir) and thus favoring membrane depolarization. Concomitantly, OXT signaling also diminishes inward current mediated by hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels (I h), providing a hyperpolarizing drive. The combined reduction in both I Kir and I h synergistically elevate the membrane resistance and favor dendritic integration while the membrane potential is restrained from quickly depolarizing from rest. As a result, the responsiveness of CA2 PYRs to synaptic inputs is highly sharpened during OXTR activation. Unexpectedly, OXTR signaling also strongly enhances a tetrodotoxin-resistant (TTX-R), voltage-gated sodium current that helps drive the membrane potential to spike threshold and thus promote rhythmic firing. This novel array of OXTR-stimulated ionic mechanisms operates in close coordination and underpins OXT-induced burst firing, a key step in CA2 PYRs' contribution to hippocampal information processing and broader influence on brain circuitry. Our study deepens our understanding of underpinnings of OXT-promoted social memory and general neuropeptidergic control of cognitive states.SIGNIFICANCE STATEMENT Oxytocin (OXT) plays key roles in reproduction, parenting and social and emotional behavior, and deficiency in OXT receptor (OXTR) signaling may contribute to neuropsychiatric disorders. We identified a novel array of OXTR-modulated ion channels that operate in close coordination to retune hippocampal CA2 pyramidal neurons, enhancing responsiveness to synaptic inputs and sculpting output. OXTR signaling inhibits both potassium conductance (I Kir) and mixed cation conductance (I h), engaging opposing influences on membrane potential, stabilizing it while synergistically elevating membrane resistance and electrotonic spread. OXT signaling also facilitates a tetrodotoxin-resistant (TTX-R) Na+ current, not previously described in hippocampus (HP), engaged on further depolarization. This TTX-R current lowers the spike threshold and supports rhythmic depolarization and burst firing, a potent driver of downstream circuitry.
Assuntos
Ocitocina , Canais de Potássio Corretores do Fluxo de Internalização , Masculino , Feminino , Camundongos , Animais , Ocitocina/metabolismo , Tetrodotoxina , Receptores de Ocitocina/metabolismo , Células Piramidais/metabolismo , PotássioRESUMO
Therapies that promote neuroprotection and axonal survival by enhancing myelin regeneration are an unmet need to prevent disability progression in multiple sclerosis. Numerous potentially beneficial compounds have originated from phenotypic screenings but failed in clinical trials. It is apparent that current cell- and animal-based disease models are poor predictors of positive treatment options, arguing for novel experimental approaches. Here we explore the experimental power of humanized zebrafish to foster the identification of pro-remyelination compounds via specific inhibition of GPR17. Using biochemical and imaging techniques, we visualize the expression of zebrafish (zf)-gpr17 during the distinct stages of oligodendrocyte development, thereby demonstrating species-conserved expression between zebrafish and mammals. We also demonstrate species-conserved function of zf-Gpr17 using genetic loss-of-function and rescue techniques. Finally, using GPR17-humanized zebrafish, we provide proof of principle for in vivo analysis of compounds acting via targeted inhibition of human GPR17. We anticipate that GPR17-humanized zebrafish will markedly improve the search for effective pro-myelinating pharmacotherapies.
Assuntos
Oligodendroglia , Pró-Fármacos , Animais , Humanos , Peixe-Zebra/metabolismo , Pró-Fármacos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Diferenciação Celular , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Modelos Animais de Doenças , Mamíferos/metabolismoRESUMO
OBJECTIVE: To determine contributions and functions of autoantibodies (Abs) directed to the angiotensin receptor type 1 (AT1R), which are suggested to be involved in the pathogenesis of AT1R Abs-related diseases such as systemic sclerosis (SSc). METHODS: C57BL/6J mice were immunised with membrane-embedded human AT1R or empty membrane as control. Mice deficient for CD4+ or CD8+ T cells and B cells were immunised with membrane-embedded AT1R or an AT1R peptide proposed to be a dominant T cell epitope. A monoclonal (m)AT1R Ab was generated by hybridoma technique and transferred into C57BL/6J and AT1Ra/b knockout mice. The induced phenotype was examined by histology, immunohistochemistry, immunofluorescence, apoptosis assay and ELISA. In vitro, Abs responses towards AT1R were measured in cells of different origins and species. RESULTS: AT1R-immunised mice developed perivascular skin and lung inflammation, lymphocytic alveolitis, weak lung endothelial apoptosis and skin fibrosis accompanied by Smad2/3 signalling, not present in controls or mice deficient for CD4+ T and B cells. The AT1R peptide 149-172 provoked lung inflammation. Application of the mAT1R Ab induced skin and lung inflammation, not observed in AT1Ra/b knockout mice. In vitro, AT1R Abs activated rat cardiomyocytes and human monocytes, enhanced angiotensin II-mediated AT1R activation in AT1R-transfected HEK293 cells via AT1R binding and mAT1R Ab-activated monocytes mediated the induction of profibrotic markers in dermal fibroblasts. CONCLUSION: Our immunisation strategy successfully induced AT1R Abs, contributing to inflammation and, possibly, to fibrosis via activation of AT1R. Therefore, AT1R Abs are valuable targets for future therapies of SSc and other AT1R Ab-related diseases.
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Gq proteins are universally important for signal transduction in mammalian cells. The underlying kinetics and transformation from extracellular stimuli into intracellular signaling, however could not be investigated in detail so far. Here we present the human Neuropsin (hOPN5) for specific and repetitive manipulation of Gq signaling in vitro and in vivo with high spatio-temporal resolution. Properties and G protein specificity of hOPN5 are characterized by UV light induced IP3 generation, Ca2+ transients and inhibition of GIRK channel activity in HEK cells. In adult hearts from a transgenic animal model, light increases the spontaneous beating rate. In addition, we demonstrate light induced contractions in the small intestine, which are not detectable after pharmacological Gq protein block. All-optical high-throughput screening for TRPC6 inhibitors is more specific and sensitive than conventional pharmacological screening. Thus, we demonstrate specific Gq signaling of hOPN5 and unveil its potential for optogenetic applications.
Assuntos
Optogenética , Transdução de Sinais , Animais , Humanos , Luz , Mamíferos , Transdução de Sinais/fisiologia , Canal de Cátion TRPC6RESUMO
Obesity is the major driver of the global epidemic in type 2 diabetes (T2D). In individuals with obesity, impaired insulin action leads to increased lipolysis in adipocytes, resulting in elevated plasma free fatty acid (FFA) levels that promote peripheral insulin resistance, a hallmark of T2D. Here we show, by using a combined genetic/biochemical/pharmacologic approach, that increased adipocyte lipolysis can be prevented by selective activation of adipocyte Gq signaling in vitro and in vivo (in mice). Activation of this pathway by a Gq-coupled designer receptor or by an agonist acting on an endogenous adipocyte Gq-coupled receptor (CysLT2 receptor) greatly improved glucose and lipid homeostasis in obese mice or in mice with adipocyte insulin receptor deficiency. Our findings identify adipocyte Gq signaling as an essential regulator of whole-body glucose and lipid homeostasis and should inform the development of novel classes of GPCR-based antidiabetic drugs.