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2.
PLoS Comput Biol ; 16(4): e1007394, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32275713

RESUMO

The development of novel analgesics with improved safety profiles to combat the opioid epidemic represents a central question to G protein coupled receptor structural biology and pharmacology: What chemical features dictate G protein or ß-arrestin signaling? Here we use adaptively biased molecular dynamics simulations to determine how fentanyl, a potent ß-arrestin biased agonist, binds the µ-opioid receptor (µOR). The resulting fentanyl-bound pose provides rational insight into a wealth of historical structure-activity-relationship on its chemical scaffold. Following an in-silico derived hypothesis we found that fentanyl and the synthetic opioid peptide DAMGO require M153 to induce ß-arrestin coupling, while M153 was dispensable for G protein coupling. We propose and validate an activation mechanism where the n-aniline ring of fentanyl mediates µOR ß-arrestin through a novel M153 "microswitch" by synthesizing fentanyl-based derivatives that exhibit complete, clinically desirable, G protein biased coupling. Together, these results provide molecular insight into fentanyl mediated ß-arrestin biased signaling and a rational framework for further optimization of fentanyl-based analgesics with improved safety profiles.


Assuntos
Fentanila/farmacologia , beta-Arrestinas/metabolismo , beta-Arrestinas/ultraestrutura , Analgésicos Opioides/química , Analgésicos Opioides/farmacologia , Fentanila/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Humanos , Simulação de Dinâmica Molecular , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Opioides mu/química , Receptores Opioides mu/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , beta-Arrestinas/agonistas
3.
Mol Pharmacol ; 97(6): 365-376, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32234808

RESUMO

Proteinase-activated receptors (PARs) are a four-member family of G-protein-coupled receptors that are activated via proteolysis. PAR4 is a member of this family that is cleaved and activated by serine proteinases such as thrombin, trypsin, and cathepsin-G. PAR4 is expressed in a variety of tissues and cell types, including platelets, vascular smooth muscle cells, and neuronal cells. In studying PAR4 signaling and trafficking, we observed dynamic changes in the cell membrane, with spherical membrane protrusions that resemble plasma membrane blebbing. Since nonapoptotic membrane blebbing is now recognized as an important regulator of cell migration, cancer cell invasion, and vesicular content release, we sought to elucidate the signaling pathway downstream of PAR4 activation that leads to such events. Using a combination of pharmacological inhibition and CRISPR/CRISPR-associated protein 9 (Cas9)-mediated gene editing approaches, we establish that PAR4-dependent membrane blebbing occurs independently of the Gα q/11- and Gα i-signaling pathways and is dependent on signaling via the ß-arrestin-1/2 and Ras homolog family member A (RhoA) signaling pathways. Together these studies provide further mechanistic insight into PAR4 regulation of cellular function. SIGNIFICANCE STATEMENT: We find that the thrombin receptor PAR4 triggers cell membrane blebbing in a RhoA-and ß-arrestin-dependent manner. In addition to identifying novel cellular responses mediated by PAR4, these data provide further evidence for biased signaling in PAR4 since membrane blebbing was dependent on some, but not all, signaling pathways activated by PAR4.


Assuntos
Membrana Celular/metabolismo , Membrana Celular/patologia , Receptores de Trombina/metabolismo , beta-Arrestinas/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Sistemas CRISPR-Cas , Forma Celular , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Músculo Liso Vascular/metabolismo , Ratos , Ratos Endogâmicos WKY , Receptores Acoplados a Proteínas-G/metabolismo , Receptores de Trombina/agonistas , Transdução de Sinais
4.
PLoS Biol ; 18(4): e3000656, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32271748

RESUMO

Chemokines and their receptors are orchestrators of cell migration in humans. Because dysregulation of the receptor-chemokine system leads to inflammation and cancer, both chemokines and receptors are highly sought therapeutic targets. Yet one of the barriers for their therapeutic targeting is the limited understanding of the structural principles behind receptor-chemokine recognition and selectivity. The existing structures do not include CXC subfamily complexes and lack information about the receptor distal N-termini, despite the importance of the latter in signaling, regulation, and bias. Here, we report the discovery of the geometry of the complex between full-length CXCR4, a prototypical CXC receptor and driver of cancer metastasis, and its endogenous ligand CXCL12. By comprehensive disulfide cross-linking, we establish the existence and the structure of a novel interface between the CXCR4 distal N-terminus and CXCL12 ß1-strand, while also recapitulating earlier findings from nuclear magnetic resonance, modeling and crystallography of homologous receptors. A cross-linking-informed high-resolution model of the CXCR4-CXCL12 complex pinpoints the interaction determinants and reveals the occupancy of the receptor major subpocket by the CXCL12 proximal N terminus. This newly found positioning of the chemokine proximal N-terminus provides a structural explanation of CXC receptor-chemokine selectivity against other subfamilies. Our findings challenge the traditional two-site understanding of receptor-chemokine recognition, suggest the possibility of new affinity and signaling determinants, and fill a critical void on the structural map of an important class of therapeutic targets. These results will aid the rational design of selective chemokine-receptor targeting small molecules and biologics with novel pharmacology.


Assuntos
Quimiocina CXCL12/química , Quimiocina CXCL12/metabolismo , Receptores CXCR4/química , Receptores CXCR4/metabolismo , Animais , Sítios de Ligação , Western Blotting , Quimiocina CXCL12/genética , Cisteína/química , Cisteína/genética , Dissulfetos/química , Citometria de Fluxo , Células HEK293 , Humanos , Insetos/citologia , Modelos Moleculares , Mutação , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores CXCR4/genética , beta-Arrestinas/metabolismo
5.
Proc Natl Acad Sci U S A ; 117(13): 7455-7460, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32184323

RESUMO

cAMP production upon activation of Gs by G protein-coupled receptors has classically been considered to be plasma membrane-delimited, but a shift in this paradigm has occurred in recent years with the identification of several receptors that continue to signal from early endosomes after internalization. The molecular mechanisms regulating this aspect of signaling remain incompletely understood. Here, we investigated the role of Gq/11 activation by the parathyroid hormone (PTH) type 1 receptor (PTHR) in mediating endosomal cAMP responses. Inhibition of Gq/11 signaling by FR900359 markedly reduced the duration of PTH-induced cAMP production, and this effect was mimicked in cells lacking endogenous Gαq/11 We determined that modulation of cAMP generation by Gq/11 occurs at the level of the heterotrimeric G protein via liberation of cell surface Gßγ subunits, which, in turn, act in a phosphoinositide-3 kinase-dependent manner to promote the assembly of PTHR-ßarrestin-Gßγ signaling complexes that mediate endosomal cAMP responses. These results unveil insights into the spatiotemporal regulation of Gs-dependent cAMP signaling.


Assuntos
AMP Cíclico/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Animais , Arrestinas/metabolismo , Membrana Celular/metabolismo , Depsipeptídeos/farmacologia , Endossomos/metabolismo , Células HEK293 , Humanos , Camundongos , Osteoblastos/metabolismo , Hormônio Paratireóideo/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Cultura Primária de Células , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/fisiologia , beta-Arrestinas/metabolismo
6.
Cell ; 180(6): 1041-1043, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32169216

RESUMO

ß-arrestins (ßarrs) play multifaceted roles in the signaling and regulation of G-protein-coupled receptors (GPCRs) including their desensitization and endocytosis. Recently determined cryo-EM structures of two different GPCRs in complex with ßarr1 provide the first glimpse of GPCR-ßarr engagement and a structural framework to understand their interaction.


Assuntos
Receptores Acoplados a Proteínas-G/ultraestrutura , beta-Arrestinas/metabolismo , beta-Arrestinas/ultraestrutura , Arrestinas/metabolismo , Endocitose/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Humanos , Fosforilação , Ligação Proteica , Isoformas de Proteínas/ultraestrutura , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/fisiologia , Relação Estrutura-Atividade , beta-Arrestina 1/metabolismo , beta-Arrestina 2/metabolismo
7.
Adv Cancer Res ; 145: 139-156, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32089163

RESUMO

ß-Arrestins (ßarrs) are multifunctional intracellular proteins with an ability to directly interact with a large number of cellular partners including the G protein-coupled receptors (GPCRs). ßarrs contribute to multiple aspects of GPCR signaling, trafficking and downregulation. Considering the central involvement of GPCR signaling in the onset and progression of diverse types of cancers, ßarrs have also emerged as key players in the context of investigating cancer phenotypes, and as potential therapeutic targets. In this chapter, we first provide a brief account of structure and function of ßarrs and then highlight recent discoveries unfolding novel functional attributes of ßarrs in breast cancer. We also underscore the recent paradigms of modulating ßarr functions in cellular context and potential therapeutic opportunities going forward.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Terapia de Alvo Molecular , beta-Arrestinas/química , beta-Arrestinas/metabolismo , Animais , Neoplasias da Mama/patologia , Feminino , Humanos , Transdução de Sinais
8.
J Biol Chem ; 295(8): 2520-2540, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-31892516

RESUMO

Proteinase-activated receptor (PAR)-4 is a member of the proteolytically-activated PAR family of G-protein-coupled receptors (GPCR) that represents an important target in the development of anti-platelet therapeutics. PARs are activated by proteolytic cleavage of their receptor N terminus by enzymes such as thrombin, trypsin, and cathepsin-G. This reveals the receptor-activating motif, termed the tethered ligand that binds intramolecularly to the receptor and triggers signaling. However, PARs are also activated by exogenous application of synthetic peptides derived from the tethered-ligand sequence. To better understand the molecular basis for PAR4-dependent signaling, we examined PAR4-signaling responses to a peptide library derived from the canonical PAR4-agonist peptide, AYPGKF-NH2, and we monitored activation of the Gαq/11-coupled calcium-signaling pathway, ß-arrestin recruitment, and mitogen-activated protein kinase (MAPK) pathway activation. We identified peptides that are poor activators of PAR4-dependent calcium signaling but were fully competent in recruiting ß-arrestin-1 and -2. Peptides that were unable to stimulate PAR4-dependent calcium signaling could not trigger MAPK activation. Using in silico docking and site-directed mutagenesis, we identified Asp230 in the extracellular loop-2 as being critical for PAR4 activation by both agonist peptide and the tethered ligand. Probing the consequence of biased signaling on platelet activation, we found that a peptide that cannot activate calcium signaling fails to cause platelet aggregation, whereas a peptide that is able to stimulate calcium signaling and is more potent for ß-arrestin recruitment triggered greater levels of platelet aggregation compared with the canonical PAR4 agonist peptide. These findings uncover molecular determinants critical for agonist binding and biased signaling through PAR4.


Assuntos
Receptores de Trombina/metabolismo , Transdução de Sinais , Trombina/metabolismo , Alanina/genética , Substituição de Aminoácidos , Cálcio/metabolismo , Sinalização do Cálcio , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Isomerismo , Sistema de Sinalização das MAP Quinases , Metilação , Simulação de Acoplamento Molecular , Proteínas Mutantes/metabolismo , Mutação/genética , Peptídeos/metabolismo , Fosforilação , Agregação Plaquetária , Receptores de Trombina/agonistas , Homologia Estrutural de Proteína , beta-Arrestinas/metabolismo
9.
Nat Chem Biol ; 16(2): 179-187, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31844302

RESUMO

Augmenting live cells with new signal transduction capabilities is a key objective in genetic engineering and synthetic biology. We showed earlier that two-component signaling pathways could function in mammalian cells, albeit while losing their ligand sensitivity. Here, we show how to transduce small-molecule ligands in a dose-dependent fashion into gene expression in mammalian cells using two-component signaling machinery. First, we engineer mutually complementing truncated mutants of a histidine kinase unable to dimerize and phosphorylate the response regulator. Next, we fuse these mutants to protein domains capable of ligand-induced dimerization, which restores the phosphoryl transfer in a ligand-dependent manner. Cytoplasmic ligands are transduced by facilitating mutant dimerization in the cytoplasm, while extracellular ligands trigger dimerization at the inner side of a plasma membrane. These findings point to the potential of two-component regulatory systems as enabling tools for orthogonal signaling pathways in mammalian cells.


Assuntos
Histidina Quinase/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologia , Biologia Sintética/métodos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Histidina Quinase/genética , Humanos , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Mutação , Fosforilação/genética , Domínios Proteicos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Multimerização Proteica/genética , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteína 1A de Ligação a Tacrolimo/genética , Proteína 1A de Ligação a Tacrolimo/metabolismo , beta-Arrestinas/genética , beta-Arrestinas/metabolismo
10.
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
11.
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
12.
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
13.
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
14.
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
15.
Ann N Y Acad Sci ; 1456(1): 26-43, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31502283

RESUMO

Mechanisms of activation, signaling, and trafficking of adhesion G protein-coupled receptors (aGPCRs) have remained largely unknown. Several aGPCRs, including GPR56/ADGRG1 and GPR64/ADGRG2, show increased activity in the absence of their N-terminal fragment (NTF). This constitutive signaling is plausibly caused by the binding of extracellular N-terminal 15-25 amino acid-long tethered agonist to extracellular domains of the cognate aGPCRs. To test the role of NTF and tethered agonist in GPR64 signaling and endocytosis, we generated mutants that lack either NTF alone (ΔNTF) or NTF and tethered agonist (P622). We discover that unlike full-length GPR64, ΔNTF and P622 mutants interact with ß-arrestin1 and ß-arrestins2 and are constitutively internalized in steady states. However, only ΔNTF shows exaggerated basal activation of the Gαs -cAMP-CRE signaling cascade. Neither ΔNTF nor P622 shows constitutive activation of the Gα13 -SRE pathway, but both mutants respond to exogenously added agonistic peptide via CRE and SRE. GPCR kinases and dynamin mediate the constitutive internalization of ΔNTF and P622 to early endosomes, where ΔNTF constantly induces CRE. These data suggest that NTF not only shields the tethered agonist to prevent G protein signaling but also confers a conformation that inhibits the interaction with ß-arrestins and the consequent endocytosis and sustained signaling from endosomes.


Assuntos
Proteínas Quinases/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais , beta-Arrestinas/metabolismo , Dinaminas/metabolismo , Endocitose , Endossomos/metabolismo , Células HEK293 , Humanos , Transporte Proteico
16.
Clin Sci (Lond) ; 133(18): 1977-1992, 2019 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-31519790

RESUMO

Inflammatory bowel disease (IBD) is a chronic intestinal inflammation, but the accurate etiology remains to be elucidated. Increasing evidence has shown that macrophages polarize to different phenotypes depending on the intestinal microenvironment and are associated with the progression of IBD. In the present study, we investigated the effect of oxytocin, a neuroendocrinal, and pro-health peptide, on the modulation of macrophages polarization and the progression of experimental colitis. Our data demonstrated that oxytocin decreased the sensitivity of macrophages to lipopolysaccharide stimulation with lower expression of inflammatory cytokines, like IL-1ß, IL-6, and TNF-α, but increased the sensitivity to IL-4 stimulation with enhanced expression of M2-type genes, arginase I (Arg1), CD206, and chitinase-like 3 (Chil3). This bidirectional modulation was partly due to the up-regulation of ß-arrestin2 and resulted in the inhibition of NF-κB signaling and reinforcement of Signal transducer and activator of transcription (STAT) 6 phosphorylation. Moreover, oxytocin receptor (OXTR) myeloid deficiency mice were more susceptible to dextran sulfate sodium (DSS) intervention compared with the wild mice. For the first time, we reveal that oxytocin-oxytocin receptor system participates in modulating the polarization of macrophages to an anti-inflammatory phenotype and alleviates experimental colitis. These findings provide new potential insights into the pathogenesis and therapy of IBD.


Assuntos
Colite/patologia , Inflamação/patologia , Intestinos/patologia , Macrófagos/patologia , Ocitocina/farmacologia , Adulto , Idoso , Animais , Polaridade Celular/efeitos dos fármacos , Sulfato de Dextrana , Feminino , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-4/metabolismo , Lipopolissacarídeos , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Modelos Biológicos , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Células RAW 264.7 , Receptores de Ocitocina/metabolismo , Fator de Transcrição STAT6/metabolismo , Células THP-1 , beta-Arrestinas/metabolismo
17.
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
18.
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
19.
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
20.
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
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