RESUMEN
G protein-coupled receptors (GPCRs) regulate every aspect of kidney function by mediating the effects of various endogenous and exogenous substances. A key concept in GPCR function is biased signalling, whereby certain ligands may selectively activate specific pathways within the receptor's signalling repertoire. For example, different agonists may induce biased signalling by stabilizing distinct active receptor conformations - a concept that is supported by advances in structural biology. However, the processes underlying functional selectivity in receptor signalling are extremely complex, involving differences in subcellular compartmentalization and signalling dynamics. Importantly, the molecular mechanisms of spatiotemporal bias, particularly its connection to ligand binding kinetics, have been detailed for GPCRs critical to kidney function, such as the AT1 angiotensin receptor (AT1R), V2 vasopressin receptor (V2R) and the parathyroid hormone 1 receptor (PTH1R). This expanding insight into the multifaceted nature of biased signalling paves the way for innovative strategies for targeting GPCR functions; the development of novel biased agonists may represent advanced pharmacotherapeutic approaches to the treatment of kidney diseases and related systemic conditions, such as hypertension, diabetes and heart failure.
RESUMEN
The stabilization of different active conformations of G protein-coupled receptors is thought to underlie the varying efficacies of biased and balanced agonists. Here, profiling the activation of signal transducers by angiotensin II type 1 receptor (AT1R) agonists revealed that the extent and kinetics of ß-arrestin binding exhibited substantial ligand-dependent differences, which were lost when receptor internalization was inhibited. When AT1R endocytosis was prevented, even weak partial agonists of the ß-arrestin pathway acted as full or near-full agonists, suggesting that receptor conformation did not exclusively determine ß-arrestin recruitment. The ligand-dependent variance in ß-arrestin translocation was much larger at endosomes than at the plasma membrane, showing that ligand efficacy in the ß-arrestin pathway was spatiotemporally determined. Experimental investigations and mathematical modeling demonstrated how multiple factors concurrently shaped the effects of agonists on endosomal receptor-ß-arrestin binding and thus determined the extent of functional selectivity. Ligand dissociation rate and G protein activity had particularly strong, internalization-dependent effects on the receptor-ß-arrestin interaction. We also showed that endocytosis regulated the agonist efficacies of two other receptors with sustained ß-arrestin binding: the V2 vasopressin receptor and a mutant ß2-adrenergic receptor. In the absence of endocytosis, the agonist-dependent variance in ß-arrestin2 binding was markedly diminished. Our results suggest that endocytosis determines the spatiotemporal bias in GPCR signaling and can aid in the development of more efficacious, functionally selective compounds.
Asunto(s)
Endocitosis , Receptor de Angiotensina Tipo 1 , Transducción de Señal , beta-Arrestinas , Endocitosis/fisiología , Humanos , Receptor de Angiotensina Tipo 1/metabolismo , Receptor de Angiotensina Tipo 1/genética , beta-Arrestinas/metabolismo , beta-Arrestinas/genética , Células HEK293 , Receptores de Vasopresinas/metabolismo , Receptores de Vasopresinas/genética , Receptores Adrenérgicos beta 2/metabolismo , Receptores Adrenérgicos beta 2/genética , Endosomas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Animales , Ligandos , Unión Proteica , Transporte de ProteínasRESUMEN
The binding and function of ß-arrestins are regulated by specific phosphorylation motifs present in G protein-coupled receptors (GPCRs). However, the exact arrangement of phosphorylated amino acids responsible for establishing a stable interaction remains unclear. We employ a 1D sequence convolution model trained on GPCRs with established ß-arrestin-binding properties. With this approach, amino acid motifs characteristic of GPCRs that form stable interactions with ß-arrestins can be identified, a pattern that we name "arreSTick." Intriguingly, the arreSTick pattern is also present in numerous non-receptor proteins. Using proximity biotinylation assay and mass spectrometry analysis, we demonstrate that the arreSTick motif controls the interaction between many non-receptor proteins and ß-arrestin2. The HIV-1 Tat-specific factor 1 (HTSF1 or HTATSF1), a nuclear transcription factor, contains the arreSTick pattern, and its subcellular localization is influenced by ß-arrestin2. Our findings unveil a broader role for ß-arrestins in phosphorylation-dependent interactions, extending beyond GPCRs to encompass non-receptor proteins as well.
Asunto(s)
Secuencias de Aminoácidos , Unión Proteica , beta-Arrestinas , Fosforilación , Humanos , beta-Arrestinas/metabolismo , Células HEK293 , Arrestina beta 2/metabolismo , Secuencia de Aminoácidos , Estabilidad ProteicaRESUMEN
ß-arrestins, which have multiple cellular functions, were initially described as proteins that desensitize rhodopsin and other G protein-coupled receptors. The cytoskeletal system plays a role in various cellular processes, including intracellular transport, cell division, organization of organelles, and cell cycle. The interactome of ß-arrestins includes the major proteins of the three main cytoskeletal systems: tubulins for microtubules, actins for the actin filaments, and vimentin for intermediate filaments. ß-arrestins bind to microtubules and regulate their activity by recruiting signaling proteins and interacting with assembly proteins that regulate the actin cytoskeleton and the intermediate filaments. Altered regulation of the cytoskeletal system plays an essential role in the development of Alzheimer's, Parkinson's and other neurodegenerative diseases. Thus, ß-arrestins, which interact with the cytoskeleton, were implicated in the pathogenesis progression of these diseases and are potential targets for the treatment of neurodegenerative disorders in the future.
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Citoesqueleto , Enfermedades Neurodegenerativas , Humanos , beta-Arrestinas/metabolismo , Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Actinas/metabolismo , beta-Arrestina 1/metabolismo , Enfermedades Neurodegenerativas/metabolismoRESUMEN
Angiotensin II (AngII) is a vasoactive peptide hormone, which, under pathological conditions, contributes to the development of cardiovascular diseases. Oxysterols, including 25-hydroxycholesterol (25-HC), the product of cholesterol-25-hydroxylase (CH25H), also have detrimental effects on vascular health by affecting vascular smooth muscle cells (VSMCs). We investigated AngII-induced gene expression changes in VSMCs to explore whether AngII stimulus and 25-HC production have a connection in the vasculature. RNA-sequencing revealed that Ch25h is significantly upregulated in response to AngII stimulus. The Ch25h mRNA levels were elevated robustly (~50-fold) 1 h after AngII (100 nM) stimulation compared to baseline levels. Using inhibitors, we specified that the AngII-induced Ch25h upregulation is type 1 angiotensin II receptor- and Gq/11 activity-dependent. Furthermore, p38 MAPK has a crucial role in the upregulation of Ch25h. We performed LC-MS/MS to identify 25-HC in the supernatant of AngII-stimulated VSMCs. In the supernatants, 25-HC concentration peaked 4 h after AngII stimulation. Our findings provide insight into the pathways mediating AngII-induced Ch25h upregulation. Our study elucidates a connection between AngII stimulus and 25-HC production in primary rat VSMCs. These results potentially lead to the identification and understanding of new mechanisms in the pathogenesis of vascular impairments.
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Angiotensina II , Músculo Liso Vascular , Esteroide Hidroxilasas , Animales , Ratas , Angiotensina II/metabolismo , Células Cultivadas , Cromatografía Liquida , Expresión Génica , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/metabolismo , Espectrometría de Masas en Tándem , Esteroide Hidroxilasas/genéticaRESUMEN
Background: Osteoclasts play a crucial role in the maintenance, repair, and remodeling of bones of the adult vertebral skeleton due to their bone resorption capability. Rheumatoid arthritis (RA) and psoriatic arthritis (PsA) are associated with increased activity of osteoclasts. Objectives: Our study aimed to investigate the dynamic proteomic changes during osteoclast differentiation in healthy donors, in RA, and PsA. Methods: Blood samples of healthy donors, RA, and PsA patients were collected, and monocytes were isolated and differentiated into osteoclasts in vitro using macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANK-L). Mass spectrometry-based proteomics was used to analyze proteins from cell lysates. The expression changes were analyzed with Gene Set Enrichment Analysis (GSEA). Results: The analysis of the proteomic changes revealed that during the differentiation of the human osteoclasts, expression of the proteins involved in metabolic activity, secretory function, and cell polarity is increased; by contrast, signaling pathways involved in the immune functions are downregulated. Interestingly, the differences between cells of healthy donors and RA/PsA patients are most pronounced after the final steps of differentiation to osteoclasts. In addition, both in RA and PsA the differentiation is characterized by decreased metabolic activity, associated with various immune pathway activities; furthermore by accelerated cytokine production in RA. Conclusions: Our results shed light on the characteristic proteomic changes during human osteoclast differentiation and expression differences in RA and PsA, which reveal important pathophysiological insights in both diseases.
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Artritis Psoriásica , Artritis Reumatoide , Resorción Ósea , Adulto , Humanos , Osteoclastos/metabolismo , ProteómicaRESUMEN
The urine concentrating function of the kidney is essential to maintain the water homeostasis of the human body. It is mainly regulated by the arginine-vasopressin (AVP), which targets the type 2 vasopressin receptor (V2R) in the kidney. The inability of V2R to respond to AVP stimulation leads to decreased urine concentration and congenital nephrogenic diabetes insipidus (NDI). NDI is characterized by polyuria, polydipsia, and hyposthenuria. In this study, we identified a point mutation (S127F) in the AVPR2 gene of an NDI patient, and we characterized the impaired function of the V2R mutant in HEK293 cells. Based on our data, the S127F-V2R mutant is almost exclusively located intracellularly in the endoplasmic reticulum (ER), and very few receptors were detected at the cell surface, where the receptor can bind to AVP. The overexpressed S127F-V2R mutant receptor has negligible cAMP generation capability compared to the wild-type receptor in response to AVP stimulation. Since certain misfolded mutant proteins, that are retained in the ER, can be rescued by pharmacological chaperones, we examined the potential rescue effects of two pharmacochaperones on the S127F-V2R. We found that pretreatment with both tolvaptan (an established V2R inverse agonist) and MCF14 compound (a cell-permeable high-affinity agonist for the V2R) were capable of partially restoring the cAMP generating function of the receptor in response to vasopressin stimulation. According to our data, both cell permeant agonists and antagonists can function as pharmacochaperones, and serve as the starting compounds to develop medicines for patients carrying the S127F mutation.
RESUMEN
The protease caspase-3 is a key mediator of apoptotic programmed cell death. But weak or transient caspase activity can contribute to neuronal differentiation, axonal pathfinding, and synaptic long-term depression. Despite the importance of sublethal, or nonapoptotic, caspase activity in neurodevelopment and neural plasticity, there has been no simple method for mapping and quantifying nonapoptotic caspase activity (NACA) in rodent brains. We therefore generated a transgenic mouse expressing a highly sensitive and specific fluorescent reporter of caspase activity, with peak signal localized to the nucleus. As a proof of concept, we first obtained evidence that NACA influences neurophysiology in an amygdalar circuit. Then focusing on the amygdala, we were able to quantify a sex-specific persistent elevation in caspase activity in females after restraint stress. This simple in vivo caspase activity reporter will facilitate systems-level studies of apoptotic and nonapoptotic phenomena in behavioral and pathologic models.
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Apoptosis , Encéfalo , Masculino , Femenino , Ratones , Animales , Apoptosis/fisiología , Ratones Transgénicos , Plasticidad Neuronal , Caspasa 9RESUMEN
Activation of the type I angiotensin receptor (AT1-R) in vascular smooth muscle cells (VSMCs) plays a crucial role in the regulation of blood pressure; however, it is also responsible for the development of pathological conditions such as vascular remodeling, hypertension and atherosclerosis. Stimulation of the VSMC by angiotensin II (AngII) promotes a broad variety of biological effects, including gene expression changes. In this paper, we have taken an integrated approach in which an analysis of AngII-induced gene expression changes has been combined with the use of small-molecule inhibitors and lentiviral-based gene silencing, to characterize the mechanism of signal transduction in response to AngII stimulation in primary rat VSMCs. We carried out Affymetrix GeneChip experiments to analyze the effects of AngII stimulation on gene expression; several genes, including DUSP5, DUSP6, and DUSP10, were identified as upregulated genes in response to stimulation. Since various dual-specificity MAPK phosphatase (DUSP) enzymes are important in the regulation of mitogen-activated protein kinase (MAPK) signaling pathways, these genes have been selected for further analysis. We investigated the kinetics of gene-expression changes and the possible signal transduction processes that lead to altered expression changes after AngII stimulation. Our data shows that the upregulated genes can be stimulated through multiple and synergistic signal transduction pathways. We have also found in our gene-silencing experiments that epidermal growth factor receptor (EGFR) transactivation is not critical in the AngII-induced expression changes of the investigated genes. Our data can help us understand the details of AngII-induced long-term effects and the pathophysiology of AT1-R. Moreover, it can help to develop potential interventions for those symptoms that are induced by the over-functioning of this receptor, such as vascular remodeling, cardiac hypertrophy or atherosclerosis.
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Regulación Enzimológica de la Expresión Génica , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/genética , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/enzimología , Receptor de Angiotensina Tipo 1/metabolismo , Angiotensina II/farmacología , Animales , Línea Celular , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Cinética , Lentivirus/metabolismo , Masculino , Metaloproteinasas de la Matriz/metabolismo , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , ARN Interferente Pequeño/metabolismo , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Regulación hacia Arriba/genéticaRESUMEN
ß-arrestins are partners of the G protein-coupled receptors (GPCRs), regulating their intracellular trafficking and signaling. Development of biased GPCR agonists, selectively targeting either G protein or ß-arrestin pathways, are in the focus of interest due to their therapeutic potential in different pathological conditions. The CB2 cannabinoid receptor (CB2R) is a GPCR involved in various functions in the periphery and the central nervous system. Two common occurring variants of CB2R, harboring Q63R or L133I missense mutations, have been implicated in the development of a diverse set of disorders. To evaluate the effect of these mutations, we characterized the binding profile of these mutant CB2 receptors to G proteins and ß-arrestin2. Although their ability to inhibit cAMP signaling was similar, the Q63R mutant had increased, whereas the L133I mutant receptor had decreased ß-arrestin2 binding. In line with these observations, the variants also had altered intracellular trafficking. Our results show that two common variants of the CB2 receptor have biased signaling properties, which may contribute to the pathogenesis of the associated disorders and may offer CB2R as a target for further development of biased receptor activation strategies.
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Mutación Missense , Receptor Cannabinoide CB2/metabolismo , beta-Arrestinas/metabolismo , Células HEK293 , Humanos , Unión Proteica , Transporte de Proteínas , Receptor Cannabinoide CB2/química , Receptor Cannabinoide CB2/genética , beta-Arrestinas/genéticaRESUMEN
(1) Adeno-associated viruses (AAV) are safe and efficient gene therapy vectors with promising results in the treatment of several diseases. Extracellular vesicles (EV) are phospholipid bilayer-surrounded structures carrying several types of lipids, proteins, and nucleic acids with the ability to cross biological barriers. EV-associated AAVs might serve as new and efficient gene therapy vectors considering that they carry the benefits of both AAVs and EVs. (2) We tested vesicle-associated AAVs and vesicles mixed with AAVs on two major cell types of the central nervous system: a neural cell line (N2A) and primary astrocyte cells. (3) In contrast to previously published in vivo observations, the extracellular vesicle packaging did not improve but, in the case of primary astrocyte cells, even inhibited the infection capacity of the AAV particles. The observed effect was not due to the inhibitory effects of the vesicles themselves, since mixing the AAVs with extracellular vesicles did not change the effectiveness. (4) Our results suggest that improvement of the in vivo efficacy of the EV-associated AAV particles is not due to the enhanced interaction between the AAV and the target cells, but most likely to the improved delivery of the AAVs through tissue barriers and to the shielding of AAVs from neutralizing antibodies.
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Astrocitos/metabolismo , Dependovirus/metabolismo , Vesículas Extracelulares/metabolismo , Animales , Línea Celular , Células Cultivadas , Vectores Genéticos/genética , Humanos , Transducción Genética/métodosRESUMEN
Reliable measurement of ligand binding to cell surface receptors is of outstanding biological and pharmacological importance. Resonance energy transfer-based assays are powerful approaches to achieve this goal, but the currently available methods are hindered by the necessity of receptor tagging, which can potentially alter ligand binding properties. Therefore, we developed a tag-free system to measure ligandâreceptor interactions in live cells using the Gaussia luciferase (GLuc) as a bioluminescence resonance energy transfer donor. GLuc is as small as the commonly applied Nanoluciferase but has enhanced brightness, and its proper substrate is the frequently used coelenterazine. In our assay, bystander bioluminescence resonance energy transfer is detected between a GLuc-based extracellular surface biosensor and fluorescent ligands bound to their unmodified receptors. The broad spectrum of applications includes equilibrium and kinetic ligand binding measurements for both labeled and competitive unlabeled ligands, and the assay can be utilized for different classes of plasma membrane receptors. Furthermore, the assay is suitable for high-throughput screening, as evidenced by the identification of novel α1 adrenergic receptor ligands. Our data demonstrate that GLuc-based biosensors provide a simple, sensitive, and cost-efficient platform for drug characterization and development.
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Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Luciferasas/química , Luciferasas/metabolismo , Bioensayo , Membrana Celular/metabolismo , Transferencia de Energía , Células HEK293 , Humanos , Cinética , Ligandos , Unión Proteica , Transporte de Proteínas , Receptores Acoplados a Proteínas G/metabolismo , Transducción de SeñalRESUMEN
AT1 angiotensin receptor plays important physiological and pathophysiological roles in the cardiovascular system. Renin-angiotensin system represents a target system for drugs acting at different levels. The main effects of ATR1 stimulation involve activation of Gq proteins and subsequent IP3, DAG, and calcium signaling. It has become evident in recent years that besides the well-known G protein pathways, AT1R also activates a parallel signaling pathway through ß-arrestins. ß-arrestins were originally described as proteins that desensitize G protein-coupled receptors, but they can also mediate receptor internalization and G protein-independent signaling. AT1R is one of the most studied receptors, which was used to unravel the newly recognized ß-arrestin-mediated pathways. ß-arrestin-mediated signaling has become one of the most studied topics in recent years in molecular pharmacology and the modulation of these pathways of the AT1R might offer new therapeutic opportunities in the near future. In this paper, we review the recent advances in the field of ß-arrestin signaling of the AT1R, emphasizing its role in cardiovascular regulation and heart failure.
RESUMEN
AT1 angiotensin receptor (AT1R), a prototypical G protein-coupled receptor (GPCR), is the main receptor, which mediates the effects of the renin-angiotensin system (RAS). AT1R plays a crucial role in the regulation of blood pressure and salt-water homeostasis, and in the development of pathological conditions, such as hypertension, heart failure, cardiovascular remodeling, renal fibrosis, inflammation, and metabolic disorders. Stimulation of AT1R leads to pleiotropic signal transduction pathways generating arrays of complex cellular responses. Growing amount of evidence shows that AT1R is a versatile GPCR, which has multiple unique faces with distinct conformations and signaling properties providing new opportunities for functionally selective pharmacological targeting of the receptor. Biased ligands of AT1R have been developed to selectively activate the ß-arrestin pathway, which may have therapeutic benefits compared to the conventional angiotensin converting enzyme inhibitors and angiotensin receptor blockers. In this review, we provide a summary about the most recent findings and novel aspects of the AT1R function, signaling, regulation, dimerization or oligomerization and its cross-talk with other receptors, including epidermal growth factor (EGF) receptor, adrenergic receptors and CB1 cannabinoid receptor. Better understanding of the mechanisms and structural aspects of AT1R activation and cross-talk can lead to the development of novel type of drugs for the treatment of cardiovascular and other diseases.
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Receptor Cross-Talk , Receptor de Angiotensina Tipo 1/fisiología , Receptores Acoplados a Proteínas G/fisiología , Transducción de Señal/fisiología , Animales , Humanos , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/fisiología , Receptor Cross-Talk/fisiologíaRESUMEN
ß-Arrestins are key regulators and signal transducers of G protein-coupled receptors (GPCRs). The interaction between receptors and ß-arrestins is generally believed to require both receptor activity and phosphorylation by GPCR kinases. In this study, we investigated whether ß-arrestins are able to bind second messenger kinase-phosphorylated, but inactive receptors as well. Because heterologous phosphorylation is a common phenomenon among GPCRs, this mode of ß-arrestin activation may represent a novel mechanism of signal transduction and receptor cross-talk. Here we demonstrate that activation of protein kinase C (PKC) by phorbol myristate acetate, Gq/11-coupled GPCR, or epidermal growth factor receptor stimulation promotes ß-arrestin2 recruitment to unliganded AT1 angiotensin receptor (AT1R). We found that this interaction depends on the stability lock, a structure responsible for the sustained binding between GPCRs and ß-arrestins, formed by phosphorylated serine-threonine clusters in the receptor's C terminus and two conserved phosphate-binding lysines in the ß-arrestin2 N-domain. Using improved FlAsH-based serine-threonine clusters ß-arrestin2 conformational biosensors, we also show that the stability lock not only stabilizes the receptor-ß-arrestin interaction, but also governs the structural rearrangements within ß-arrestins. Furthermore, we found that ß-arrestin2 binds to PKC-phosphorylated AT1R in a distinct active conformation, which triggers MAPK recruitment and receptor internalization. Our results provide new insights into the activation of ß-arrestins and reveal their novel role in receptor cross-talk.
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Proteínas Quinasas Activadas por Mitógenos/metabolismo , beta-Arrestinas/metabolismo , Angiotensina II/metabolismo , Animales , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Immunoblotting , Microscopía Confocal , Fosforilación , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
It is still not possible to predict whether a given molecule will have a perceived odor or what olfactory percept it will produce. We therefore organized the crowd-sourced DREAM Olfaction Prediction Challenge. Using a large olfactory psychophysical data set, teams developed machine-learning algorithms to predict sensory attributes of molecules based on their chemoinformatic features. The resulting models accurately predicted odor intensity and pleasantness and also successfully predicted 8 among 19 rated semantic descriptors ("garlic," "fish," "sweet," "fruit," "burnt," "spices," "flower," and "sour"). Regularized linear models performed nearly as well as random forest-based ones, with a predictive accuracy that closely approaches a key theoretical limit. These models help to predict the perceptual qualities of virtually any molecule with high accuracy and also reverse-engineer the smell of a molecule.
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Odorantes , Percepción Olfatoria , Olfato , Adulto , Conjuntos de Datos como Asunto , Humanos , Masculino , Modelos BiológicosRESUMEN
Heterodimerization between angiotensin type 1A receptor (AT1R) and ß2-adrenergic receptor (ß2AR) has been shown to modulate G protein-mediated effects of these receptors. Activation of G protein-coupled receptors (GPCRs) leads to ß-arrestin binding, desensitization, internalization and G protein-independent signaling of GPCRs. Our aim was to study the effect of heterodimerization on ß-arrestin coupling. We found that ß-arrestin binding of ß2AR is affected by activation of AT1Rs. Costimulation with angiotensin II and isoproterenol markedly enhanced the interaction between ß2AR and ß-arrestins, by prolonging the lifespan of ß2AR-induced ß-arrestin2 clusters at the plasma membrane. While candesartan, a conventional AT1R antagonist, had no effect on the ß-arrestin2 binding to ß2AR, TRV120023, a ß-arrestin biased agonist, enhanced the interaction. These findings reveal a new crosstalk mechanism between AT1R and ß2AR, and suggest that enhanced ß-arrestin2 binding to ß2AR can contribute to the pharmacological effects of biased AT1R agonists.
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Receptor de Angiotensina Tipo 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , beta-Arrestinas/metabolismo , Angiotensina II/metabolismo , Animales , Bencimidazoles/farmacología , Compuestos de Bifenilo , Células CHO , Células COS , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Chlorocebus aethiops , Cricetulus , Dimerización , Proteínas de Unión al GTP/metabolismo , Células HEK293 , Humanos , Oligopéptidos/farmacología , Unión Proteica/efectos de los fármacos , Receptor de Angiotensina Tipo 1/agonistas , Transducción de Señal/efectos de los fármacos , Tetrazoles/farmacologíaRESUMEN
Activation of G protein-coupled receptors (GPCRs) can induce vasoconstriction via calcium signal-mediated and Rho-dependent pathways. Earlier reports have shown that diacylglycerol produced during calcium signal generation can be converted to an endocannabinoid, 2-arachidonoylglycerol (2-AG). Our aim was to provide evidence that GPCR signaling-induced 2-AG production and activation of vascular type1 cannabinoid receptors (CB1R) is capable of reducing agonist-induced vasoconstriction and hypertension. Rat and mouse aortic rings were examined by myography. Vascular expression of CB1R was demonstrated with immunohistochemistry. Rat aortic vascular smooth muscle cells (VSMCs) were cultured for calcium measurements and 2-AG-determination. Inhibition or genetic loss of CB1Rs enhanced vasoconstriction induced by angiotensin II (AngII) or phenylephrine (Phe), but not by prostaglandin(PG)F2α. AngII-induced vasoconstriction was augmented by inhibition of diacylglycerol lipase (tetrahydrolipstatin) and was attenuated by inhibition of monoacylglycerol lipase (JZL184) suggesting a functionally relevant role for endogenously produced 2-AG. In Gαq/11-deficient mice vasoconstriction was absent to AngII or Phe, which activate Gq/11-coupled receptors, but was maintained in response to PGF2α. In VSMCs, AngII-stimulated 2-AG-formation was inhibited by tetrahydrolipstatin and potentiated by JZL184. CB1R inhibition increased the sustained phase of AngII-induced calcium signal. Pharmacological or genetic loss of CB1R function augmented AngII-induced blood pressure rise in mice. These data demonstrate that vasoconstrictor effect of GPCR agonists is attenuated via Gq/11-mediated vascular endocannabinoid formation. Agonist-induced endocannabinoid-mediated CB1R activation is a significant physiological modulator of vascular tone. Thus, the selective modulation of GPCR signaling-induced endocannabinoid release has a therapeutic potential in case of increased vascular tone and hypertension.
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Aorta/efectos de los fármacos , Ácidos Araquidónicos/farmacología , Endocannabinoides/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Glicéridos/farmacología , Hipertensión/metabolismo , Receptor Cannabinoide CB1/metabolismo , Vasoconstricción/efectos de los fármacos , Angiotensina II/farmacología , Animales , Benzodioxoles/farmacología , Calcio/metabolismo , Señalización del Calcio , Dinoprost/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/deficiencia , Regulación de la Expresión Génica , Hipertensión/tratamiento farmacológico , Hipertensión/genética , Hipertensión/fisiopatología , Lactonas/farmacología , Lipoproteína Lipasa/antagonistas & inhibidores , Lipoproteína Lipasa/genética , Lipoproteína Lipasa/metabolismo , Masculino , Ratones , Ratones Noqueados , Monoacilglicerol Lipasas/antagonistas & inhibidores , Monoacilglicerol Lipasas/genética , Monoacilglicerol Lipasas/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Orlistat , Fenilefrina/farmacología , Piperidinas/farmacología , Ratas , Ratas Wistar , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptor Cannabinoide CB1/genética , Técnicas de Cultivo de TejidosRESUMEN
The role of the highly conserved 'DRY' motif in the signaling of the CB1 cannabinoid receptor (CB1R) was investigated by inducing single-, double-, and triple-alanine mutations into this site of the receptor. We found that the CB1R-R3.50A mutant displays a partial decrease in its ability to activate heterotrimeric Go proteins (â¼80% of WT CB1R (CB1R-WT)). Moreover, this mutant showed an enhanced basal ß-arrestin2 (ß-arr2) recruitment. More strikingly, the double-mutant CB1R-D3.49A/R3.50A was biased toward ß-arrs, as it gained a robustly increased ß-arr1 and ß-arr2 recruitment ability compared with the WT receptor, while its G-protein activation was decreased. In contrast, the double-mutant CB1R-R3.50A/Y3.51A proved to be G-protein-biased, as it was practically unable to recruit ß-arrs in response to agonist stimulus, while still activating G-proteins, although at a reduced level (â¼70% of CB1R-WT). Agonist-induced ERK1/2 activation of the CB1R mutants showed a good correlation with their ß-arr recruitment ability but not with their G-protein activation or inhibition of cAMP accumulation. Our results suggest that G-protein activation and ß-arr binding of the CB1R are mediated by distinct receptor conformations, and the conserved 'DRY' motif plays different roles in the stabilization of these conformations, thus mediating both G-protein- and ß-arr-mediated functions of CB1R.
Asunto(s)
Receptor Cannabinoide CB1/genética , Secuencias de Aminoácidos , Animales , Arrestinas/metabolismo , Células CHO , Cricetinae , Cricetulus , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Células HeLa , Humanos , Mutación Missense , Transporte de Proteínas , Ratas , Receptor Cannabinoide CB1/metabolismo , beta-Arrestina 1 , Arrestina beta 2 , beta-ArrestinasRESUMEN
CB1 cannabinoid receptor (CB1R) undergoes both constitutive and agonist-induced internalization, but the underlying mechanisms of these processes and the role of ß-arrestins in the regulation of CB1R function are not completely understood. In this study, we followed CB1R internalization using confocal microscopy and bioluminescence resonance energy transfer measurements in HeLa and Neuro-2a cells. We found that upon activation CB1R binds ß-arrestin2 (ß-arr2), but not ß-arrestin1. Furthermore, both the expression of dominant-negative ß-arr2 (ß-arr2-V54D) and siRNA-mediated knock-down of ß-arr2 impaired the agonist-induced internalization of CB1R. In contrast, neither ß-arr2-V54D nor ß-arr2-specific siRNA had a significant effect on the constitutive internalization of CB1R. However, both constitutive and agonist-induced internalization of CB1R were impaired by siRNA-mediated depletion of clathrin heavy chain. We conclude that although clathrin is required for both constitutive and agonist-stimulated internalization of CB1R, ß-arr2 binding is only required for agonist-induced internalization of the receptor suggesting that the molecular mechanisms underlying constitutive and agonist-induced internalization of CB1R are different.