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1.
Int J Mol Sci ; 24(12)2023 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-37373182

RESUMEN

A class-A GPCR dopamine D2 receptor (D2R) plays a critical role in the proper functioning of neuronal circuits through the downstream activation of both G-protein- and ß-arrestin-dependent signaling pathways. Understanding the signaling pathways downstream of D2R is critical for developing effective therapies with which to treat dopamine (DA)-related disorders such as Parkinson's disease and schizophrenia. Extensive studies have focused on the regulation of D2R-mediated extracellular-signal-regulated kinase (ERK) 1/2 signaling; however, the manner in which ERKs are activated upon the stimulation of a specific signaling pathway of D2R remains unclear. The present study conducted a variety of experimental techniques, including loss-of-function experiments, site-directed mutagenesis, and the determination of protein interactions, in order to investigate the mechanisms underlying ß-arrestin-biased signaling-pathway-mediated ERK activation. We found that the stimulation of the D2R ß-arrestin signaling pathway caused Mdm2, an E3 ubiquitin ligase, to move from the nucleus to the cytoplasm and interact with tyrosine phosphorylated G-protein-coupled receptor kinase 2 (GRK2), which was facilitated by Src, a non-receptor tyrosine kinase. This interaction led to the ubiquitination of GRK2, which then moved to the plasma membrane and interacted with activated D2R, followed by the phosphorylation of D2R as well as the mediation of ERK activation. In conclusion, Mdm2-mediated GRK2 ubiquitination, which is selectively triggered by the stimulation of the D2R ß-arrestin signaling pathway, is necessary for GRK2 membrane translocation and its interaction with D2R, which in turn mediates downstream ERK signaling. This study is primarily novel and provides essential information with which to better understand the detailed mechanisms of D2R-dependent signaling.


Asunto(s)
Quinasa 2 del Receptor Acoplado a Proteína-G , Transducción de Señal , beta-Arrestinas/metabolismo , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Transducción de Señal/fisiología , beta-Arrestina 1/metabolismo , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Fosforilación/fisiología , Dopamina , Ubiquitinación
2.
Sci Rep ; 13(1): 7707, 2023 05 12.
Artículo en Inglés | MEDLINE | ID: mdl-37173348

RESUMEN

Clinical scenario 1 (CS1) is acute heart failure (HF) characterized by transient systolic blood pressure (SBP) elevation and pulmonary congestion. Although it is managed by vasodilators, the molecular mechanism remains unclear. The sympathetic nervous system plays a key role in HF, and desensitization of cardiac ß-adrenergic receptor (AR) signaling due to G protein-coupled receptor kinase 2 (GRK2) upregulation is known. However, vascular ß-AR signaling that regulates cardiac afterload remains unelucidated in HF. We hypothesized that upregulation of vascular GRK2 leads to pathological conditions similar to CS1. GRK2 was overexpressed in vascular smooth muscle (VSM) of normal adult male mice by peritoneally injected adeno-associated viral vectors driven by the myosin heavy chain 11 promoter. Upregulation of GRK2 in VSM of GRK2 overexpressing mice augmented the absolute increase in SBP (+ 22.5 ± 4.3 mmHg vs. + 36.0 ± 4.0 mmHg, P < 0.01) and lung wet weight (4.28 ± 0.05 mg/g vs. 4.76 ± 0.15 mg/g, P < 0.01) by epinephrine as compared to those in control mice. Additionally, the expression of brain natriuretic peptide mRNA was doubled in GRK2 overexpressing mice as compared to that in control mice (P < 0.05). These findings were similar to CS1. GRK2 overexpression in VSM may cause inappropriate hypertension and HF, as in CS1.


Asunto(s)
Insuficiencia Cardíaca , Hipertensión , Ratones , Masculino , Animales , Músculo Liso Vascular/metabolismo , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Hipertensión/genética , Corazón , Receptores Adrenérgicos beta
3.
Int Immunopharmacol ; 117: 109957, 2023 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-37012864

RESUMEN

OBJECTIVE: In cases of heart failure, cardiac hypertrophy may be caused by the upregulation of G-protein-coupled receptor kinase 2 (GRK2). Both NLRP3 inflammasome and oxidative stress contribute to cardiovascular disease. In this study, we clarified the effect of GRK2 on cardiac hypertrophy in H9c2 cells induced by isoproterenol (ISO) and examined the underlying mechanisms. METHODS: We randomly categorized H9c2 cells into five groups: an ISO group, a paroxetine plus ISO group, a GRK2 small-interfering RNA (siRNA) plus ISO group, a GRK2 siRNA combined with ML385 plus ISO group, and a control group. To determine the effect of GRK2 on cardiac hypertrophy induced by ISO, we carried out CCK8 assays, RT-PCR, TUNEL staining, ELISA assay, DCFH-DA staining, immunofluorescence staining, and western blotting. RESULTS: By using paroxetine or siRNA to inhibit GRK2, we significantly decreased cell viability; reduced the mRNA levels of ANP, BNP, and ß-MHC; and limited the apoptosis rate and protein levels of cleaved caspase-3 and cytochrome c in H9c2 cells treated with ISO. We also found that oxidative stress induced by ISO could be mitigated with paroxetine or GRK2 siRNA. This result was validated by decreased activities of the antioxidant enzymes CAT, GPX, and SOD and increased MDA levels and ROS production. We observed that the protein expression of NLRP3, ASC, and caspase-1 and the intensity of NLRP3 could be inhibited by paroxetine or GRK2 siRNA. Both paroxetine and GRK2 siRNA were able to abolish the increase in GRK2 expression induced by ISO. They also could increase protein levels of HO-1, nuclear Nrf2, and Nrf2 immunofluorescence intensity; however, they could not change the protein level of cytoplasmic Nrf2. By combining treatment with ML385, we were able to reverse GRK2 inhibition on H9c2 cells treated with ISO. CONCLUSION: According to the results of this study, GRK2 participated in cardiac hypertrophy induced by ISO by mitigating NLRP3 inflammasome and oxidative stress through the signaling of Nrf2 in H9c2 cells.


Asunto(s)
Quinasa 2 del Receptor Acoplado a Proteína-G , Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Cardiomegalia/inducido químicamente , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Inflamasomas/metabolismo , Isoproterenol , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Estrés Oxidativo , Paroxetina/farmacología , ARN Interferente Pequeño/metabolismo , Animales , Ratas
4.
Int Immunopharmacol ; 113(Pt A): 109271, 2022 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-36461590

RESUMEN

G-protein coupled receptor (GPCR) kinases (GRKs) and hypoxia-inducible factor-1α (HIF-1α) play key roles in rheumatoid arthritis (RA). Several studies have demonstrated that HIF-1α expression is positively regulated by GRK2, suggesting its posttranscriptional effects on HIF-1α. In this study, we review the role of HIF-1α and GRK2 in RA pathophysiology, focusing on their proinflammatory roles in immune cells and fibroblast-like synoviocytes (FLS).We then introduce several drugs that inhibit GRK2 and HIF-1α, and briefly outline their molecular mechanisms. We conclude by presenting gaps in knowledge and our prospects for the pharmacological potential of targeting these proteins and the relevant downstream signaling pathways.Future research is warranted and paramount for untangling these novel and promising roles for GRK2 and HIF-1α in RA.


Asunto(s)
Artritis Reumatoide , Quinasa 2 del Receptor Acoplado a Proteína-G , Subunidad alfa del Factor 1 Inducible por Hipoxia , Sinoviocitos , Humanos , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Hipoxia/genética , Hipoxia/inmunología , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/inmunología , Inflamación/tratamiento farmacológico , Inflamación/genética , Inflamación/inmunología , Sinoviocitos/inmunología , Quinasa 2 del Receptor Acoplado a Proteína-G/antagonistas & inhibidores , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Quinasa 2 del Receptor Acoplado a Proteína-G/inmunología
5.
Oxid Med Cell Longev ; 2022: 4566851, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35132350

RESUMEN

Hypoxia is an important factor in the development of synovitis in rheumatoid arthritis (RA). The previous study of the research group found that monomeric derivatives of paeoniflorin (MDP) can alleviate joint inflammation in adjuvant-induced arthritis (AA) rats by inhibiting macrophage pyroptosis. This study revealed increased levels of hypoxia-inducible factor- (HIF-) 1α and N-terminal p30 fragment of GSDMD (GSDMD-N) in fibroblast-like synoviocytes (FLS) of RA patients and AA rats, while MDP significantly inhibited their expression. Subsequently, FLS were exposed to a hypoxic environment or treated with cobalt ion in vitro. Western blot and immunofluorescence analysis showed increased expression of G protein-coupled receptor kinase 2 (GRK2), HIF-1α, nucleotide-binding oligomerization segment-like receptor family 3 (NLRP3), ASC, caspase-1, cleaved-caspase-1, and GSDMD-N. Electron microscopy revealed FLS pyroptosis after exposure in hypoxia. Next, corresponding shRNAs were transferred into FLS to knock down hypoxia-inducible factor- (HIF-) 1α, and in turn, NLRP3 and western blot results confirmed the same. The enhanced level of GSDMD was reversed under hypoxia by inhibiting NLRP3 expression. Knockdown and overexpression of GRK2 in FLS revealed GRK2 to be a positive regulator of HIF-1α. Levels of GRK2 and HIF-1α were inhibited by eliminating excess reactive oxygen species (ROS). Furthermore, MDP reduced FLS pyroptosis through targeted inhibition of GRK2 phosphorylation. According to these findings, hypoxia induces FLS pyroptosis through the ROS/GRK2/HIF-1α/NLRP3 pathway, while MDP regulates this pathway to reduce FLS pyroptosis.


Asunto(s)
Artritis Experimental/metabolismo , Artritis Reumatoide/metabolismo , Fibroblastos/metabolismo , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Glucósidos/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Monoterpenos/farmacología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Piroptosis/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Sinoviocitos/metabolismo , Animales , Artritis Experimental/patología , Artritis Reumatoide/patología , Células Cultivadas , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Fosforilación/efectos de los fármacos , Fosforilación/genética , Piroptosis/genética , ARN Interferente Pequeño/genética , Ratas , Ratas Sprague-Dawley , Transducción de Señal/genética , Transfección
6.
Am J Physiol Cell Physiol ; 322(1): C63-C72, 2022 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-34852209

RESUMEN

Pulmonary fibrosis is a chronic, progressive, and irreversible interstitial lung disease. Transforming growth factor-ß1 (TGF-ß1) plays a major role in lung fibroblast cell differentiation to myofibroblast cells and production of extracellular matrix, which are hallmarks of pulmonary fibrosis. G protein-coupled receptor kinase-2 (GRK2) has been shown to play controversial roles in TGF-ß1-induced signal transduction in different cell types; however, the role of GRK2 in TGF-ß1-induced activation of lung fibroblast cells and development of pulmonary fibrosis has not been revealed. In this study, we found that GRK2 levels were increased in lungs and isolated fibroblast cells in a murine model of pulmonary fibrosis, as well as TGF-ß1-treated lung fibroblasts. GRK2 levels were not changed in lungs in the injury phase of pulmonary fibrosis. Posttreatment with GRK2 inhibitor reduced extracellular matrix (ECM) accumulation in lungs in bleomycin-challenged mice, suggesting that GRK2 activation contributes to the progressive phase of pulmonary fibrosis. Inhibition or downregulation of GRK2 attenuates fibronectin, collagen, and α-smooth muscle actin expression in TGF-ß1-induced lung fibroblast cells or myofibroblast cells isolated from patients with pulmonary fibrosis. Furthermore, we showed that GRK2 regulates Smad3 expression, indicating that inhibition of GRK2 attenuates ECM accumulation through downregulation of Smad3 expression. This study reveals that GRK2 is a therapeutic target in treating pulmonary fibrosis and inhibition of GRK2 dampens pulmonary fibrosis by suppression of Smad3 expression, eventually attenuating TGF-ß1 signal pathway and ECM accumulation.


Asunto(s)
Fibroblastos/metabolismo , Quinasa 2 del Receptor Acoplado a Proteína-G/biosíntesis , Pulmón/metabolismo , Fibrosis Pulmonar/metabolismo , Proteína smad3/biosíntesis , Animales , Bleomicina/toxicidad , Línea Celular , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Quinasa 2 del Receptor Acoplado a Proteína-G/antagonistas & inhibidores , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Expresión Génica , Humanos , Pulmón/efectos de los fármacos , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/patología , Proteína smad3/antagonistas & inhibidores , Proteína smad3/genética
7.
Anesth Analg ; 134(1): 204-215, 2022 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-34652301

RESUMEN

BACKGROUND: The main symptoms of chemotherapy-induced peripheral neuropathy (CIPN) include pain and numbness. Neuronal G protein-coupled receptor kinase 2 (GRK2) plays an important role in various pain models. Cisplatin treatment can induce the activation of proinflammatory microglia in spinal cord. The purpose of this study was to investigate the role of spinal neuronal GRK2 in cisplatin-induced CIPN and in the prevention of CIPN by electroacupuncture (EA). METHODS: The pain and sensory deficit behaviors of mice were examined by von Frey test and adhesive removal test. The expression of neuronal GRK2 in the spinal cord is regulated by intraspinal injection of adeno-associated virus (AAV) containing neuron-specific promoters. The protein levels of GRK2, triggering receptor expressed on myeloid cells 2 (TREM2), and DNAX-activating protein of 12 kDa (DAP12) in spinal dorsal horn were detected by Western blot, the density of intraepidermal nerve fibers (IENFs) was detected by immunofluorescence, and microglia activation were evaluated by real-time polymerase chain reaction (PCR). RESULTS: In this study, cisplatin treatment led to the decrease of GRK2 expression in the dorsal horn of spinal cord. Overexpression of neuronal GRK2 in spinal cord by intraspinal injection of an AAV vector expressing GRK2 with human synapsin (hSyn) promotor significantly inhibited the loss of IENFs and alleviated the mechanical pain and sensory deficits induced by cisplatin. Real-time PCR analysis showed that the overexpression of neuronal GRK2 significantly inhibited the messenger RNA (mRNA) upregulation of proinflammatory cytokine interleukin (IL)-1ß, IL-6, inducible nitric oxide synthase (iNOS), and M1 microglia marker cluster of differentiation (CD)16 induced by cisplatin. Furthermore, the TREM2 and DAP12, which has been demonstrated to play a role in microglia activation and in the development of CIPN, were also downregulated by overexpression of neuronal GRK2 in this study. Interestingly, preventive treatment with EA completely mimics the effect of overexpression of neuronal GRK2 in the spinal cord in this mouse model of cisplatin-induced CIPN. EA increased GRK2 level in spinal dorsal horn after cisplatin treatment. Intraspinal injection of AAV vector specifically downregulated neuronal GRK2, completely reversed the regulatory effect of EA on CIPN and microglia activation. All these indicated that the neuronal GRK2 mediated microglial activation contributed to the process of CIPN. CONCLUSIONS: Neuronal GRK2 in the spinal cord contributed to the preventive effect of EA on CIPN. The neuronal GRK2 may be a potential target for CIPN intervention.


Asunto(s)
Cisplatino , Electroacupuntura , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Médula Espinal/patología , Animales , Conducta Animal , Dependovirus , Humanos , Hiperalgesia/metabolismo , Inflamación , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/metabolismo , Fibras Nerviosas , Neuralgia/metabolismo , Neuronas/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Dolor , Asta Dorsal de la Médula Espinal/metabolismo , Factores de Tiempo
8.
Int J Mol Sci ; 22(18)2021 Sep 13.
Artículo en Inglés | MEDLINE | ID: mdl-34576061

RESUMEN

The role of nintedanib, a multiple tyrosine kinase inhibitor, in the treatment of sepsis-induced acute lung injury (ALI) remains unclear. Lipopolysaccharide (LPS), also known as endotoxin, has been used to induce ALI. The goal of this study was to assess the effect of nintedanib in attenuating the histopathological changes of LPS-induced ALI. Nintedanib was administered via oral gavage to male C57BL/6 mice 24 h and 10 min before intratracheal endotoxin instillation. Lung histopathological characteristics, adhesion molecule expression, and the regulatory signaling pathways of neutrophil chemotaxis were analyzed after 24 h. We found that nintedanib significantly reduced histopathological changes and neutrophil recruitment in LPS-induced ALI. The number of neutrophils in bronchoalveolar lavage fluid (BALF) was reduced in nintedanib-treated relative to untreated mice with ALI. Nintedanib mediated the downregulation of the chemotactic response to LPS by reducing the expression of adhesion molecules and the phosphorylated p38:total p38 mitogen-activated protein kinase (MAPK) ratio in the lungs of mice with ALI. Nintedanib also reduced the expression of lymphocyte antigen 6 complex locus G6D (Ly6G) and very late antigen 4 (VLA-4) in BALF neutrophils and mediated the downregulation of chemokine (C-X-C motif) receptor 2 (CXCR2) and upregulation of G protein-coupled receptor kinase 2 (GRK2) activity in peripheral blood neutrophils in mice with LPS-induced ALI. Nintedanib improved the histopathological changes of LPS-induced ALI by reducing neutrophil chemotaxis. These effects were mediated by the inhibition of adhesion molecules via the activation of GRK2 and the inhibition of p38 MAPK and CXCR2.


Asunto(s)
Lesión Pulmonar Aguda/tratamiento farmacológico , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Indoles/farmacología , Receptores de Interleucina-8B/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/genética , Animales , Antígenos Ly/genética , Líquido del Lavado Bronquioalveolar , Modelos Animales de Enfermedad , Endotoxinas/toxicidad , Integrina alfa4beta1/genética , Lipopolisacáridos/toxicidad , Neutrófilos/metabolismo , Neutrófilos/patología , Sepsis/inducido químicamente , Sepsis/complicaciones , Sepsis/tratamiento farmacológico , Sepsis/patología
9.
Am J Physiol Heart Circ Physiol ; 321(5): H850-H864, 2021 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-34477461

RESUMEN

Molecular mechanisms underlying cardiac dysfunction and subsequent heart failure in diabetic cardiomyopathy are incompletely understood. Initially we intended to test the role of G protein-coupled receptor kinase 2 (GRK2), a potential mediator of cardiac dysfunction in diabetic cardiomyopathy, but found that control animals on HFD did not develop cardiomyopathy. Cardiac function was preserved in both wild-type and GRK2 knockout animals fed high-fat diet as indicated by preserved left ventricular ejection fraction (LVEF) although heart mass was increased. The absence of cardiac dysfunction led us to rigorously evaluate the utility of diet-induced obesity to model diabetic cardiomyopathy in mice. Using pure C57BL/6J animals and various diets formulated with different sources of fat-lard (32% saturated fat, 68% unsaturated fat) or hydrogenated coconut oil (95% saturated fat), we consistently observed left ventricular hypertrophy, preserved LVEF, and preserved contractility measured by invasive hemodynamics in animals fed high-fat diet. Gene expression patterns that characterize pathological hypertrophy were not induced, but a modest induction of various collagen isoforms and matrix metalloproteinases was observed in heart with high-fat diet feeding. PPARα-target genes that enhance lipid utilization such as Pdk4, CD36, AcadL, and Cpt1b were induced, but mitochondrial energetics was not impaired. These results suggest that although long-term fat feeding in mice induces cardiac hypertrophy and increases cardiac fatty acid metabolism, it may not be sufficient to activate pathological hypertrophic mechanisms that impair cardiac function or induce cardiac fibrosis. Thus, additional factors that are currently not understood may contribute to the cardiac abnormalities previously reported by many groups.NEW & NOTEWORTHY Dietary fat overload (DFO) is widely used to model diabetic cardiomyopathy but the utility of this model is controversial. We comprehensively characterized cardiac contractile and mitochondrial function in C57BL6/J mice fed with lard-based or saturated fat-enriched diets initiated at two ages. Despite cardiac hypertrophy, contractile and mitochondrial function is preserved, and molecular adaptations likely limit lipotoxicity. The resilience of these hearts to DFO underscores the need to develop robust alternative models of diabetic cardiomyopathy.


Asunto(s)
Cardiomiopatías Diabéticas/etiología , Dieta Alta en Grasa , Hipertrofia Ventricular Izquierda/etiología , Obesidad/complicaciones , Volumen Sistólico , Disfunción Ventricular Izquierda/etiología , Función Ventricular Izquierda , Factores de Edad , Animales , Cardiomiopatías Diabéticas/enzimología , Cardiomiopatías Diabéticas/patología , Cardiomiopatías Diabéticas/fisiopatología , Modelos Animales de Enfermedad , Metabolismo Energético , Femenino , Fibrosis , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Hipertrofia Ventricular Izquierda/enzimología , Hipertrofia Ventricular Izquierda/patología , Hipertrofia Ventricular Izquierda/fisiopatología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias Cardíacas/enzimología , Mitocondrias Cardíacas/patología , Miocardio/enzimología , Miocardio/patología , Disfunción Ventricular Izquierda/enzimología , Disfunción Ventricular Izquierda/patología , Disfunción Ventricular Izquierda/fisiopatología , Remodelación Ventricular
10.
Science ; 372(6548)2021 06 18.
Artículo en Inglés | MEDLINE | ID: mdl-34140358

RESUMEN

Neutrophils communicate with each other to form swarms in infected organs. Coordination of this population response is critical for the elimination of bacteria and fungi. Using transgenic mice, we found that neutrophils have evolved an intrinsic mechanism to self-limit swarming and avoid uncontrolled aggregation during inflammation. G protein-coupled receptor (GPCR) desensitization acts as a negative feedback control to stop migration of neutrophils when they sense high concentrations of self-secreted attractants that initially amplify swarming. Interference with this process allows neutrophils to scan larger tissue areas for microbes. Unexpectedly, this does not benefit bacterial clearance as containment of proliferating bacteria by neutrophil clusters becomes impeded. Our data reveal how autosignaling stops self-organized swarming behavior and how the finely tuned balance of neutrophil chemotaxis and arrest counteracts bacterial escape.


Asunto(s)
Quimiotaxis de Leucocito , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Ganglios Linfáticos/microbiología , Neutrófilos/fisiología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/crecimiento & desarrollo , Animales , Agregación Celular , Quimiocina CXCL2 , Eosinófilos/fisiología , Femenino , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Inflamación , Leucotrieno B4/metabolismo , Masculino , Ratones , Ratones Transgénicos , Neutrófilos/inmunología , Infecciones por Pseudomonas/microbiología , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Piel/inmunología , Piel/lesiones , Piel/patología
11.
Eur J Pain ; 25(9): 2039-2049, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34101933

RESUMEN

BACKGROUND: Previous studies have indicated a negative correlation between GRK2 expression and pain development and transmission. Here, we investigated whether G-protein-coupled receptor kinase 2 (GRK2) was involved in regulating diabetic mechanical hyperalgesia (DMH). METHODS: The adeno-associated viral vectors containing the GRK2 gene (AAV-GRK2) were used to up-regulate GRK2 protein expression. The expression of GRK2 and exchange protein directly activated by cyclic adenosine monophosphate 1 (Epac1) in the dorsal root ganglion (DRG) of lumbar 4-6 was detected via immunoblotting and immunohistochemistry, and the transfection of the GRK2 gene was detected by immunofluorescence. RESULTS: Low levels of GRK2 were able to sustain STZ-induced pain in DMH rats. Intrathecal injection of AAV-GRK2 vector up-regulated GRK2 expression, providing pain rain to rats with DMH. With an increase in DMH duration, there was a decrease in paw withdrawal threshold (PWT) value, aggravating the pain, resulting in a decreasing pattern in GRK2 protein expression over time, whereas Epac1 protein expression showed an opposite trend. CONCLUSION: GRK2 expression regulated DMH progression and is expected to play a role in the development of targeted therapy for DMH. GRK2 and Epac1 expressions play a vital role in maintaining pain in DMH rats.


Asunto(s)
Diabetes Mellitus , Hiperalgesia , Animales , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Ganglios Espinales , Hiperalgesia/genética , Dolor , Ratas
12.
Sci Rep ; 11(1): 11129, 2021 05 27.
Artículo en Inglés | MEDLINE | ID: mdl-34045505

RESUMEN

Diabetes is a metabolic syndrome rooted in impaired insulin and/or glucagon secretory responses within the pancreatic islets of Langerhans (islets). Insulin secretion is primarily regulated by two key factors: glucose-mediated ATP production and G-protein coupled receptors (GPCRs) signaling. GPCR kinase 2 (GRK2), a key regulator of GPCRs, is reported to be downregulated in the pancreas of spontaneously obesogenic and diabetogenic mice (ob/ob). Moreover, recent studies have shown that GRK2 non-canonically localizes to the cardiac mitochondrion, where it can contribute to glucose metabolism. Thus, islet GRK2 may impact insulin secretion through either mechanism. Utilizing Min6 cells, a pancreatic ß-cell model, we knocked down GRK2 and measured glucose-mediated intracellular calcium responses and insulin secretion. Silencing of GRK2 attenuated calcium responses, which were rescued by pertussis toxin pre-treatment, suggesting a Gαi/o-dependent mechanism. Pancreatic deletion of GRK2 in mice resulted in glucose intolerance with diminished insulin secretion. These differences were due to diminished insulin release rather than decreased insulin content or gross differences in islet architecture. Furthermore, a high fat diet feeding regimen exacerbated the metabolic phenotype in this model. These results suggest a new role for pancreatic islet GRK2 in glucose-mediated insulin responses that is relevant to type 2 diabetes disease progression.


Asunto(s)
Calcio/metabolismo , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Intolerancia a la Glucosa/metabolismo , Glucosa/metabolismo , Secreción de Insulina/fisiología , Células Secretoras de Insulina/metabolismo , Animales , Línea Celular , AMP Cíclico , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Dieta Alta en Grasa , Regulación hacia Abajo , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Glucagón/metabolismo , Intolerancia a la Glucosa/genética , Prueba de Tolerancia a la Glucosa , Islotes Pancreáticos/metabolismo , Peroxidación de Lípido/fisiología , Ratones , Ratones Noqueados , Obesidad/genética , Obesidad/metabolismo
13.
Biomolecules ; 11(3)2021 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-33802765

RESUMEN

G protein-coupled receptors (GPCRs), which regulate a vast number of eukaryotic processes, are desensitized by various mechanisms but, most importantly, by the GPCR kinases (GRKs). Ever since GRKs were first identified, investigators have sought to determine which structural features of GRKs are used to select for the agonist-bound states of GPCRs and how this binding event in turn enhances GRK catalytic activity. Despite a wealth of molecular information from high-resolution crystal structures of GRKs, the mechanisms driving activation have remained elusive, in part because the GRK N-terminus and active site tether region, previously proposed to serve as a receptor docking site and to be key to kinase domain closure, are often disordered or adopt inconsistent conformations. However, two recent studies have implicated other regions of GRKs as being involved in direct interactions with active GPCRs. Atomic resolution structures of GPCR-GRK complexes would help refine these models but are, so far, lacking. Here, we assess three distinct models for how GRKs recognize activated GPCRs, discuss limitations in the approaches used to generate them, and then experimentally test a hypothetical GPCR interaction site in GRK2 suggested by the two newest models.


Asunto(s)
Quinasa 2 del Receptor Acoplado a Proteína-G/química , Dominios Proteicos , Receptores Adrenérgicos beta 2/química , Receptores Acoplados a Proteínas G/química , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Células COS , Chlorocebus aethiops , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Fosforilación , Unión Proteica , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Homología de Secuencia de Aminoácido
14.
BMC Biol ; 19(1): 40, 2021 03 03.
Artículo en Inglés | MEDLINE | ID: mdl-33658023

RESUMEN

BACKGROUND: Insulin secretion from the pancreatic ß-cell is finely modulated by different signals to allow an adequate control of glucose homeostasis. Incretin hormones such as glucagon-like peptide-1 (GLP-1) act as key physiological potentiators of insulin release through binding to the G protein-coupled receptor GLP-1R. Another key regulator of insulin signaling is the Ser/Thr kinase G protein-coupled receptor kinase 2 (GRK2). However, whether GRK2 affects insulin secretion or if GRK2 can control incretin actions in vivo remains to be analyzed. RESULTS: Using GRK2 hemizygous mice, isolated pancreatic islets, and model ß-cell lines, we have uncovered a relevant physiological role for GRK2 as a regulator of incretin-mediated insulin secretion in vivo. Feeding, oral glucose gavage, or administration of GLP-1R agonists in animals with reduced GRK2 levels (GRK2+/- mice) resulted in enhanced early phase insulin release without affecting late phase secretion. In contrast, intraperitoneal glucose-induced insulin release was not affected. This effect was recapitulated in isolated islets and correlated with the increased size or priming efficacy of the readily releasable pool (RRP) of insulin granules that was observed in GRK2+/- mice. Using nanoBRET in ß-cell lines, we found that stimulation of GLP-1R promoted GRK2 association to this receptor and that GRK2 protein and kinase activity were required for subsequent ß-arrestin recruitment. CONCLUSIONS: Overall, our data suggest that GRK2 is an important negative modulator of GLP-1R-mediated insulin secretion and that GRK2-interfering strategies may favor ß-cell insulin secretion specifically during the early phase, an effect that may carry interesting therapeutic applications.


Asunto(s)
Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Regulación de la Expresión Génica , Receptor del Péptido 1 Similar al Glucagón/genética , Secreción de Insulina/genética , Animales , Línea Celular , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Masculino , Ratones
15.
J Mol Cell Cardiol ; 154: 137-153, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33548241

RESUMEN

G protein-coupled receptor (GPCR) kinase 2 (GRK2) expression and activity are elevated early on in response to several forms of cardiovascular stress and are a hallmark of heart failure. Interestingly, though, in addition to its well-characterized role in regulating GPCRs, mounting evidence suggests a GRK2 "interactome" that underlies a great diversity in its functional roles. Several such GRK2 interacting partners are important for adaptive and maladaptive myocyte growth; therefore, an understanding of domain-specific interactions with signaling and regulatory molecules could lead to novel targets for heart failure therapy. Herein, we subjected transgenic mice with cardiac restricted expression of a short, amino terminal fragment of GRK2 (ßARKnt) to pressure overload and found that unlike their littermate controls or previous GRK2 fragments, they exhibited an increased left ventricular wall thickness and mass prior to cardiac stress that underwent proportional hypertrophic growth to controls after acute pressure overload. Importantly, despite this enlarged heart, ßARKnt mice did not undergo the expected transition to heart failure observed in controls. Further, ßARKnt expression limited adverse left ventricular remodeling and increased cell survival signaling. Proteomic analysis to identify ßARKnt binding partners that may underlie the improved cardiovascular phenotype uncovered a selective functional interaction of both endogenous GRK2 and ßARKnt with AKT substrate of 160 kDa (AS160). AS160 has emerged as a key downstream regulator of insulin signaling, integrating physiological and metabolic cues to couple energy demand to membrane recruitment of Glut4. Our preliminary data indicate that in ßARKnt mice, cardiomyocyte insulin signaling is improved during stress, with a coordinate increase in spare respiratory activity and ATP production without metabolite switching. Surprisingly, these studies also revealed a significant decrease in gonadal fat weight, equivalent to human abdominal fat, in male ßARKnt mice at baseline and following cardiac stress. These data suggest that the enhanced AS160-mediated signaling in the ßARKnt mice may ameliorate pathological cardiac remodeling through direct modulation of insulin signaling within cardiomyocytes, and translate these to beneficial effects on systemic metabolism.


Asunto(s)
Cardiomegalia/etiología , Cardiomegalia/fisiopatología , Quinasa 2 del Receptor Acoplado a Proteína-G/química , Péptidos/genética , Dominios y Motivos de Interacción de Proteínas , Animales , Biomarcadores , Cardiomegalia/diagnóstico , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Expresión Génica , Ratones , Ratones Transgénicos , Péptidos/metabolismo , Fenotipo , Unión Proteica , Transducción de Señal , Remodelación Ventricular
16.
J Biol Chem ; 296: 100216, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33465377

RESUMEN

For most G protein-coupled receptors, the third intracellular loop (IL3) and carboxy-terminal tail (CT) are sites for G protein-coupled receptor kinase (GRK)-mediated phosphorylation, leading to ß-arrestin binding and agonist-specific desensitization. These regions of bitter taste receptors (TAS2Rs) are extremely short compared with the superfamily, and their function in desensitization is unknown. TAS2R14 expressed on human airway smooth muscle cells relax the cell, suggesting a novel target for bronchodilators. To assess IL3 and CT in agonist-promoted TAS2R14 desensitization (tachyphylaxis), we generated fusion proteins of both the WT sequence and Ala substituted for Ser/Thr in the IL3 and CT sequences. In vitro, activated GRK2 phosphorylated WT IL3 and WT CT proteins but not Ala-substituted forms. TAS2R14s with mutations in IL3 (IL-5A), CT (CT-5A), and in both regions (IL/CT-10A) were expressed in human embryonic kidney 293T cells. IL/CT-10A and CT-5A failed to undergo desensitization of the intracellular calcium response compared with WT, indicating that functional desensitization by GRK phosphorylation is at residues in the CT. Desensitization of TAS2R14 was blocked by GRK2 knockdown in human airway smooth muscle cells. Receptor:ß-arrestin binding was absent in IL/CT-10A and CT-5A and reduced in IL-5A, indicating a role for IL3 phosphorylation in the ß-arrestin interaction for this function. Agonist-promoted internalization of IL-5A and CT-5A receptors was impaired, and they failed to colocalize with early endosomes. Thus, agonist-promoted functional desensitization of TAS2R14 occurs by GRK phosphorylation of CT residues and ß-arrestin binding. However, ß-arrestin function in the internalization and trafficking of the receptor also requires GRK phosphorylation of IL3 residues.


Asunto(s)
Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Miocitos del Músculo Liso/metabolismo , Procesamiento Proteico-Postraduccional , ARN Interferente Pequeño/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sustitución de Aminoácidos , Bronquios/citología , Bronquios/metabolismo , Calcio/metabolismo , Difenhidramina/farmacología , Endosomas/metabolismo , Quinasa 2 del Receptor Acoplado a Proteína-G/antagonistas & inhibidores , Quinasa 2 del Receptor Acoplado a Proteína-G/química , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Mutación , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Fosforilación/efectos de los fármacos , Unión Proteica , ARN Interferente Pequeño/genética , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Taquifilaxis/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , beta-Arrestinas/genética , beta-Arrestinas/metabolismo
17.
Exp Cell Res ; 399(2): 112482, 2021 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-33434531

RESUMEN

IL-6-triggered Th17 cell expansion is responsible for the pathogenesis of many immune diseases including rheumatoid arthritis (RA). Traditionally, IL-6 induces Th17 cell differentiation through JAK-STAT3 signaling. In the present work, PKA inhibition reduces in vitro induction of Th17 cells, while IL-6 stimulation of T cells facilitates the internalization of A3AR and increased cAMP production in a GRK2 dependent manner. Inhibition of GRK2 by paroxetine (PAR) or genetic depletion of GRK2 restored A3AR distribution and prevented Th17 cell differentiation. Furthermore, in vivo PAR treatment effectively reduced the splenic Th17 cell proportion in a rat model of collagen-induced arthritis (CIA) which was accompanied by a significant improvement in clinical manifestations. These results indicate that IL-6-induced Th17 cell differentiation not only occurs through JAK-STAT3-RORγt but is also mediated through GRK2-A3AR-cAMP-PKA-CREB/ICER-RORγt. This elucidates the significance of GRK2-controlled cAMP signaling in the differentiation of Th17 cells and its potential application in treating Th17-driven immune diseases such as RA.


Asunto(s)
Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Interleucina-6/farmacología , Receptor de Adenosina A3/metabolismo , Células Th17/fisiología , Animales , Artritis Experimental/genética , Artritis Experimental/inmunología , Artritis Experimental/metabolismo , Artritis Experimental/patología , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Interleucina-6/fisiología , Masculino , Ratas , Ratas Transgénicas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Células Th17/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genética
18.
Mol Cell Biochem ; 476(3): 1505-1516, 2021 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-33392923

RESUMEN

G protein-coupled receptor kinases (GRKs), in addition to their role in modulating signal transduction mechanisms associated with activated G protein-coupled receptors (GPCRs), can also interact with many non-GPCR proteins to mediate cellular responses to chemotherapeutics. The rationale for this study is based on the presumption that GRK2 modulates the responses of cancer cells to the chemotherapeutic cisplatin. In this report, we show that GRK2 modulates the responses of cancer cells to cisplatin. Cervical cancer HeLa cells stably transfected with GRK2 shRNA, to decrease GRK2 protein expression, show increased sensitivity to cisplatin. Of interest, these cells also show increased accumulation of NADPH, associating with decreased NADP buildup, at low concentrations of cisplatin tested. These changes in NADPH and NADP levels are also observed in the breast cancer MDA MB 231 cells, which has lower endogenous GRK2 protein expression levels, but not BT549, a breast cancer cell line with higher GRK2 protein expression. This effect of NADPH accumulation may be associated with a decrease in NADPH oxidase 4 (NOX4) protein expression, which is found to correlate with GRK2 protein expression in cancer cells-a relationship which mimics that observed in cardiomyocytes. Furthermore, like in cardiomyocytes, GRK2 and NOX4 interact to form complexes in cancer cells. Collectively, these results suggest that GRK2 interacts with NOX4 to modify cisplatin sensitivity in cancer cells and may also factor into the success of cisplatin-based regimens.


Asunto(s)
Cisplatino/farmacología , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , NADPH Oxidasa 4/genética , NADPH Oxidasa 4/metabolismo , Antineoplásicos/farmacología , Apoptosis , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Quinasa 3 del Receptor Acoplado a Proteína-G/metabolismo , Quinasa 5 del Receptor Acoplado a Proteína-G/metabolismo , Proteínas de Unión al GTP/metabolismo , Perfilación de la Expresión Génica , Células HeLa , Humanos , Neoplasias/metabolismo , Fosforilación , Unión Proteica , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Factores de Tiempo
19.
Int J Mol Sci ; 22(2)2021 Jan 07.
Artículo en Inglés | MEDLINE | ID: mdl-33430208

RESUMEN

Cardiac diseases including heart failure (HF), are the leading cause of morbidity and mortality globally. Among the prominent characteristics of HF is the loss of ß-adrenoceptor (AR)-mediated inotropic reserve. This is primarily due to the derangements in myocardial regulatory signaling proteins, G protein-coupled receptor (GPCR) kinases (GRKs) and ß-arrestins (ß-Arr) that modulate ß-AR signal termination via receptor desensitization and downregulation. GRK2 and ß-Arr2 activities are elevated in the heart after injury/stress and participate in HF through receptor inactivation. These GPCR regulators are modulated profoundly by nitric oxide (NO) produced by NO synthase (NOS) enzymes through S-nitrosylation due to receptor-coupled NO generation. S-nitrosylation, which is NO-mediated modification of protein cysteine residues to generate an S-nitrosothiol (SNO), mediates many effects of NO independently from its canonical guanylyl cyclase/cGMP/protein kinase G signaling. Herein, we review the knowledge on the NO system in the heart and S-nitrosylation-dependent modifications of myocardial GPCR signaling components GRKs and ß-Arrs.


Asunto(s)
Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Óxido Nítrico/genética , Receptores Adrenérgicos beta/genética , beta-Arrestinas/genética , GMP Cíclico/genética , Proteínas Quinasas Dependientes de GMP Cíclico/genética , Humanos , Óxido Nítrico Sintasa/genética , S-Nitrosotioles/metabolismo , Transducción de Señal/genética
20.
Cells ; 10(1)2021 01 14.
Artículo en Inglés | MEDLINE | ID: mdl-33466800

RESUMEN

ß-adrenergic receptors (ß-ARs) play a major role in the physiological regulation of cardiac function through signaling routes tightly controlled by G protein-coupled receptor kinases (GRKs). Although the acute stimulation of ß-ARs and the subsequent production of cyclic AMP (cAMP) have beneficial effects on cardiac function, chronic stimulation of ß-ARs as observed under sympathetic overdrive promotes the development of pathological cardiac remodeling and heart failure (HF), a leading cause of mortality worldwide. This is accompanied by an alteration in cAMP compartmentalization and the activation of the exchange protein directly activated by cAMP 1 (Epac1) signaling. Among downstream signals of ß-ARs, compelling evidence indicates that GRK2, GRK5, and Epac1 represent attractive therapeutic targets for cardiac disease. Here, we summarize the pathophysiological roles of GRK2, GRK5, and Epac1 in the heart. We focus on their signalosome and describe how under pathological settings, these proteins can cross-talk and are part of scaffolded nodal signaling systems that contribute to a decreased cardiac function and HF development.


Asunto(s)
Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Quinasa 5 del Receptor Acoplado a Proteína-G/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Insuficiencia Cardíaca/metabolismo , Miocardio/metabolismo , Remodelación Ventricular , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Quinasa 5 del Receptor Acoplado a Proteína-G/genética , Factores de Intercambio de Guanina Nucleótido/genética , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/patología , Humanos , Miocardio/patología
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