RESUMEN
Obesity impairs tissue insulin sensitivity and signaling, promoting type-2 diabetes. Although improving insulin signaling is key to reversing diabetes, the multi-organ mechanisms regulating this process are poorly defined. Here, we screen the secretome and receptome in Drosophila to identify the hormonal crosstalk affecting diet-induced insulin resistance and obesity. We discover a complex interplay between muscle, neuronal, and adipose tissues, mediated by Bone Morphogenetic Protein (BMP) signaling and the hormone Bursicon, that enhances insulin signaling and sugar tolerance. Muscle-derived BMP signaling, induced by sugar, governs neuronal Bursicon signaling. Bursicon, through its receptor Rickets, a Leucine-rich-repeat-containing G-protein coupled receptor (LGR), improves insulin secretion and insulin sensitivity in adipose tissue, mitigating hyperglycemia. In mouse adipocytes, loss of the Rickets ortholog LGR4 blunts insulin responses, showing an essential role of LGR4 in adipocyte insulin sensitivity. Our findings reveal a muscle-neuronal-fat-tissue axis driving metabolic adaptation to high-sugar conditions, identifying LGR4 as a critical mediator in this regulatory network.
Asunto(s)
Tejido Adiposo , Resistencia a la Insulina , Obesidad , Receptores Acoplados a Proteínas G , Transducción de Señal , Animales , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Tejido Adiposo/metabolismo , Ratones , Obesidad/metabolismo , Insulina/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Adipocitos/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Músculos/metabolismo , Masculino , Músculo Esquelético/metabolismo , Drosophila melanogaster/metabolismo , Dieta Alta en Grasa/efectos adversos , Neuronas/metabolismo , Ratones Endogámicos C57BLRESUMEN
Positive allosteric modulation of metabotropic glutamate subtype 5 (mGlu5) receptor has emerged as a potential new therapeutic strategy for the treatment of schizophrenia and cognitive impairments. However, positive allosteric modulator (PAM) agonist activity has been associated with adverse side effects, and neurotoxicity has also been observed for pure PAMs. The structural and pharmacological basis of therapeutic versus adverse mGlu5 PAM in vivo effects remains unknown. Thus, gaining insights into the signaling fingerprints, as well as the binding kinetics of structurally diverse mGlu5 PAMs, may help in the rational design of compounds with desired properties. We assessed the binding and signaling profiles of N-methyl-5-(phenylethynyl)pyrimidin-2-amine (MPPA), 3-cyano-N-(2,5-diphenylpyrazol-3-yl)benzamide (CDPPB), and 1-[4-(4-chloro-2-fluoro-phenyl)piperazin-1-yl]-2-(4-pyridylmethoxy)ethenone [compound 2c, a close analog of 1-(4-(2-chloro-4-fluorophenyl)piperazin-1-yl)-2-(pyridin-4-ylmethoxy)ethanone] in human embryonic kidney 293A cells stably expressing mGlu5 using Ca2+ mobilization, inositol monophosphate (IP1) accumulation, extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, and receptor internalization assays. Of the three allosteric ligands, only CDPPB had intrinsic agonist efficacy, and it also had the longest receptor residence time and highest affinity. MPPA was a biased PAM, showing higher positive cooperativity with orthosteric agonists in ERK1/2 phosphorylation and Ca2+ mobilization over IP1 accumulation and receptor internalization. In primary cortical neurons, all three PAMs showed stronger positive cooperativity with (S)-3,5-dihydroxyphenylglycine (DHPG) in Ca2+ mobilization over IP1 accumulation. Our characterization of three structurally diverse mGlu5 PAMs provides further molecular pharmacological insights and presents the first assessment of PAM-mediated mGlu5 internalization. SIGNIFICANCE STATEMENT: Enhancing metabotropic glutamate receptor subtype 5 (mGlu5) activity is a promising strategy to treat cognitive and positive symptoms in schizophrenia. It is increasingly evident that positive allosteric modulators (PAMs) of mGlu5 are not all equal in preclinical models; there remains a need to better understand the molecular pharmacological properties of mGlu5 PAMs. This study reports detailed characterization of the binding and functional pharmacological properties of mGlu5 PAMs and is the first study of the effects of mGlu5 PAMs on receptor internalization.
Asunto(s)
Regulación Alostérica/efectos de los fármacos , Receptor del Glutamato Metabotropico 5/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Benzamidas/farmacología , Línea Celular , Ácidos Grasos/farmacología , Femenino , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Fosforilación/efectos de los fármacos , Pirazoles/farmacología , RatasRESUMEN
VGF is a peptide precursor expressed in neuroendocrine cells that is suggested to play a role in the regulation of energy homeostasis. VGF is proteolytically cleaved to yield multiple bioactive peptides. However, the specific actions of VGF-derived peptides on energy homeostasis remain unclear. The aim of the present work was to investigate the role of VGF-derived peptides in energy homeostasis and explore the pharmacological actions of VGF-derived peptides on body weight in preclinical animal models. VGF-derived peptides (NERP-1, NERP-2, PGH-NH2, PGH-OH, NERP-4, TLQP-21, TLQP-30, TLQP-62, HHPD-41, AQEE-30, and LQEQ-19) were synthesized and screened for their ability to affect neuronal activity in vitro on hypothalamic brain slices and modulate food intake and energy expenditure after acute central administration in vivo. In addition, the effects of NERP-1, NERP-2, PGH-NH2, TLQP-21, TLQP-62, and HHPD-41 on energy homeostasis were studied after chronic central infusion. NERP-1, PGH-NH2, HHPD-41, and TLQP-62 increased the functional activity of hypothalamic neuronal networks. However, none of the peptides altered energy homeostasis after either acute or chronic ICV administration. The present data do not support the potential use of the tested VGF-derived peptides as novel anti-obesity drug candidates.
Asunto(s)
Fármacos Antiobesidad/farmacología , Neuropéptidos/genética , Neuropéptidos/farmacología , Obesidad/tratamiento farmacológico , Animales , Peso Corporal/efectos de los fármacos , Modelos Animales de Enfermedad , Metabolismo Energético/efectos de los fármacos , Humanos , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Hipotálamo/patología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Obesidad/genética , Obesidad/patología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/farmacología , RatasRESUMEN
Organisms adapt their metabolism and growth to the availability of nutrients and oxygen, which are essential for development, yet the mechanisms by which this adaptation occurs are not fully understood. Here we describe an RNAi-based body-size screen in Drosophila to identify such mechanisms. Among the strongest hits is the fibroblast growth factor receptor homolog breathless necessary for proper development of the tracheal airway system. Breathless deficiency results in tissue hypoxia, sensed primarily in this context by the fat tissue through HIF-1a prolyl hydroxylase (Hph). The fat relays its hypoxic status through release of one or more HIF-1a-dependent humoral factors that inhibit insulin secretion from the brain, thereby restricting systemic growth. Independently of HIF-1a, Hph is also required for nutrient-dependent Target-of-rapamycin (Tor) activation. Our findings show that the fat tissue acts as the primary sensor of nutrient and oxygen levels, directing adaptation of organismal metabolism and growth to environmental conditions.
Asunto(s)
Proteínas de Drosophila/metabolismo , Animales , Proteínas de Unión al ADN/metabolismo , Drosophila , Proteínas de Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , Secreción de Insulina/genética , Secreción de Insulina/fisiología , Oxígeno/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The myogenic response (MR) and myogenic tone (MT) in resistance vessels is crucial for maintaining peripheral vascular resistance and blood flow autoregulation. Development of MT involves G protein-coupled receptors, and may be affected by aging. AIMS: (1) to estimate the mesenteric blood flow in myogenically active small mesenteric arteries; (2) to investigate the signaling from Gαq/11 and/or Gα12 activation to MT development; (3) to investigate the role of Rho-kinase 2 and aging on MT in mesenteric resistance arteries. METHODS: we used pressure myography, quantitative real-time PCR, and immunolocalization to study small (<200 µm) mesenteric arteries (SMA) from young, mature adult, and middle aged mice. RESULTS: Poiseuille flow calculations indicated autoregulation of blood flow at 60-120 mm Hg arterial pressure. Gαq/11 and Gα12 were abundantly expressed at the mRNA and protein levels in SMA. The Gαq/11 inhibitor YM-254890 suppressed MT development, and the Phosholipase C inhibitors U73122 and ET-18-OCH3 robustly inhibited it. We found an age-dependent increase in ROCK2 mRNA expression, and in basal MT. The specific ROCK2 inhibitor KD025 robustly inhibited MT in SMAs in all mice with an age-dependent variation in KD025 sensitivity. The inhibitory effect of KD025 was not prevented by the L-type Ca2+ channel activator BayK 8644. KD025 reversibly inhibited MT and endothelin-1 vasoconstriction in small pial arteries from Göttingen minipigs. CONCLUSIONS: MT development in SMAs occurs through a Gαq/11 /PLC/Ca2+ -dependent pathway, and is maintained via ROCK2-mediated Ca2+ sensitization. Increased MT at mature adulthood can be explained by increased ROCK2 expression/activity.
Asunto(s)
Envejecimiento/fisiología , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Arterias Mesentéricas/metabolismo , Músculo Liso Vascular/fisiología , Transducción de Señal , Quinasas Asociadas a rho/metabolismo , Envejecimiento/metabolismo , Animales , Canales de Calcio Tipo L/metabolismo , Subunidades alfa de la Proteína de Unión al GTP/antagonistas & inhibidores , Subunidades alfa de la Proteína de Unión al GTP/genética , Masculino , Arterias Mesentéricas/crecimiento & desarrollo , Arterias Mesentéricas/fisiología , Ratones , Ratones Endogámicos C57BL , Tono Muscular , Músculo Liso Vascular/crecimiento & desarrollo , Músculo Liso Vascular/metabolismo , Porcinos , Porcinos Enanos , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/genéticaRESUMEN
Hypersecretion of norepinephrine (NE) and angiotensin II (AngII) is a hallmark of major prevalent cardiovascular diseases that contribute to cardiac pathophysiology and morbidity. Herein, we explore whether heterodimerization of presynaptic AngII AT1 receptor (AT1-R) and NE α2C-adrenergic receptor (α2C-AR) could underlie their functional cross-talk to control NE secretion. Multiple bioluminescence resonance energy transfer and protein complementation assays allowed us to accurately probe the structures and functions of the α2C-AR-AT1-R dimer promoted by ligand binding to individual protomers. We found that dual agonist occupancy resulted in a conformation of the heterodimer different from that induced by active individual protomers and triggered atypical Gs-cAMP-PKA signaling. This specific pharmacological signaling unit was identified in vivo to promote not only NE hypersecretion in sympathetic neurons but also sympathetic hyperactivity in mice. Thus, we uncovered a new process by which GPCR heterodimerization creates an original functional pharmacological entity and that could constitute a promising new target in cardiovascular therapeutics.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Receptor de Angiotensina Tipo 1/agonistas , Transducción de Señal , Agonistas alfa-Adrenérgicos/química , Animales , Biofisica , Enfermedades Cardiovasculares/metabolismo , AMP Cíclico/metabolismo , Dimerización , Diseño de Fármacos , Proteínas de Unión al GTP/metabolismo , Células HEK293 , Humanos , Ligandos , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Norepinefrina/química , Células PC12 , Fosforilación , Conformación Proteica , Ratas , Receptores Adrenérgicos alfa 2/química , Sistema Nervioso Simpático/efectos de los fármacosRESUMEN
Recent progress in the understanding of seven-transmembrane receptor (7TMR) signalling has promoted the development of a new generation of pathway selective ligands. The angiotensin II type I receptor (AT1aR) is one of the most studied 7TMRs with respect to selective activation of the ß-arrestin dependent signalling. Two complimentary global phosphoproteomics studies have analyzed the complex signalling induced by the AT1aR. Here we integrate the data sets from these studies and perform a joint analysis using a novel method for prediction of differential kinase activity from phosphoproteomics data. The method builds upon NetworKIN, which applies sophisticated linear motif analysis in combination with contextual network modelling to predict kinase-substrate associations with high accuracy and sensitivity. These predictions form the basis for subsequently nonparametric statistical analysis to identify likely activated kinases. This suggested that AT1aR-dependent signalling activates 48 of the 285 kinases detected in HEK293 cells. Of these, Aurora B, CLK3 and PKG1 have not previously been described in the pathway whereas others, such as PKA, PKB and PKC, are well known. In summary, we have developed a new method for kinase-centric analysis of phosphoproteomes to pinpoint differential kinase activity in large-scale data sets.
Asunto(s)
Receptores de Angiotensina/metabolismo , Transducción de Señal/fisiología , Bases de Datos de Proteínas , Células HEK293 , Humanos , Fosforilación , Estructura Terciaria de Proteína , Análisis de Secuencia de ProteínaRESUMEN
OBJECTIVES: To study the role of microRNAs in hypertension-induced vascular pathology before the onset of symptoms of severe cardiovascular disease. BACKGROUND: MicroRNAs play a crucial role in cardiovascular disease. However, microRNAs are often studied in full-blown cardiovascular disease models, not during development of cardiovascular pathology. METHODS: Angiotensin II was infused into healthy adult rats, inducing chronic hypertension, and microRNA expression profiles were obtained. The most prominently regulated microRNA, miR-487b, was further investigated, using primary cultures of rat aortic and human umbilical cord arterial cells. RESULTS: MiR-487b is predicted to target insulin receptor substrate 1 (IRS1). IRS1 plays an important role in both insulin signaling and cell proliferation and survival. IRS1 mRNA and protein levels were downregulated in aortae of hypertensive rats. MiR-487b binds directly to both rat and human IRS1 3'UTR and inhibits reporter gene expression in vitro. In primary rat and human arterial adventitial fibroblasts, inhibition of miR-487b leads to upregulation of IRS1 expression. Upregulation of miR-487b had the opposite effect, confirming direct targeting of IRS1 by miR-487b.Immunohistochemistry of aortic cross sections and rt/qPCR analyses of the separate aortic wall layers showed that both IRS1 and miR-487b were present mainly in the adventitia and less or not at all in the intima and tunica media. IRS1 expression in adventitial fibroblasts was predominantly nuclear and nuclear IRS1 is known to have antiapoptotic effects. Indeed, inhibition of miR-487b protected adventitial fibroblasts, and also medial smooth muscle cells, against serum starvation-induced apoptosis and increased cell survival. CONCLUSIONS: Angiotensin II-induced hypertension leads to upregulation of miR-487b, which targets IRS1. Via downregulation of IRS1, miR-487b can contribute to cell death and loss of adventitial and medial integrity during hypertension-induced vascular pathology.
Asunto(s)
Aorta/metabolismo , Hipertensión/metabolismo , Proteínas Sustrato del Receptor de Insulina/metabolismo , MicroARNs/metabolismo , Angiotensina II/farmacología , Animales , Aorta/patología , Apoptosis , Western Blotting , Proliferación Celular , Supervivencia Celular , Femenino , Fibroblastos/metabolismo , Glucosa/metabolismo , Humanos , Hipertensión/patología , Inmunohistoquímica , Luciferasas/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Células Tumorales Cultivadas , Cordón Umbilical/irrigación sanguíneaRESUMEN
Functional selectivity of G protein-coupled receptor (GPCR) ligands toward different downstream signals has recently emerged as a general hallmark of this receptor class. However, pleiotropic and crosstalk signaling of GPCRs makes functional selectivity difficult to decode. To look from the initial active receptor point of view, we developed new, highly sensitive and direct bioluminescence resonance energy transfer-based G protein activation probes specific for all G protein isoforms, and we used them to evaluate the G protein-coupling activity of [(1)Sar(4)Ile(8)Ile]-angiotensin II (SII), previously described as an angiotensin II type 1 (AT(1)) receptor-biased agonist that is G protein independent but ß-arrestin selective. By multiplexing assays sensing sequential signaling events, from receptor conformations to downstream signaling, we decoded SII as an agonist stabilizing a G protein-dependent AT(1A) receptor signaling module different from that of the physiological agonist angiotensin II, both in recombinant and primary cells. Thus, a biased agonist does not necessarily select effects from the physiological agonist but may instead stabilize and create a new distinct active pharmacological receptor entity.
Asunto(s)
Receptor de Angiotensina Tipo 1/metabolismo , Técnicas Biosensibles , Línea Celular , Proteínas de Unión al GTP/metabolismo , Humanos , Conformación Proteica , Receptor de Angiotensina Tipo 1/agonistas , Receptor de Angiotensina Tipo 1/químicaRESUMEN
BACKGROUND: We hypothesized that single nucleotide polymorphisms (SNPs) located in microRNA (miR) binding sites in genes of the renin angiotensin aldosterone system (RAAS) can influence blood pressure and risk of myocardial infarction. METHODS: Using online databases dbSNP and TargetScan, we identified 10 SNPs in potential miR binding sites in eight RAAS-related genes, common in Caucasians. We genotyped a large case-control study on myocardial infarctions, the Study of Myocardial Infarctions LEiden (SMILE) for these 10 SNPs and found nine SNPs, in seven genes, to be prevalent. Functionality of each SNP in interfering with mRNA/miR binding was tested using a dual luciferase reporter gene system. RESULTS: Of these nine SNPs, four SNPs, located in the arginine vasopressin 1A receptor (AVPR1A), bradykinin 2 receptor (BDKRB2), and thromboxane A2 receptor (TBXA2R) genes were associated with blood pressure. The rare allele of the AVPR1A SNP rs11174811, was associated with increased blood pressure whereas the rare alleles of the two linked BDKRB2 SNPs rs5225 and rs2069591 and of the TBXA2R SNP rs13306046 were associated with decreased blood pressure. Although not associated with blood pressure, the rare allele of the mineralocorticoid receptor (NR3C2) SNP rs5534, was associated with a twofold increased risk of myocardial infarction in men younger than 50 years. For all of these five SNPs, except rs2069591, we could demonstrate a reduction in miR-induced repression of gene expression. CONCLUSIONS: Common SNPs in miR binding sites of RAAS-related genes can influence both blood pressure and risk of myocardial infarction. These results may imply an important role for SNPs in miR target sites in human disease.
Asunto(s)
MicroARNs/genética , MicroARNs/metabolismo , Sistema Renina-Angiotensina/genética , Regiones no Traducidas 3'/genética , Regiones no Traducidas 3'/fisiología , Sitios de Unión/genética , Presión Sanguínea/genética , Presión Sanguínea/fisiología , Factor VIII/genética , Humanos , Infarto del Miocardio/etiología , Infarto del Miocardio/genética , Polimorfismo de Nucleótido Simple , Receptor de Bradiquinina B2/genética , Receptores de Tromboxano A2 y Prostaglandina H2/genética , Receptores de Vasopresinas/genética , Sistema Renina-Angiotensina/fisiología , Factor de von Willebrand/genéticaRESUMEN
The angiotensin II type 1 receptor (AT(1)R) is known to signal through heterotrimeric G proteins, and Gαq protein-independent signalling has only recently gained appreciation for profound impact on a diverse range of biological functions. ß-Arrestins, among other central mediators of Gαq protein-independent signalling from the AT(1)R interact with transcriptional regulators and promote phosphorylation of nuclear proteins. However, the relative contribution of Gαq protein-independent signalling in AT(1)R mediated transcriptional regulation remains elusive. We here present a comprehensive comparative analysis of Gαq protein-dependent and -independent regulation of AT(1)R mediated gene expression. We found angiotensin II to regulate 212 genes, whereas Gαq-independent signalling obtained with the biased agonist, SII angiotensin II only regulated few genes. Interestingly, SII angiotensin II, like Ang II vastly potentiated ß2-adrenergic receptor-stimulated gene expression. These novel findings indicate that the Gαq protein-independent signalling mainly modifies the transcriptional response governed by other signalling pathways, while direct induction of gene expression by the AT(1)R is dependent on classical Gαq protein activation.
Asunto(s)
Regulación de la Expresión Génica , Receptor de Angiotensina Tipo 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Transducción de Señal/genética , Transcripción Genética , Angiotensina II/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Reacción en Cadena de la Polimerasa , Receptor de Angiotensina Tipo 1/genética , Reproducibilidad de los Resultados , Elementos de Respuesta/genética , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
Delta like 1 homolog (Dlk1) exists in both transmembrane and soluble molecular forms, and is implicated in cellular growth and plays multiple roles in development, tissue regeneration, and cancer. Thus, DLK1 levels are critical for cell function, and abnormal DLK1 expression can be lethal; however, little is known about the underlying mechanisms. We here report that miR-15a modulates DLK1 levels in preadipocytes thus providing a mechanism for DLK1 regulation that further links it to cell cycle arrest and cancer since miR-15a is deregulated in these processes. In preadipocytes, miR-15a increases with cell density, and peaks at the same stage where membrane DLK1(M) and soluble DLK1(S) are found at maximum levels. Remarkably, miR-15a represses the amount of all Dlk1 variants at the mRNA level but also the level of DLK1(M) protein while it increases the amount of DLK1(S) supporting a direct repression of DLK1 and a parallel effect on the protease that cleaves off the DLK1 from the membrane. In agreement with previous studies, we found that miR-15a represses cell numbers, but additionally, we report that miR-15a also increases cell size. Conversely, anti-miR-15a treatment decreases cell size while increasing cell numbers, scenarios that were completely rescued by addition of purified DLK1(S). Our data thus imply that miR-15a regulates cell size and proliferation by fine-tuning Dlk1 among others, and further emphasize miR-15a and DLK1 levels to play important roles in growth signaling networks.
Asunto(s)
Adipocitos/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Regiones no Traducidas 3' , Células 3T3-L1 , Adipocitos/citología , Adipogénesis/genética , Adipogénesis/fisiología , Animales , Secuencia de Bases , Sitios de Unión/genética , Proteínas de Unión al Calcio , Ciclo Celular , Proliferación Celular , Tamaño de la Célula , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
Seven-transmembrane receptors (7TMRs) signal through the well described heterotrimeric G proteins but can also activate G protein-independent signaling pathways of which the impact and complexity are less understood. The angiotensin II type 1 receptor (AT(1)R) is a prototypical 7TMR and an important drug target in cardiovascular diseases. "Biased agonists" with intrinsic "functional selectivity" that simultaneously blocks Galpha(q) protein activity and activates G protein-independent pathways of the AT(1)R confer important perspectives in treatment of cardiovascular diseases. In this study, we performed a global quantitative phosphoproteomics analysis of the AT(1)R signaling network. We analyzed ligand-stimulated SILAC (stable isotope labeling by amino acids in cell culture) cells by high resolution (LTQ-Orbitrap) MS and compared the phosphoproteomes of the AT(1)R agonist angiotensin II and the biased agonist [Sar(1),Ile(4),Ile(8)]angiotensin II (SII angiotensin II), which only activates the Galpha(q) protein-independent signaling. We quantified more than 10,000 phosphorylation sites of which 1183 were regulated by angiotensin II or its analogue SII angiotensin II. 36% of the AT(1)R-regulated phosphorylations were regulated by SII angiotensin II. Analysis of phosphorylation site patterns showed a striking distinction between protein kinases activated by Galpha(q) protein-dependent and -independent mechanisms, and we now place protein kinase D as a key protein involved in both Galpha(q)-dependent and -independent AT(1)R signaling. This study provides substantial novel insight into angiotensin II signal transduction and is the first study dissecting the differences between a full agonist and a biased agonist from a 7TMR on a systems-wide scale. Importantly, it reveals a previously unappreciated diversity and quantity of Galpha(q) protein-independent signaling and uncovers novel signaling pathways. We foresee that the amount and diversity of G protein-independent signaling may be more pronounced than previously recognized for other 7TMRs as well. Quantitative mass spectrometry is a promising tool for evaluation of the signaling properties of biased agonists to other receptors in the future.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Fosfoproteínas/análisis , Proteoma/análisis , Receptor de Angiotensina Tipo 1 , Secuencia de Aminoácidos , Línea Celular , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/antagonistas & inhibidores , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Humanos , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Receptor de Angiotensina Tipo 1/agonistas , Receptor de Angiotensina Tipo 1/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Residues that mediate helix-helix interactions within the seven transmembranes (TM) of G protein-coupled receptors are important for receptor biogenesis and the receptor switch mechanism. By contrast, the residues directly contacting the lipid bilayer have only recently garnered attention as potential receptor dimerization interfaces. In the present study, we aimed to determine the contributions of these lipid-facing residues to receptor function and oligomerization by systemically generating chimeric complement factor 5a receptors in which the entire lipid-exposed surface of a single TM helix was exchanged with the cognate residues from the angiotensin type 1 receptor. Disulfide-trapping and bioluminescence resonance energy transfer (BRET) studies demonstrated robust homodimerization of both complement factor 5a receptor and angiotensin type 1 receptor, but no evidence for heterodimerization. Despite relatively conservative substitutions, the lipid-facing chimeras (TM1, TM2, TM4, TM5, TM6 or TM7) were retained in the endoplasmic reticulum/cis-Golgi network. With the exception of the TM7 chimera that did not bind ligand, the lipid-facing chimeras bound ligand with low affinity, but similar to wild-type complement factor 5a receptors trapped in the endoplasmic reticulum with brefeldin A. These results suggest that the chimeric receptors were properly folded; moreover, native complement factor 5a receptors are not fully competent to bind ligand when present in the endoplasmic reticulum. BRET oligomerization studies demonstrated energy transfer between the wild-type complement factor 5a receptor and the lipid-facing chimeras, suggesting that the lipid-facing residues within a single TM segment are not essential for oligomerization. These studies highlight the importance of the lipid-facing residues in the complement factor 5a receptor for transport competence.
Asunto(s)
Membrana Dobles de Lípidos , Receptor de Anafilatoxina C5a/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Biopolímeros , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , Transferencia de Energía , Humanos , Luminiscencia , Modelos Moleculares , Datos de Secuencia Molecular , Receptor de Anafilatoxina C5a/química , Homología de Secuencia de AminoácidoRESUMEN
The Renin-Angiotensin System (RAS) is important for the regulation of cardiovascular physiology, where it controls blood pressure, and salt- and water homeostasis. Dysregulation of RAS can lead to severe diseases including hypertension, diabetic nephropathy, and cardiac arrhythmia, and -failure. The importance of the RAS is clearly emphasised by the widespread use of drugs targeting this system in clinical practice. These include, renin inhibitors, angiotensin II receptor type I blockers, and inhibitors of the angiotensin converting enzyme. Some of the important effectors within the system are 7 transmembrane (7TM) receptors (or G-protein-coupled receptors) such as the angiotensin II Receptors type I and II (AT1R and AT2R) and the MAS-oncogene receptor. Several findings indicate that the 7TM receptors can form both homo- and heterodimers, or higher orders of oligomers. Furthermore, dimerization may be important for receptor function, and in the development of cardiovascular diseases. This is very significant, since "dimers" may provide pharmacologists with novel targets for improved drug therapy. However, we know that 7TM receptors can mediate signals as monomeric units, and so far it has been very difficult to establish if our observations reflect actual well-defined dimerization or merely reflect close proximity between the receptors and/or various types of functional interaction. In this review, we will present and critically discuss the current data on 7TM receptor dimerization with a clear focus on the RAS, and delineate future challenges within the field.
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Receptor Cross-Talk , Receptores Acoplados a Proteínas G/metabolismo , Sistema Renina-Angiotensina , Humanos , Multimerización de Proteína , Receptores de Angiotensina/metabolismo , Receptores de Bradiquinina/metabolismoRESUMEN
It has been suggested previously ( AbdAlla, S., Lother, H., and Quitterer, U. (2000) Nature 407, 94-98 ) that the angiotensin II type 1 receptor (AT1R) and the bradykinin B2 receptor (B2R) form constitutive heterodimers. Furthermore they demonstrate that AT1R signaling significantly increases in the presence of the B2R. These findings suggest that heterodimerization and potentiation of AT1R signaling is a universal phenomenon that occurs as a natural consequence of simultaneous expression of the two receptors. Hence this potential interaction is of great pharmacological and biological interest that adds an additional layer of complexity to the understanding of the cross-talk between the renin-angiotensin and kallikrein-kinin systems. Given the remarkable significance of this finding, scientists from four independent research groups have set out to reproduce and further examine the potential AT1R/B2R interaction. We have investigated functional potentiation by the B2R of AT1R signaling in three different cell lines using multiple assays including phosphoinositide hydrolysis, ERK activation, beta-arrestin recruitment, and receptor selection and amplification technology, and we have examined dimerization using bioluminescence resonance energy transfer and regulated secretion/aggregation technology. However, although both the AT1Rs and B2Rs were functional in our systems and the systems were fine tuned to detect small changes in receptor function, we failed to detect any functional modulation by or physical interaction between the two receptor proteins. In contrast to the previous observations, our data collectively suggest that AT1R/B2R heterodimerization does not occur as a natural consequence of their simultaneous expression in the same cell nor does the B2R influence the AT1R signaling.
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Receptor de Angiotensina Tipo 1/metabolismo , Receptor de Bradiquinina B2/metabolismo , Animales , Células COS , Chlorocebus aethiops , Dimerización , Regulación de la Expresión Génica/fisiología , Humanos , Sistema Calicreína-Quinina/fisiología , Ratones , Células 3T3 NIH , Ratas , Receptor de Angiotensina Tipo 1/genética , Receptor de Bradiquinina B2/genética , Sistema Renina-Angiotensina/fisiología , Transducción de Señal/fisiologíaRESUMEN
Multiple reports have focused on S100A4's role in cancer progression, specifically its ability to enhance metastasis. However, recent studies have linked S100A4 to several diseases besides cancer, including kidney fibrosis, cirrhosis, pulmonary disease, cardiac hypertrophy and fibrosis, arthritis and neuronal injuries. Common to all these diseases is the involvement of fibrotic and inflammatory processes, i.e. processes greatly dependent on tissue remodelling, cell motility and epithelial-mesenchymal transition. Therefore, the basic biological mechanisms behind S100A4's effects are emerging. S100A4 belongs to the S100 family of proteins that contain two Ca2+-binding sites including a canonical EF-hand motif. S100A4 is involved in the regulation of a wide range of biological effects including cell motility, survival, differentiation and contractility. S100A4 has both intracellular and extracellular effects. Hence, S100A4 interacts with cytoskeletal proteins and enhances metastasis of several types of cancer cells. In addition, S100A4 is secreted by unknown mechanisms, thus, paracrinely stimulating a variety of cellular responses, including angiogenesis and neuronal growth. Although many cellular effects of S100A4 are well described, the molecular mechanisms whereby S100A4 elicits these responses remain largely unknown. However, it is likely that the intracellular and the extracellular effects involve distinct mechanisms. In this review, we explore the possible roles of S100A4 in non-cancer diseases and employ this knowledge to describe underlying biological mechanisms including a change in cellular phenotype towards less tightly adherent cells and activation of fibrotic processes that may explain this protein's involvement in multiple pathologies.
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Enfermedad , Epitelio/patología , Mesodermo/patología , Regeneración , Proteínas S100/metabolismo , Animales , Fibrosis , HumanosRESUMEN
To analyze the interaction between the neurokinin-1 (NK-1) receptor and G-protein coupled receptor kinases (GRKs), we performed bioluminescence resonance energy transfer(2) (BRET(2)) measurements between the family A NK-1 receptor and GRK2 and GRK5 as well as their respective kinase-inactive mutants. We observed agonist induced interaction of both GRK5 and GRK2 with the activated NK-1 receptor. In saturation experiments, we observed GRK5 to interact with the activated receptor in a monophasic manner while GRK2 interacted in a biphasic manner with the low affinity phase corresponding to receptor affinity for GRK5. Agonist induced GRK5 interaction with the receptor was dependent on intact kinase-activity, whereas the high affinity phase of GRK2 interaction was independent of kinase activity. We were surprised to find that the BRET(2) saturation experiments indicated that before receptor activation, the full-length NK-1 receptor, but not a functional C-terminal tail-truncated receptor, is preassociated with GRK5 in a relatively low-affinity state. We demonstrate that GRK5 can compete for agonist induced GRK2 interaction with the NK-1 receptor, whereas GRK2 does not compete for receptor interaction with GRK5. We suggest that GRK5 is preassociated with the NK-1 receptor and that GRK5, rather than GRK2, is a key player in competitive regulation of GRK subtype specific interaction with the NK-1 receptor.
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Transferencia de Energía/fisiología , Quinasas de Receptores Acoplados a Proteína-G/metabolismo , Proteínas Luminiscentes/metabolismo , Receptores de Neuroquinina-1/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Quinasas de Receptores Acoplados a Proteína-G/análisis , Humanos , Proteínas Luminiscentes/análisis , Datos de Secuencia Molecular , Unión Proteica/fisiología , Receptores de Neuroquinina-1/análisis , RenillaRESUMEN
Two main pieces of data have created a new field in cardiac research. First, the traditional view on the heart as a postmitotic organ has been challenged by the finding of small dividing cells in the heart expressing cardiac contractile proteins with stem cell properties and, second, cellular therapy of the diseased heart using a variety of different cells has shown encouraging effects on cardiac function. These findings immediately raise questions like "what is the identity and origin of the cardiac progenitor cells?","which molecular factors are involved in their mobilization and differentiation?", and "can these cells repair the damaged heart?" This review will address the state of current answers to these questions. Emerging evidence suggests that several subpopulations of cardiac stem or progenitor cells (CPCs) reside within the adult heart. CPCs with the ability to differentiate into all the constituent cells in the adult heart including cardiac myocytes, vascular smooth muscle and endothelial cells have been identified. Valuable knowledge has been obtained from the large number of animal studies and a number of small clinical trials that have utilized a variety of adult stem cells for regenerating infarcted hearts. However, contradictory reports on the regenerative potential of the CPCs exist, and the mechanisms behind the reported hemodynamic effects are intensely debated. Besides directly replenishing cardiac tissue, CPCs could also function by stimulating angiogenesis and improving survival of existing cells by secretion of paracrine factors. With this review we suggest that a better understanding of CPC biology will be pivotal for progressing therapeutic cardiac regeneration. This includes an extended knowledge of the molecular mechanisms behind their mobilization, differentiation, survival and integration in the myocardium.
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Células Madre Adultas/fisiología , Corazón/fisiología , Miocardio/citología , Regeneración/fisiología , Animales , Diferenciación Celular/fisiología , Humanos , Isquemia Miocárdica/terapia , Trasplante de Células MadreRESUMEN
The G protein-coupled receptor kinase 2 (GRK2) phosphorylates and shuts down signaling from 7-transmembrane receptors (7TMs). Although, receptor activity controls GRK2 expression levels, the underlying molecular mechanisms are poorly understood. We have previously shown that extracellular signal-regulated kinase (ERK1/2) activation increases GRK2 expression [J. Theilade, J. Lerche Hansen, S. Haunso, S.P. Sheikh, Extracellular signal-regulated kinases control expression of G protein-coupled receptor kinase 2 (GRK2), FEBS Lett. 518 (2002) 195-199]. In the present study, we found that ERK1/2 regulates GRK2 degradation rather than synthesis. ERK1/2 blockade using PD98059 decreased GRK2 cellular levels to 0.25-fold of control in Cos7 cells. This effect was due to enhanced degradation of the GRK2 protein, since proteasome blockade prevented down-regulation of GRK2 protein levels in the presence of PD98059. Further, ERK blockade had no effect on GRK2 synthesis as probed using a reporter construct carrying the GRK2 promoter upstream of the luciferase gene. We predict ERK1/2 mediated GRK2 protection could be a general phenomenon as proteasome inhibition increased GRK2 expression in two other cell lines, HEK293 and NIH3T3.