Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 34
Filtrar
1.
Brain ; 147(2): 649-664, 2024 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-37703312

RESUMO

The unfolded protein response (UPR) is rapidly gaining momentum as a therapeutic target for protein misfolding neurodegenerative diseases, in which its overactivation results in sustained translational repression leading to synapse loss and neurodegeneration. In mouse models of these disorders, from Alzheimer's to prion disease, modulation of the pathway-including by the licensed drug, trazodone-restores global protein synthesis rates with profound neuroprotective effects. However, the precise nature of the translational impairment, in particular the specific proteins affected in disease, and their response to therapeutic UPR modulation are poorly understood. We used non-canonical amino acid tagging (NCAT) to measure de novo protein synthesis in the brains of prion-diseased mice with and without trazodone treatment, in both whole hippocampus and cell-specifically. During disease the predominant nascent proteome changes occur in synaptic, cytoskeletal and mitochondrial proteins in both hippocampal neurons and astrocytes. Remarkably, trazodone treatment for just 2 weeks largely restored the whole disease nascent proteome in the hippocampus to that of healthy, uninfected mice, predominantly with recovery of proteins involved in synaptic and mitochondrial function. In parallel, trazodone treatment restored the disease-associated decline in synapses and mitochondria and their function to wild-type levels. In conclusion, this study increases our understanding of how translational repression contributes to neurodegeneration through synaptic and mitochondrial toxicity via depletion of key proteins essential for their function. Further, it provides new insights into the neuroprotective mechanisms of trazodone through reversal of this toxicity, relevant for the treatment of neurodegenerative diseases via translational modulation.


Assuntos
Doença de Alzheimer , Doenças Neurodegenerativas , Doenças Priônicas , Príons , Trazodona , Camundongos , Animais , Príons/metabolismo , Proteoma/metabolismo , Proteoma/farmacologia , Trazodona/farmacologia , Trazodona/uso terapêutico , Trazodona/metabolismo , Doenças Priônicas/tratamento farmacológico , Doenças Priônicas/metabolismo , Doenças Neurodegenerativas/metabolismo , Sinapses/metabolismo , Doença de Alzheimer/metabolismo
2.
J Biol Chem ; 298(3): 101655, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35101446

RESUMO

G protein-coupled receptor 35 (GPR35) is poorly characterized but nevertheless has been revealed to have diverse roles in areas including lower gut inflammation and pain. The development of novel reagents and tools will greatly enhance analysis of GPR35 functions in health and disease. Here, we used mass spectrometry, mutagenesis, and [32P] orthophosphate labeling to identify that all five hydroxy-amino acids in the C-terminal tail of human GPR35a became phosphorylated in response to agonist occupancy of the receptor and that, apart from Ser294, each of these contributed to interactions with arretin-3, which inhibits further G protein-coupled receptor signaling. We found that Ser303 was key to such interactions; the serine corresponding to human GPR35a residue 303 also played a dominant role in arrestin-3 interactions for both mouse and rat GPR35. We also demonstrated that fully phospho-site-deficient mutants of human GPR35a and mouse GPR35 failed to interact effectively with arrestin-3, and the human phospho-deficient variant was not internalized from the surface of cells in response to agonist treatment. Even in cells stably expressing species orthologues of GPR35, a substantial proportion of the expressed protein(s) was determined to be immature. Finally, phospho-site-specific antisera targeting the region encompassing Ser303 in human (Ser301 in mouse) GPR35a identified only the mature forms of GPR35 and provided effective sensors of the activation status of the receptors both in immunoblotting and immunocytochemical studies. Such antisera may be useful tools to evaluate target engagement in drug discovery and target validation programs.


Assuntos
Receptores Acoplados a Proteínas G , Animais , Humanos , Soros Imunes/farmacologia , Camundongos , Fosforilação , Ratos , Receptores Acoplados a Proteínas G/metabolismo , Serina/metabolismo , beta-Arrestina 2/metabolismo
3.
J Biol Chem ; 298(5): 101932, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35427647

RESUMO

GPR84 is an immune cell-expressed, proinflammatory receptor currently being assessed as a therapeutic target in conditions including fibrosis and inflammatory bowel disease. Although it was previously shown that the orthosteric GPR84 activators 2-HTP and 6-OAU promoted its interactions with arrestin-3, a G protein-biased agonist DL-175 did not. Here, we show that replacement of all 21 serine and threonine residues within i-loop 3 of GPR84, but not the two serines in the C-terminal tail, eliminated the incorporation of [32P] and greatly reduced receptor-arrestin-3 interactions promoted by 2-HTP. GPR84 was phosphorylated constitutively on residues Ser221 and Ser224, while various other amino acids are phosphorylated in response to 2-HTP. Consistent with this, an antiserum able to identify pSer221/pSer224 recognized GPR84 from cells treated with and without activators, whereas an antiserum able to identify pThr263/pThr264 only recognized GPR84 after exposure to 2-HTP and not DL-175. Two distinct GPR84 antagonists as well as inhibition of G protein-coupled receptor kinase 2/3 prevented phosphorylation of pThr263/pThr264, but neither strategy affected constitutive phosphorylation of Ser221/Ser224. Furthermore, mutation of residues Thr263 and Thr264 to alanine generated a variant of GPR84 also limited in 2-HTP-induced interactions with arrestin-2 and -3. By contrast, this mutant was unaffected in its capacity to reduce cAMP levels. Taken together, these results define a key pair of threonine residues, regulated only by subsets of GPR84 small molecule activators and by GRK2/3 that define effective interactions with arrestins and provide novel tools to monitor the phosphorylation and functional status of GPR84.


Assuntos
Arrestinas , Treonina , Arrestinas/metabolismo , Humanos , Ligantes , Mutação , Fosforilação , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Serina/metabolismo , Treonina/metabolismo , beta-Arrestina 2/metabolismo
4.
Int J Mol Sci ; 23(4)2022 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-35216136

RESUMO

Dementia is a global medical and societal challenge; it has devastating personal, social and economic costs, which will increase rapidly as the world's population ages. Despite this, there are no disease-modifying treatments for dementia; current therapy modestly improves symptoms but does not change the outcome. Therefore, new treatments are urgently needed-particularly any that can slow down the disease's progression. Many of the neurodegenerative diseases that lead to dementia are characterised by common pathological responses to abnormal protein production and misfolding in brain cells, raising the possibility of the broad application of therapeutics that target these common processes. The unfolded protein response (UPR) is one such mechanism. The UPR is a highly conserved cellular stress response to abnormal protein folding and is widely dysregulated in neurodegenerative diseases. In this review, we describe the basic machinery of the UPR, as well as the evidence for its overactivation and pathogenicity in dementia, and for the marked neuroprotective effects of its therapeutic manipulation in murine models of these disorders. We discuss drugs identified as potential UPR-modifying therapeutic agents-in particular the licensed antidepressant trazodone-and we review epidemiological and trial data from their use in human populations. Finally, we explore future directions for investigating the potential benefit of using trazodone or similar UPR-modulating compounds for disease modification in patients with dementia.


Assuntos
Demência/patologia , Resposta a Proteínas não Dobradas/fisiologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Demência/tratamento farmacológico , Humanos , Trazodona/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos
5.
Nat Chem Biol ; 15(5): 489-498, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30992568

RESUMO

Differentiating actions of short chain fatty acids (SCFAs) at free fatty acid receptor 2 (FFA2) from other free fatty acid-responsive receptors and from non-receptor-mediated effects has been challenging. Using a novel chemogenetic and knock-in strategy, whereby an engineered variant of FFA2 (FFA2-DREADD) that is unresponsive to natural SCFAs but is instead activated by sorbic acid replaced the wild-type receptor, we determined that activation of FFA2 in differentiated adipocytes and colonic crypt enteroendocrine cells of mouse accounts fully for SCFA-regulated lipolysis and release of the incretin glucagon-like peptide-1 (GLP-1), respectively. In vivo studies confirmed the specific role of FFA2 in GLP-1 release and also demonstrated a direct role for FFA2 in accelerating gut transit. Thereby, we establish the general principle that such a chemogenetic knock-in strategy can successfully define novel G-protein-coupled receptor (GPCR) biology and provide both target validation and establish therapeutic potential of a 'hard to target' GPCR.


Assuntos
Ácidos Graxos Voláteis/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Humanos , Camundongos , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G/genética
6.
Proc Natl Acad Sci U S A ; 113(16): 4524-9, 2016 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-27071102

RESUMO

G protein-coupled receptors (GPCRs) are known to initiate a plethora of signaling pathways in vitro. However, it is unclear which of these pathways are engaged to mediate physiological responses. Here, we examine the distinct roles of Gq/11-dependent signaling and receptor phosphorylation-dependent signaling in bronchial airway contraction and lung function regulated through the M3-muscarinic acetylcholine receptor (M3-mAChR). By using a genetically engineered mouse expressing a G protein-biased M3-mAChR mutant, we reveal the first evidence, to our knowledge, of a role for M3-mAChR phosphorylation in bronchial smooth muscle contraction in health and in a disease state with relevance to human asthma. Furthermore, this mouse model can be used to distinguish the physiological responses that are regulated by M3-mAChR phosphorylation (which include control of lung function) from those responses that are downstream of G protein signaling. In this way, we present an approach by which to predict the physiological/therapeutic outcome of M3-mAChR-biased ligands with important implications for drug discovery.


Assuntos
Brônquios/metabolismo , Músculo Liso/metabolismo , Receptor Muscarínico M3/metabolismo , Transdução de Sinais/fisiologia , Animais , Brônquios/citologia , Humanos , Camundongos , Camundongos Knockout , Músculo Liso/citologia , Fosforilação/fisiologia , Receptor Muscarínico M3/genética
7.
Proc Natl Acad Sci U S A ; 112(49): E6818-24, 2015 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-26598688

RESUMO

G protein-coupled receptors (GPCRs) regulate virtually all physiological functions including the release of insulin from pancreatic ß-cells. ß-Cell M3 muscarinic receptors (M3Rs) are known to play an essential role in facilitating insulin release and maintaining proper whole-body glucose homeostasis. As is the case with other GPCRs, M3R activity is regulated by phosphorylation by various kinases, including GPCR kinases and casein kinase 2 (CK2). At present, it remains unknown which of these various kinases are physiologically relevant for the regulation of ß-cell activity. In the present study, we demonstrate that inhibition of CK2 in pancreatic ß-cells, knockdown of CK2α expression, or genetic deletion of CK2α in ß-cells of mutant mice selectively augmented M3R-stimulated insulin release in vitro and in vivo. In vitro studies showed that this effect was associated with an M3R-mediated increase in intracellular calcium levels. Treatment of mouse pancreatic islets with CX4945, a highly selective CK2 inhibitor, greatly reduced agonist-induced phosphorylation of ß-cell M3Rs, indicative of CK2-mediated M3R phosphorylation. We also showed that inhibition of CK2 greatly enhanced M3R-stimulated insulin secretion in human islets. Finally, CX4945 treatment protected mice against diet-induced hyperglycemia and glucose intolerance in an M3R-dependent fashion. Our data demonstrate, for the first time to our knowledge, the physiological relevance of CK2 phosphorylation of a GPCR and suggest the novel concept that kinases acting on ß-cell GPCRs may represent novel therapeutic targets.


Assuntos
Caseína Quinase II/fisiologia , Insulina/metabolismo , Receptor Muscarínico M3/fisiologia , Animais , Células COS , Chlorocebus aethiops , Feminino , Células HEK293 , Humanos , Secreção de Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Naftiridinas/farmacologia , Fenazinas
8.
J Biol Chem ; 291(17): 8862-75, 2016 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-26826123

RESUMO

Establishing the in vivo activation status of G protein-coupled receptors would not only indicate physiological roles of G protein-coupled receptors but would also aid drug discovery by establishing drug/receptor engagement. Here, we develop a phospho-specific antibody-based biosensor to detect activation of the M1 muscarinic acetylcholine receptor (M1 mAChR) in vitro and in vivo Mass spectrometry phosphoproteomics identified 14 sites of phosphorylation on the M1 mAChR. Phospho-specific antibodies to four of these sites established that serine at position 228 (Ser(228)) on the M1 mAChR showed extremely low levels of basal phosphorylation that were significantly up-regulated by orthosteric agonist stimulation. In addition, the M1 mAChR-positive allosteric modulator, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, enhanced acetylcholine-mediated phosphorylation at Ser(228) These data supported the hypothesis that phosphorylation at Ser(228) was an indicator of M1 mAChR activation. This was further supported in vivo by the identification of phosphorylated Ser(228) on the M1 mAChR in the hippocampus of mice following administration of the muscarinic ligands xanomeline and 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid. Finally, Ser(228) phosphorylation was seen to increase in the CA1 region of the hippocampus following memory acquisition, a response that correlated closely with up-regulation of CA1 neuronal activity. Thus, determining the phosphorylation status of the M1 mAChR at Ser(228) not only provides a means of establishing receptor activation following drug treatment both in vitro and in vivo but also allows for the mapping of the activation status of the M1 mAChR in the hippocampus following memory acquisition thereby establishing a link between M1 mAChR activation and hippocampus-based memory and learning.


Assuntos
Anticorpos Monoclonais Murinos/química , Técnicas Biossensoriais/métodos , Região CA1 Hipocampal/metabolismo , Aprendizagem/fisiologia , Memória/fisiologia , Fosfoproteínas/metabolismo , Receptor Muscarínico M1/metabolismo , Animais , Região CA1 Hipocampal/citologia , Células CHO , Cricetinae , Cricetulus , Camundongos , Fosfoproteínas/genética , Fosforilação/fisiologia , Receptor Muscarínico M1/genética
9.
Biochem J ; 473(22): 4173-4192, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27623777

RESUMO

The parathyroid hormone receptor 1 (PTH1R) is a member of family B of G-protein-coupled receptors (GPCRs), predominantly expressed in bone and kidney where it modulates extracellular Ca2+ homeostasis and bone turnover. It is well established that phosphorylation of GPCRs constitutes a key event in regulating receptor function by promoting arrestin recruitment and coupling to G-protein-independent signaling pathways. Mapping phosphorylation sites on PTH1R would provide insights into how phosphorylation at specific sites regulates cell signaling responses and also open the possibility of developing therapeutic agents that could target specific receptor functions. Here, we have used mass spectrometry to identify nine sites of phosphorylation in the C-terminal tail of PTH1R. Mutational analysis revealed identified two clusters of serine and threonine residues (Ser489-Ser495 and Ser501-Thr506) specifically responsible for the majority of PTH(1-34)-induced receptor phosphorylation. Mutation of these residues to alanine did not affect negatively on the ability of the receptor to couple to G-proteins or activate extracellular-signal-regulated kinase 1/2. Using fluorescence resonance energy transfer and bioluminescence resonance energy transfer to monitor PTH(1-34)-induced interaction of PTH1R with arrestin3, we show that the first cluster Ser489-Ser495 and the second cluster Ser501-Thr506 operated in concert to mediate both the efficacy and potency of ligand-induced arrestin3 recruitment. We further demonstrate that Ser503 and Thr504 in the second cluster are responsible for 70% of arrestin3 recruitment and are key determinants for interaction of arrestin with the receptor. Our data are consistent with the hypothesis that the pattern of C-terminal tail phosphorylation on PTH1R may determine the signaling outcome following receptor activation.


Assuntos
Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Sequência de Aminoácidos , Arrestinas/metabolismo , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Ensaio de Imunoadsorção Enzimática , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Imunoprecipitação , Espectrometria de Massas , Dados de Sequência Molecular , Fosforilação , Receptor Tipo 1 de Hormônio Paratireóideo/química , Receptores Acoplados a Proteínas G/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais
10.
Mol Pharmacol ; 89(5): 505-20, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26873857

RESUMO

It is established that long-chain free fatty acids includingω-3 fatty acids mediate an array of biologic responses through members of the free fatty acid (FFA) receptor family, which includes FFA4. However, the signaling mechanisms and modes of regulation of this receptor class remain unclear. Here, we employed mass spectrometry to determine that phosphorylation of mouse (m)FFAR4 occurs at five serine and threonine residues clustered in two separable regions of the C-terminal tail, designated cluster 1 (Thr(347), Thr(349), and Ser(350)) and cluster 2 (Ser(357)and Ser(361)). Mutation of these phosphoacceptor sites to alanine completely prevented phosphorylation of mFFA4 but did not limit receptor coupling to extracellular signal regulated protein kinase 1 and 2 (ERK1/2) activation. Rather, an inhibitor of Gq/11proteins completely prevented receptor signaling to ERK1/2. By contrast, the recruitment of arrestin 3, receptor internalization, and activation of Akt were regulated by mFFA4 phosphorylation. The analysis of mFFA4 phosphorylation-dependent signaling was extended further by selective mutations of the phosphoacceptor sites. Mutations within cluster 2 did not affect agonist activation of Akt but instead significantly compromised receptor internalization and arrestin 3 recruitment. Distinctly, mutation of the phosphoacceptor sites within cluster 1 had no effect on receptor internalization and had a less extensive effect on arrestin 3 recruitment but significantly uncoupled the receptor from Akt activation. These unique observations define differential effects on signaling mediated by phosphorylation at distinct locations. This hallmark feature supports the possibility that the signaling outcome of mFFA4 activation can be determined by the pattern of phosphorylation (phosphorylation barcode) at the C terminus of the receptor.


Assuntos
Membrana Celular/metabolismo , Sistema de Sinalização das MAP Quinases , Processamento de Proteína Pós-Traducional , Receptores Acoplados a Proteínas G/metabolismo , Serina/metabolismo , Treonina/metabolismo , Substituição de Aminoácidos , Animais , Arrestinas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células CHO , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Cricetulus , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Moduladores de Transporte de Membrana/farmacologia , Camundongos , Mutação , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/agonistas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo
11.
Mol Pharmacol ; 87(2): 349-62, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25425623

RESUMO

G protein-coupled receptor phosphorylation plays a major role in receptor desensitization and arrestin binding. It is, however, unclear how distinct receptor phosphorylation patterns may influence arrestin binding and subsequent trafficking. Here we engineer phosphorylation sites into the C-terminal tail of the ß2-adrenoceptor (ß2AR) and demonstrate that this mutant, termed ß2AR(SSS), showed increased isoprenaline-stimulated phosphorylation and differences in arrestin-3 affinity and trafficking. By measuring arrestin-3 recruitment and the stability of arrestin-3 receptor complexes in real time using fluorescence resonance energy transfer and fluorescence recovery after photobleaching, we demonstrate that arrestin-3 dissociated quickly and almost completely from the ß2AR, whereas the interaction with ß2AR(SSS) was 2- to 4-fold prolonged. In contrast, arrestin-3 interaction with a ß2-adrenoceptor fused to the carboxyl-terminal tail of the vasopressin type 2 receptor was nearly irreversible. Further analysis of arrestin-3 localization revealed that by engineering phosphorylation sites into the ß2-adrenoceptor the receptor showed prolonged interaction with arrestin-3 and colocalization with arrestin in endosomes after internalization. This is in contrast to the wild-type receptor that interacts transiently with arrestin-3 at the plasma membrane. Furthermore, ß2AR(SSS) internalized more efficiently than the wild-type receptor, whereas recycling was very similar for both receptors. Thus, we show how the interaction between arrestins and receptors can be increased with minimal receptor modification and that relatively modest increases in receptor-arrestin affinity are sufficient to alter arrestin trafficking.


Assuntos
Arrestinas/genética , Arrestinas/metabolismo , Endocitose/fisiologia , Engenharia de Proteínas/métodos , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Sequência de Aminoácidos , Células HEK293 , Humanos , Dados de Sequência Molecular , Fosforilação/fisiologia , Ligação Proteica/fisiologia , Transporte Proteico/fisiologia
12.
J Biol Chem ; 289(26): 18451-65, 2014 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-24817122

RESUMO

In addition to being nutrients, free fatty acids act as signaling molecules by activating a family of G protein-coupled receptors. Among these is FFA4, previously called GPR120, which responds to medium and long chain fatty acids, including health-promoting ω-3 fatty acids, which have been implicated in the regulation of metabolic and inflammatory responses. Here we show, using mass spectrometry, mutagenesis, and phosphospecific antibodies, that agonist-regulated phosphorylation of the human FFA4 receptor occurred primarily at five residues (Thr(347), Thr(349), Ser(350), Ser(357), and Ser(360)) in the C-terminal tail. Mutation of these residues reduced both the efficacy and potency of ligand-mediated arrestin-3 recruitment as well as affecting recruitment kinetics. Combined mutagenesis of all five of these residues was insufficient to fully abrogate interaction with arrestin-3, but further mutagenesis of negatively charged residues revealed additional structural components for the interaction with arrestin-3 within the C-terminal tail of the receptor. These elements consist of the acidic residues Glu(341), Asp(348), and Asp(355) located close to the phosphorylation sites. Receptor phosphorylation thus operates in concert with structural elements within the C-terminal tail of FFA4 to allow for the recruitment of arrestin-3. Importantly, these mechanisms of arrestin-3 recruitment operate independently from Gq/11 coupling, thereby offering the possibility that ligands showing stimulus bias could be developed that exploit these differential coupling mechanisms. Furthermore, this provides a strategy for the design of biased receptors to probe physiologically relevant signaling.


Assuntos
Arrestinas/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Arrestinas/química , Arrestinas/genética , Linhagem Celular , Humanos , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Receptores Acoplados a Proteínas G/genética
13.
Science ; 385(6712): eadp7114, 2024 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-39116259

RESUMO

Endoplasmic reticulum (ER) stress induces the repression of protein synthesis throughout the cell. Attempts to understand how localized stress leads to widespread repression have been limited by difficulties in resolving translation rates at the subcellular level. Here, using live-cell imaging of reporter mRNA translation, we unexpectedly found that during ER stress, active translation at mitochondria was significantly protected. The mitochondrial protein ATPase family AAA domain-containing protein 3A (ATAD3A) interacted with protein kinase RNA-like endoplasmic reticulum kinase (PERK) and mediated this effect on localized translation by competing for binding with PERK's target, eukaryotic initiation factor 2 (eIF2). PERK-ATAD3A interactions increased during ER stress, forming mitochondria-ER contact sites. Furthermore, ATAD3A binding attenuated local PERK signaling and rescued the expression of some mitochondrial proteins. Thus, PERK-ATAD3A interactions can control translational repression at a subcellular level, mitigating the impact of ER stress on the cell.


Assuntos
ATPases Associadas a Diversas Atividades Celulares , Estresse do Retículo Endoplasmático , Fator de Iniciação 2 em Eucariotos , Proteínas de Membrana , Proteínas Mitocondriais , Biossíntese de Proteínas , eIF-2 Quinase , Humanos , ATPases Associadas a Diversas Atividades Celulares/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , eIF-2 Quinase/metabolismo , Retículo Endoplasmático/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Células HEK293 , Células HeLa , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Ligação Proteica , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Transdução de Sinais , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo
14.
Mol Pharmacol ; 84(5): 710-25, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23979972

RESUMO

TUG-891 [3-(4-((4-fluoro-4'-methyl-[1,1'-biphenyl]-2-yl)methoxy)phenyl)propanoic acid] was recently described as a potent and selective agonist for the long chain free fatty acid (LCFA) receptor 4 (FFA4; previously G protein-coupled receptor 120, or GPR120). Herein, we have used TUG-891 to further define the function of FFA4 and used this compound in proof of principle studies to indicate the therapeutic potential of this receptor. TUG-891 displayed similar signaling properties to the LCFA α-linolenic acid at human FFA4 across various assay end points, including stimulation of Ca²âº mobilization, ß-arrestin-1 and ß-arrestin-2 recruitment, and extracellular signal-regulated kinase phosphorylation. Activation of human FFA4 by TUG-891 also resulted in rapid phosphorylation and internalization of the receptor. While these latter events were associated with desensitization of the FFA4 signaling response, removal of TUG-891 allowed both rapid recycling of FFA4 back to the cell surface and resensitization of the FFA4 Ca²âº signaling response. TUG-891 was also a potent agonist of mouse FFA4, but it showed only limited selectivity over mouse FFA1, complicating its use in vivo in this species. Pharmacologic dissection of responses to TUG-891 in model murine cell systems indicated that activation of FFA4 was able to mimic many potentially beneficial therapeutic properties previously reported for LCFAs, including stimulating glucagon-like peptide-1 secretion from enteroendocrine cells, enhancing glucose uptake in 3T3-L1 adipocytes, and inhibiting release of proinflammatory mediators from RAW264.7 macrophages, which suggests promise for FFA4 as a therapeutic target for type 2 diabetes and obesity. Together, these results demonstrate both potential but also significant challenges that still need to be overcome to therapeutically target FFA4.


Assuntos
Compostos de Bifenilo/farmacologia , Fenilpropionatos/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Células 3T3-L1 , Animais , Arrestinas/fisiologia , Cálcio/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Glucose/metabolismo , Células HEK293 , Humanos , Camundongos , Fosforilação , beta-Arrestina 1 , beta-Arrestina 2 , beta-Arrestinas
15.
J Neurochem ; 124(2): 189-99, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23106126

RESUMO

Phosphorylation is considered a key event in the signalling and regulation of the µ opioid receptor (MOPr). Here, we used mass spectroscopy to determine the phosphorylation status of the C-terminal tail of the rat MOPr expressed in human embryonic kidney 293 (HEK-293) cells. Under basal conditions, MOPr is phosphorylated on Ser(363) and Thr(370), while in the presence of morphine or [D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin (DAMGO), the COOH terminus is phosphorylated at three additional residues, Ser(356) , Thr(357) and Ser(375). Using N-terminal glutathione S transferase (GST) fusion proteins of the cytoplasmic, C-terminal tail of MOPr and point mutations of the same, we show that, in vitro, purified G protein-coupled receptor kinase 2 (GRK2) phosphorylates Ser(375), protein kinase C (PKC) phosphorylates Ser(363), while CaMKII phosphorylates Thr(370). Phosphorylation of the GST fusion protein of the C-terminal tail of MOPr enhanced its ability to bind arrestin-2 and -3. Hence, our study identifies both the basal and agonist-stimulated phospho-acceptor sites in the C-terminal tail of MOPr, and suggests that the receptor is subject to phosphorylation and hence regulation by multiple protein kinases.


Assuntos
Receptores Opioides mu/química , Receptores Opioides mu/genética , Região 5'-Flanqueadora/genética , Sequência de Aminoácidos , Animais , Células HEK293 , Humanos , Dados de Sequência Molecular , Fosforilação/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína/genética , Transporte Proteico/genética , Ratos , Receptores Opioides mu/metabolismo
16.
Proc Natl Acad Sci U S A ; 107(49): 21181-6, 2010 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-21078968

RESUMO

The activity of G protein-coupled receptors is regulated via hyper-phosphorylation following agonist stimulation. Despite the universal nature of this regulatory process, the physiological impact of receptor phosphorylation remains poorly studied. To address this question, we have generated a knock-in mouse strain that expresses a phosphorylation-deficient mutant of the M(3)-muscarinic receptor, a prototypical G(q/11)-coupled receptor. This mutant mouse strain was used here to investigate the role of M(3)-muscarinic receptor phosphorylation in the regulation of insulin secretion from pancreatic islets. Importantly, the phosphorylation deficient receptor coupled to G(q/11)-signaling pathways but was uncoupled from phosphorylation-dependent processes, such as receptor internalization and ß-arrestin recruitment. The knock-in mice showed impaired glucose tolerance and insulin secretion, indicating that M(3)-muscarinic receptors expressed on pancreatic islets regulate glucose homeostasis via receptor phosphorylation-/arrestin-dependent signaling. The mechanism centers on the activation of protein kinase D1, which operates downstream of the recruitment of ß-arrestin to the phosphorylated M(3)-muscarinic receptor. In conclusion, our findings support the unique concept that M(3)-muscarinic receptor-mediated augmentation of sustained insulin release is largely independent of G protein-coupling but involves phosphorylation-/arrestin-dependent coupling of the receptor to protein kinase D1.


Assuntos
Arrestinas/metabolismo , Insulina/metabolismo , Proteína Quinase C/metabolismo , Receptor Muscarínico M3/fisiologia , Animais , Ativação Enzimática , Glucose , Homeostase , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Mutantes , Fosforilação , Transporte Proteico , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Receptores Acoplados a Proteínas G , Transdução de Sinais , beta-Arrestinas
17.
J Biol Chem ; 286(13): 11506-18, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21177246

RESUMO

G-protein-coupled receptors are hyper-phosphorylated in a process that controls receptor coupling to downstream signaling pathways. The pattern of receptor phosphorylation has been proposed to generate a "bar code" that can be varied in a tissue-specific manner to direct physiologically relevant receptor signaling. If such a mechanism existed, receptors would be expected to be phosphorylated in a cell/tissue-specific manner. Using tryptic phosphopeptide maps, mass spectrometry, and phospho-specific antibodies, it was determined here that the prototypical G(q/11)-coupled M(3)-muscarinic receptor was indeed differentially phosphorylated in various cell and tissue types supporting a role for differential receptor phosphorylation in directing tissue-specific signaling. Furthermore, the phosphorylation profile of the M(3)-muscarinic receptor was also dependent on the stimulus. Full and partial agonists to the M(3)-muscarinic receptor were observed to direct phosphorylation preferentially to specific sites. This hitherto unappreciated property of ligands raises the possibility that one mechanism underlying ligand bias/functional selectivity, a process where ligands direct receptors to preferred signaling pathways, may be centered on the capacity of ligands to promote receptor phosphorylation at specific sites.


Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Receptor Muscarínico M3/metabolismo , Transdução de Sinais/fisiologia , Animais , Células CHO , Cricetinae , Cricetulus , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Camundongos , Fosforilação/fisiologia , Receptor Muscarínico M3/agonistas , Receptor Muscarínico M3/genética
18.
Handb Exp Pharmacol ; (208): 79-94, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22222696

RESUMO

It is now well established that G-protein coupled receptors (GPCRs) are hyper-phosphorylated following agonist occupation usually at serine and threonine residues contained on the third intracellular loop and C-terminal tail. After some 2 decades of intensive research, the nature of protein kinases involved in this process together with the signalling consequences of receptor phosphorylation has been firmly established. The major challenge that the field currently faces is placing all this information within a physiological context and determining to what extent does phosphoregulation of GPCRs impact on whole animal responses. In this chapter, we address this issue by describing how GPCR phosphorylation might vary depending on the cell type in which the receptor is expressed and how this might be employed to drive selective regulation of physiological responses.


Assuntos
Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Animais , Humanos , Ligantes , Fosforilação , Rodopsina/metabolismo
19.
J Biol Chem ; 284(25): 17147-17156, 2009 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-19332541

RESUMO

Changes in synaptic strength mediated by ionotropic glutamate N-methyl-D-asparate (NMDA) receptors is generally considered to be the molecular mechanism underlying memory and learning. NMDA receptors themselves are subject to regulation through signaling pathways that are activated by G-protein-coupled receptors (GPCRs). In this study we investigate the ability of NMDA receptors to regulate the signaling of GPCRs by focusing on the G(q/11)-coupled M(3)-muscarinic receptor expressed endogenously in mouse cerebellar granule neurons. We show that NMDA receptor activation results in the phosphorylation and desensitization of M(3)-muscarinic receptors through a mechanism dependent on NMDA-mediated calcium influx and the activity of calcium-calmodulin-dependent protein kinase II. Our study reveals a complex pattern of regulation where GPCRs (M(3)-muscarinic) and NMDA receptors can feedback on each other in a process that is likely to influence the threshold value of signaling networks involved in synaptic plasticity.


Assuntos
Cerebelo/metabolismo , Receptor Muscarínico M3/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sequência de Aminoácidos , Animais , Sinalização do Cálcio , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Cerebelo/citologia , Retroalimentação Fisiológica , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , N-Metilaspartato/farmacologia , Plasticidade Neuronal , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosfatidilinositóis/metabolismo , Fosforilação , Receptor Muscarínico M3/química , Receptor Muscarínico M3/deficiência , Receptor Muscarínico M3/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais
20.
Sci Signal ; 13(644)2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32788341

RESUMO

Chronic activation of the unfolded protein response (UPR), notably the branch comprising the kinase PERK and the translation initiation factor eIF2α, is a pathological feature of many neurodegenerative diseases caused by protein misfolding. Partial reduction of UPR signaling at the level of phosphorylated eIF2α is neuroprotective and avoids the pancreatic toxicity caused by full inhibition of PERK kinase activity. However, other stress pathways besides the UPR converge on phosphorylated eIF2α in the integrated stress response (ISR), which is critical to normal cellular function. We explored whether partial inhibition of PERK signaling may be a better therapeutic option. PERK-mediated phosphorylation of eIF2α requires its binding to the insert loop within PERK's kinase domain, which is, itself, phosphorylated at multiple sites. We found that, as expected, Akt mediates the phosphorylation of Thr799 in PERK. This phosphorylation event reduced eIF2α binding to PERK and selectively attenuated downstream signaling independently of PERK activity and the broader ISR. Induction of Thr799 phosphorylation with a small-molecule activator of Akt similarly reduced PERK signaling and increased both neuronal and animal survival without measurable pancreatic toxicity in a mouse model of prion disease. Thus, promoting PERK phosphorylation at Thr799 to partially down-regulate PERK-eIF2α signaling while avoiding widespread ISR inhibition may be a safe therapeutic approach in neurodegenerative disease.


Assuntos
Modelos Animais de Doenças , Fator de Iniciação 2 em Eucariotos/metabolismo , Doenças Priônicas/metabolismo , Transdução de Sinais , eIF-2 Quinase/metabolismo , Acetatos/farmacologia , Animais , Benzopiranos/farmacologia , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Células HEK293 , Humanos , Estimativa de Kaplan-Meier , Camundongos , Fosforilação/efeitos dos fármacos , Doenças Priônicas/tratamento farmacológico , Resposta a Proteínas não Dobradas/efeitos dos fármacos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA