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1.
Nature ; 579(7798): 297-302, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31945772

RESUMO

After activation by an agonist, G-protein-coupled receptors (GPCRs) recruit ß-arrestin, which desensitizes heterotrimeric G-protein signalling and promotes receptor endocytosis1. Additionally, ß-arrestin directly regulates many cell signalling pathways that can induce cellular responses distinct from that of G proteins2. In contrast to G proteins, for which there are many high-resolution structures in complex with GPCRs, the molecular mechanisms underlying the interaction of ß-arrestin with GPCRs are much less understood. Here we present a cryo-electron microscopy structure of ß-arrestin 1 (ßarr1) in complex with M2 muscarinic receptor (M2R) reconstituted in lipid nanodiscs. The M2R-ßarr1 complex displays a multimodal network of flexible interactions, including binding of the N domain of ßarr1 to phosphorylated receptor residues and insertion of the finger loop of ßarr1 into the M2R seven-transmembrane bundle, which adopts a conformation similar to that in the M2R-heterotrimeric Go protein complex3. Moreover, the cryo-electron microscopy map reveals that the C-edge of ßarr1 engages the lipid bilayer. Through atomistic simulations and biophysical, biochemical and cellular assays, we show that the C-edge is critical for stable complex formation, ßarr1 recruitment, receptor internalization, and desensitization of G-protein activation. Taken together, these data suggest that the cooperative interactions of ß-arrestin with both the receptor and the phospholipid bilayer contribute to its functional versatility.


Assuntos
Lipídeos/química , Modelos Moleculares , beta-Arrestinas/química , Linhagem Celular , Simulação por Computador , Microscopia Crioeletrônica , Humanos , Nanoestruturas/química , Estrutura Terciária de Proteína
2.
Nat Struct Mol Biol ; 26(12): 1123-1131, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31740855

RESUMO

Classically, G-protein-coupled receptors (GPCRs) are thought to activate G protein from the plasma membrane and are subsequently desensitized by ß-arrestin (ß-arr). However, some GPCRs continue to signal through G protein from internalized compartments, mediated by a GPCR-G protein-ß-arr 'megaplex'. Nevertheless, the molecular architecture of the megaplex remains unknown. Here, we present its cryo-electron microscopy structure, which shows simultaneous engagement of human G protein and bovine ß-arr to the core and phosphorylated tail, respectively, of a single active human chimeric ß2-adrenergic receptor with the C-terminal tail of the arginine vasopressin type 2 receptor (ß2V2R). All three components adopt their canonical active conformations, suggesting that a single megaplex GPCR is capable of simultaneously activating G protein and ß-arr. Our findings provide a structural basis for GPCR-mediated sustained internalized G protein signaling.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais , beta-Arrestinas/metabolismo , Animais , Bovinos , Microscopia Crioeletrônica , Endossomos/metabolismo , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/ultraestrutura , Humanos , Modelos Moleculares , Conformação Proteica , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Receptores Adrenérgicos beta 2/ultraestrutura , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/ultraestrutura , Receptores de Vasopressinas/química , Receptores de Vasopressinas/metabolismo , Receptores de Vasopressinas/ultraestrutura , beta-Arrestinas/química , beta-Arrestinas/ultraestrutura
3.
Nat Commun ; 10(1): 4752, 2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31628314

RESUMO

Meningococcus utilizes ß-arrestin selective activation of endothelial cell ß2 adrenergic receptor (ß2AR) to cause meningitis in humans. Molecular mechanisms of receptor activation by the pathogen and of its species selectivity remained elusive. We report that ß2AR activation requires two asparagine-branched glycan chains with terminally exposed N-acetyl-neuraminic acid (sialic acid, Neu5Ac) residues located at a specific distance in its N-terminus, while being independent of surrounding amino-acid residues. Meningococcus triggers receptor signaling by exerting direct and hemodynamic-promoted traction forces on ß2AR glycans. Similar activation is recapitulated with beads coated with Neu5Ac-binding lectins, submitted to mechanical stimulation. This previously unknown glycan-dependent mode of allosteric mechanical activation of a G protein-coupled receptor contributes to meningococcal species selectivity, since Neu5Ac is only abundant in humans due to the loss of CMAH, the enzyme converting Neu5Ac into N-glycolyl-neuraminic acid in other mammals. It represents an additional mechanism of evolutionary adaptation of a pathogen to its host.


Assuntos
Fímbrias Bacterianas/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Neisseria meningitidis/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Fímbrias Bacterianas/genética , Células HEK293 , Humanos , Lectinas/metabolismo , Microscopia Confocal , Neisseria meningitidis/fisiologia , Polissacarídeos/metabolismo , Receptores Adrenérgicos beta 2/genética , Homologia de Sequência de Aminoácidos , beta-Arrestinas/metabolismo
4.
Molecules ; 24(20)2019 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-31614728

RESUMO

Cannabinoid receptor interacting protein 1a (CRIP1a) is an important CB1 cannabinoid receptor-associated protein, first identified from a yeast two-hybrid screen to modulate CB1-mediated N-type Ca2+ currents. In this paper we review studies of CRIP1a function and structure based upon in vitro experiments and computational chemistry, which elucidate the specific mechanisms for the interaction of CRIP1a with CB1 receptors. N18TG2 neuronal cells overexpressing or silencing CRIP1a highlighted the ability of CRIP1 to regulate cyclic adenosine 3',5'monophosphate (cAMP) production and extracellular signal-regulated kinase (ERK1/2) phosphorylation. These studies indicated that CRIP1a attenuates the G protein signaling cascade through modulating which Gi/o subtypes interact with the CB1 receptor. CRIP1a also attenuates CB1 receptor internalization via ß-arrestin, suggesting that CRIP1a competes for ß-arrestin binding to the CB1 receptor. Predictions of CRIP1a secondary structure suggest that residues 34-110 are minimally necessary for association with key amino acids within the distal C-terminus of the CB1 receptor, as well as the mGlu8a metabotropic glutamate receptor. These interactions are disrupted through phosphorylation of serines and threonines in these regions. Through investigations of the function and structure of CRIP1a, new pharmacotherapies based upon the CRIP-CB1 receptor interaction can be designed to treat diseases such as epilepsy, motor dysfunctions and schizophrenia.


Assuntos
Canabinoides/metabolismo , Proteínas de Transporte/genética , Receptor CB1 de Canabinoide/genética , Canabinoides/genética , Proteínas de Transporte/química , Epilepsia/tratamento farmacológico , Epilepsia/genética , Humanos , Sistema de Sinalização das MAP Quinases , Transtornos Motores/tratamento farmacológico , Transtornos Motores/genética , Neurônios/metabolismo , Neurônios/patologia , Fosforilação/efeitos dos fármacos , Esquizofrenia/tratamento farmacológico , Esquizofrenia/genética , Transdução de Sinais/efeitos dos fármacos , beta-Arrestinas/genética , beta-Arrestinas/metabolismo
5.
Nat Commun ; 10(1): 4075, 2019 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-31501422

RESUMO

Signaling diversity of G protein-coupled (GPCR) ligands provides novel opportunities to develop more effective, better-tolerated therapeutics. Taking advantage of these opportunities requires identifying which effectors should be specifically activated or avoided so as to promote desired clinical responses and avoid side effects. However, identifying signaling profiles that support desired clinical outcomes remains challenging. This study describes signaling diversity of mu opioid receptor (MOR) ligands in terms of logistic and operational parameters for ten different in vitro readouts. It then uses unsupervised clustering of curve parameters to: classify MOR ligands according to similarities in type and magnitude of response, associate resulting ligand categories with frequency of undesired events reported to the pharmacovigilance program of the Food and Drug Administration and associate signals to side effects. The ability of the classification method to associate specific in vitro signaling profiles to clinically relevant responses was corroborated using ß2-adrenergic receptor ligands.


Assuntos
Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais , Analgésicos Opioides/metabolismo , Animais , Análise por Conglomerados , Proteínas de Ligação ao GTP/metabolismo , Cobaias , Células HEK293 , Humanos , Ligantes , Receptores Adrenérgicos beta 2/metabolismo , Receptores Opioides mu/metabolismo , beta-Arrestinas/metabolismo
6.
Int J Mol Sci ; 20(17)2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31484312

RESUMO

Opioid use disorder is classified as a chronic recurrent disease of the central nervous system (CNS) which leads to personality disorders, co-morbidities and premature death. It develops as a result of long-term administration of various abused substances, along with morphine. The pharmacological action of morphine is associated with its stimulation of opioid receptors. Opioid receptors are a group of G protein-coupled receptors and activation of these receptors by ligands induces significant molecular changes inside the cell, such as an inhibition of adenylate cyclase activity, activation of potassium channels and reductions of calcium conductance. Recent data indicate that other signalling pathways also may be involved in morphine activity. Among these are phospholipase C, mitogen-activated kinases (MAP kinases) or ß-arrestin. The present review focuses on major mechanisms which currently are considered as essential in morphine activity and dependence and may be important for further studies.


Assuntos
Adenilil Ciclases/metabolismo , Dependência de Morfina/metabolismo , Adenilil Ciclases/genética , Animais , Humanos , Dependência de Morfina/genética , Receptores Opioides/genética , Receptores Opioides/metabolismo , beta-Arrestinas/metabolismo
7.
Mol Pharmacol ; 96(4): 463-474, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31399503

RESUMO

G protein-coupled receptor (GPCR) internalization is crucial for the termination of GPCR activity, and in some cases is associated with G protein-independent signaling and endosomal receptor signaling. To date, internalization has been studied in great detail for class A GPCRs; whereas it is not well established to what extent the observations can be generalized to class C GPCRs, including the extracellular calcium-sensing receptor (CaSR). The CaSR is a prototypical class C GPCR that maintains stable blood calcium (Ca2+) levels by sensing minute changes in extracellular free Ca2+ It is thus necessary that the activity of the CaSR is tightly regulated, even while continuously being exposed to its endogenous agonist. Previous studies have used overexpression of intracellular proteins involved in GPCR trafficking, pathway inhibitors, and cell-surface expression or functional desensitization as indirect measures to investigate CaSR internalization. However, there is no general consensus on the processes involved, and the mechanism of CaSR internalization remains poorly understood. The current study provides new insights into the internalization mechanism of the CaSR. We have used a state-of-the-art time-resolved fluorescence resonance energy transfer-based internalization assay to directly measure CaSR internalization in real-time. We demonstrate that the CaSR displays both constitutive and concentration-dependent Ca2+-mediated internalization. For the first time, we conclusively show that CaSR internalization is sensitive to immediate positive and negative modulation by the CaSR-specific allosteric modulators N-(3-[2-chlorophenyl]propyl)-(R)-α-methyl-3-methoxybenzylamine (NPS R-568) and 2-chloro-6-[(2R)-2-hydroxy-3-[(2-methyl-1-naphthalen-2-ylpropan-2-yl)amino]propoxy]benzonitrile (NPS 2143), respectively. In addition, we provide compelling evidence that CaSR internalization is ß-arrestin-dependent while interestingly being largely independent of Gq/11 and Gi/o protein signaling. SIGNIFICANCE STATEMENT: A novel highly efficient cell-based real-time internalization assay to show that calcium-sensing receptor (CaSR) internalization is ß-arrestin-dependent and sensitive to modulation by allosteric ligands.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Receptores de Detecção de Cálcio/metabolismo , beta-Arrestinas/metabolismo , Regulação Alostérica , Cálcio/sangue , Transferência Ressonante de Energia de Fluorescência , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Ligantes , Mutação , Naftalenos/farmacologia , Fenetilaminas/farmacologia , Propilaminas/farmacologia , Transporte Proteico , Receptores de Detecção de Cálcio/genética
8.
Elife ; 82019 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-31373553

RESUMO

The metabotropic glutamate receptor 7 (mGlu7) is a class C G protein-coupled receptor that modulates excitatory neurotransmitter release at the presynaptic active zone. Although post-translational modification of cellular proteins with ubiquitin is a key molecular mechanism governing protein degradation and function, mGlu7 ubiquitination and its functional consequences have not been elucidated yet. Here, we report that Nedd4 ubiquitin E3 ligase and ß-arrestins regulate ubiquitination of mGlu7 in heterologous cells and rat neurons. Upon agonist stimulation, ß-arrestins recruit Nedd4 to mGlu7 and facilitate Nedd4-mediated ubiquitination of mGlu7. Nedd4 and ß-arrestins regulate constitutive and agonist-induced endocytosis of mGlu7 and are required for mGlu7-dependent MAPK signaling in neurons. In addition, Nedd4-mediated ubiquitination results in the degradation of mGlu7 by both the ubiquitin-proteasome system and the lysosomal degradation pathway. These findings provide a model in which Nedd4 and ß-arrestin act together as a complex to regulate mGlu7 surface expression and function at presynaptic terminals.


Assuntos
Ubiquitina-Proteína Ligases Nedd4/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Ubiquitinação , beta-Arrestinas/metabolismo , Animais , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Transporte Proteico , Ratos
9.
J Pharmacol Sci ; 140(2): 171-177, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31320243

RESUMO

Morphine, fentanyl, and oxycodone are widely used as analgesics, and recently hydromorphone has been approved in Japan. Although all of these are selective for µ-opioid receptors (MORs) and have similar structures, their analgesic potencies and adverse effects (AEs) are diverse. Recent molecular analyses of MOR signaling revealed that the G protein-mediated signaling pathway causes analgesic effects and the ß-arrestin-mediated signaling pathway is responsible for AEs. We used several cell-based analyses that selectively measure cellular responses activated by either G protein- or ß-arrestin-mediated pathways. GloSensor™ cAMP, CellKey™, and receptor internalization assays were performed with four different types of cells stably expressing differentially labelled MOR. EC50 values measured by cAMP and CellKey™ assays had potencies in the order fentanyl ≤ hydromorphone < morphine ≤ oxycodone, all also exhibiting full agonist responses. However, in the internalization assay, only fentanyl elicited a full agonist response. Hydromorphone had the strongest potency next to fentanyl; however, contribution of the ß-arrestin-mediated pathway was small, suggesting that its effect could be biased toward the G protein-mediated pathway. Based on these properties, hydromorphone could be chosen as an effective analgesic.


Assuntos
Analgésicos Opioides/efeitos adversos , Analgésicos Opioides/farmacologia , AMP Cíclico , Proteínas de Ligação ao GTP/metabolismo , Hidromorfona/efeitos adversos , Hidromorfona/farmacologia , Receptores Opioides mu/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , beta-Arrestinas/metabolismo , Células HEK293 , Humanos , Hidromorfona/metabolismo
10.
Nat Rev Cardiol ; 16(10): 612-622, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31186538

RESUMO

G protein-coupled receptors (GPCRs) are critical cellular sensors that mediate numerous physiological processes. In the heart, multiple GPCRs are expressed on various cell types, where they coordinate to regulate cardiac function by modulating critical processes such as contractility and blood flow. Under pathological settings, these receptors undergo aberrant changes in expression levels, localization and capacity to couple to downstream signalling pathways. Conventional therapies for heart failure work by targeting GPCRs, such as ß-adrenergic receptor and angiotensin II receptor antagonists. Although these treatments have improved patient survival, heart failure remains one of the leading causes of mortality worldwide. GPCR kinases (GRKs) are responsible for GPCR phosphorylation and, therefore, desensitization and downregulation of GPCRs. In this Review, we discuss the GPCR signalling pathways and the GRKs involved in the pathophysiology of heart disease. Given that increased expression and activity of GRK2 and GRK5 contribute to the loss of contractile reserve in the stressed and failing heart, inhibition of overactive GRKs has been proposed as a novel therapeutic approach to treat heart failure.


Assuntos
Quinases de Receptores Acoplados a Proteína G/antagonistas & inibidores , Quinases de Receptores Acoplados a Proteína G/metabolismo , Cardiopatias/tratamento farmacológico , Cardiopatias/fisiopatologia , Antagonistas Adrenérgicos beta/uso terapêutico , Animais , Catecolaminas/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Quinase 2 de Receptor Acoplado a Proteína G/antagonistas & inibidores , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Quinase 5 de Receptor Acoplado a Proteína G/antagonistas & inibidores , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/fisiopatologia , Humanos , Contração Muscular , Miócitos Cardíacos , Fragmentos de Peptídeos/genética , Receptores Adrenérgicos/metabolismo , Proteínas Recombinantes/genética , Transdução de Sinais/genética , beta-Arrestinas/metabolismo
11.
Int Rev Cell Mol Biol ; 346: 129-155, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31122393

RESUMO

Metastatic progression is strongly influenced by the connection between hyperactivated signaling pathways. G-protein coupled receptors (GPCRs) through ß-arrestins (ß-arrs), which serve as intracellular signaling molecules, integrate different pathways to control multiple aspects of metastatic process. As primary component of a core-scaffold, ß-arr-dependent signaling represents a mean to direct spatiotemporal specificity of multi-protein complexes in invasion and extracellular matrix (ECM) degradation. Under this paradigm, ß-arrs engage a growing number of signaling molecules and organizing protein networks controlling multiple pathways, and cytoskeleton modifications, permitting adaptation to the tumor microenvironment to sustain metastatic dissemination. These findings implicate GPCR/ß-arr function as a regulatory tethering hub to orchestrate diverse cellular mechanisms of cancer cell migration and invasion that are critical for metastatic progression. In this chapter, we outline the most recent findings on GPCR/ß-arr-guided molecular interactions in specific intracellular compartments to drive metastasis, while discussing new perspectives for the selection of most effective therapeutic options for a personalized medicine.


Assuntos
Citoesqueleto de Actina/metabolismo , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias , Receptores Acoplados a Proteínas-G/metabolismo , beta-Arrestinas/metabolismo , Citoesqueleto de Actina/patologia , Animais , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de Sinais
12.
J Biol Chem ; 294(24): 9416-9429, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31036565

RESUMO

The human complement component, C5a, binds two different seven-transmembrane receptors termed C5aR1 and C5aR2. C5aR1 is a prototypical G-protein-coupled receptor that couples to the Gαi subfamily of heterotrimeric G-proteins and ß-arrestins (ßarrs) following C5a stimulation. Peptide fragments derived from the C terminus of C5a can still interact with the receptor, albeit with lower affinity, and can act as agonists or antagonists. However, whether such fragments might display ligand bias at C5aR1 remains unexplored. Here, we compare C5a and a modified C-terminal fragment of C5a, C5apep, in terms of G-protein coupling, ßarr recruitment, endocytosis, and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase activation at the human C5aR1. We discover that C5apep acts as a full agonist for Gαi coupling as measured by cAMP response and extracellular signal-regulated kinase 1/2 phosphorylation, but it displays partial agonism for ßarr recruitment and receptor endocytosis. Interestingly, C5apep exhibits full-agonist efficacy with respect to inhibiting lipopolysaccharide-induced interleukin-6 secretion in human macrophages, but its ability to induce human neutrophil migration is substantially lower compared with C5a, although both these responses are sensitive to pertussis toxin treatment. Taken together, our data reveal that compared with C5a, C5apep exerts partial efficacy for ßarr recruitment, receptor trafficking, and neutrophil migration. Our findings therefore uncover functional bias at C5aR1 and also provide a framework that can potentially be extended to chemokine receptors, which also typically interact with chemokines through a biphasic mechanism.


Assuntos
Complemento C5a/metabolismo , Endocitose , Receptor da Anafilatoxina C5a/metabolismo , beta-Arrestinas/metabolismo , Sequência de Aminoácidos , Movimento Celular , Complemento C5a/genética , Células HEK293 , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neutrófilos/metabolismo , Fosforilação , Ligação Proteica , Receptor da Anafilatoxina C5a/genética , Homologia de Sequência , Transdução de Sinais , beta-Arrestinas/genética
13.
Eur J Pharmacol ; 855: 267-275, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31078517

RESUMO

FFA4 (Free Fatty Acid receptor 4, previously known as GPR120) is a G protein-coupled receptor that acts as a sensor of long-chain fatty acids, modulates metabolism, and whose dysfunction participates in endocrine disturbances. FFA4 is known to be phosphorylated and internalized in response to agonists and protein kinase C activation. In this paper report the modulation of this fatty acid receptor by activation of receptor tyrosine kinases. Cell-activation with growth factors (insulin, epidermal growth factor, insulin-like growth factor-I, and platelet-derived growth factor) increases FFA4 phosphorylation in a time- and concentration-dependent fashion. This effect was blocked by inhibitors of protein kinase C and phosphoinositide 3-kinase, suggesting the involvement of these kinases in it. FFA4 phosphorylation did not alter agonist-induced FFA4 calcium signaling, but was associated with decreased ERK 1/2 phosphorylation. In addition, insulin, insulin-like growth factor-I, epidermal growth factor, and to a lesser extent, platelet-derived growth factor, induce receptor internalization. This action of insulin, insulin-like growth factor I, and epidermal growth factor was blocked by inhibitors of protein kinase C and phosphoinositide 3-kinase. Additionally, cell treatment with these growth factors induced FFA4-ß-arrestin coimmunoprecipitation. Our results evidenced cross-talk between receptor tyrosine kinases and FFA4 and suggest roles of protein kinase C and phosphoinositide 3-kinase in such a functional interaction.


Assuntos
Ativadores de Enzimas/farmacologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , beta-Arrestinas/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Fatores de Tempo
14.
Int J Mol Sci ; 20(10)2019 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-31137460

RESUMO

The ghrelin system has received substantial recognition as a potential target for novel anti-seizure drugs. Ghrelin receptor (ghrelin-R) signaling is complex, involving Gαq/11, Gαi/o, Gα12/13, and ß-arrestin pathways. In this study, we aimed to deepen our understanding regarding signaling pathways downstream the ghrelin-R responsible for mediating anticonvulsive effects in a kindling model. Mice were administered the proconvulsive dopamine 1 receptor-agonist, SKF81297, to gradually induce a kindled state. Prior to every SKF81297 injection, mice were treated with a ghrelin-R full agonist (JMV-1843), a Gαq and Gα12 biased ligand unable to recruit ß-arrestin (YIL781), a ghrelin-R antagonist (JMV-2959), or saline. Mice treated with JMV-1843 had fewer and less severe seizures compared to saline-treated controls, while mice treated with YIL781 experienced longer and more severe seizures. JMV-2959 treatment did not lead to differences in seizure severity and number. Altogether, these results indicate that the Gαq or Gα12 signaling pathways are not responsible for mediating JMV-1843's anticonvulsive effects and suggest a possible involvement of ß-arrestin signaling in the anticonvulsive effects mediated by ghrelin-R modulation.


Assuntos
Encéfalo/metabolismo , Excitação Neurológica , Receptores de Grelina/agonistas , Animais , Benzazepinas/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/fisiologia , Agonistas de Dopamina/farmacologia , Glicina/análogos & derivados , Glicina/farmacologia , Indóis/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Piperidinas/farmacologia , Quinazolinonas/farmacologia , Receptores de Grelina/antagonistas & inibidores , Triazóis/farmacologia , Triptofano/análogos & derivados , Triptofano/farmacologia , beta-Arrestinas/farmacologia
16.
Cell ; 177(3): 597-607.e9, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-31002796

RESUMO

The melanocortin 4 receptor (MC4R) is a G protein-coupled receptor whose disruption causes obesity. We functionally characterized 61 MC4R variants identified in 0.5 million people from UK Biobank and examined their associations with body mass index (BMI) and obesity-related cardiometabolic diseases. We found that the maximal efficacy of ß-arrestin recruitment to MC4R, rather than canonical Gαs-mediated cyclic adenosine-monophosphate production, explained 88% of the variance in the association of MC4R variants with BMI. While most MC4R variants caused loss of function, a subset caused gain of function; these variants were associated with significantly lower BMI and lower odds of obesity, type 2 diabetes, and coronary artery disease. Protective associations were driven by MC4R variants exhibiting signaling bias toward ß-arrestin recruitment and increased mitogen-activated protein kinase pathway activation. Harnessing ß-arrestin-biased MC4R signaling may represent an effective strategy for weight loss and the treatment of obesity-related cardiometabolic diseases.


Assuntos
Mutação com Ganho de Função/genética , Obesidade/patologia , Receptor Tipo 4 de Melanocortina/genética , Transdução de Sinais , Adulto , Idoso , Índice de Massa Corporal , Doença da Artéria Coronariana/complicações , Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/patologia , AMP Cíclico/metabolismo , Bases de Dados Factuais , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Feminino , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Obesidade/complicações , Obesidade/metabolismo , Polimorfismo de Nucleotídeo Único , Receptor Tipo 4 de Melanocortina/química , Receptor Tipo 4 de Melanocortina/metabolismo , beta-Arrestinas/metabolismo
17.
Methods Mol Biol ; 1947: 257-267, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30969421

RESUMO

Intracellular signal transduced by G protein-coupled receptors (GPCRs) is tightly controlled by a guanine nucleotide-binding complex made of G protein Gα, Gß, and Gγ subunits, as well as a growing array of regulatory and accessory proteins such as arrestins. G protein-independent ß-arrestin recruitment at GPCRs is universally accepted as the canonical interactor system and it has been found to be a powerful tracker of most GPCRs activation. Pharmacological concepts have evolved remarkably after the finding that different ligands, binding at the same receptor, can selectively activate specific subsets of signaling pathways among all pathways activated by balanced ligands. This new paradigm referred to as functional selectivity or biased signaling, has opened new avenues for the design of tailored drugs with enhanced therapeutic efficacies and reduced side effects. Here, we describe a unique platform for the interrogation of GPCR using a transcriptional-based assay to measure transient ß-arrestin recruitment called Tango.


Assuntos
Bioensaio/métodos , Receptores Acoplados a Proteínas-G/metabolismo , beta-Arrestinas/metabolismo , Bleomicina/farmacologia , Células HEK293 , Humanos , Ligantes , Transdução de Sinais , Ativação Transcricional
18.
Methods Mol Biol ; 1947: 323-336, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30969425

RESUMO

G protein-coupled receptors (GPCRs) comprise the largest family of integral membrane proteins, which are coupled to heterotrimeric G proteins to influence cell signaling. Subsequent to G protein activation, agonist-stimulated G protein-coupled receptor kinase (GRK) phosphorylation results in the recruitment of ß-arrestin proteins, which form both stable and unstable complexes with GPCRs. ß-Arrestins when bound to GPCRs not only contribute to the uncoupling of G protein signaling but also to the redistribution of GPCRs to clathrin-coated pits via their association with both clathrin and ß2-adaptin facilitating GPCR endocytosis. This allows ß-arrestins to couple GPCRs to additional cell signaling proteins allowing a second wave of receptor signaling. Importantly, the ß-arrestin-regulated subcellular localization of these complexes also plays a critical role in regulating how these signals are transduced and which proteins are recruited. Here, we describe a methodology for assessing the GPCR subcellular localization by super-resolution microscopy and suggest that this methodology can be extended to the study of GPCR/protein complexes.


Assuntos
Clatrina/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos , Receptores Acoplados a Proteínas-G/metabolismo , beta-Arrestinas/metabolismo , Endocitose , Humanos , Transdução de Sinais
19.
Methods Mol Biol ; 1957: 3-8, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30919344

RESUMO

Arrestins have now been implicated in the actions of virtually every G protein-coupled receptor (GPCR) for which they have been examined. Originally discovered for their role in the turnoff of visual perception, their newly discovered pleotropic functions in the cellular and physiological actions of GPCRs not only illuminate new mechanisms of signal transduction but also offer new avenues for therapeutic utility. Below, in this introductory chapter, we provide a short historical description and synopsis of how arrestins conceptually became associated with the function of GPCRs.


Assuntos
beta-Arrestinas/história , beta-Arrestinas/metabolismo , Animais , História do Século XX , Humanos , beta-Arrestinas/uso terapêutico
20.
Methods Mol Biol ; 1957: 9-55, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30919345

RESUMO

The ß-arrestins (ß-arrs) were initially appreciated for the roles they play in the desensitization and endocytosis of G protein-coupled receptors (GPCRs). They are now also known to act as multifunctional adaptor proteins binding many non-receptor protein partners to control multiple signalling pathways. ß-arrs therefore act as key regulatory hubs at the crossroads of external cell inputs and functional outputs in cellular processes ranging from gene transcription to cell growth, survival, cytoskeletal regulation, polarity, and migration. An increasing number of studies have also highlighted the scaffolding roles ß-arrs play in vivo in both physiological and pathological conditions, which opens up therapeutic avenues to explore. In this introductory review chapter, we discuss the functional roles that ß-arrs exert to control GPCR function, their dynamic scaffolding roles and how this impacts signal transduction events, compartmentalization of ß-arrs, how ß-arrs are regulated themselves, and how the combination of these events culminates in cellular regulation.


Assuntos
Células/metabolismo , Transdução de Sinais , beta-Arrestinas/metabolismo , Animais , Endocitose , Humanos , Modelos Biológicos , Transporte Proteico , beta-Arrestinas/química
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