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1.
Sci Rep ; 10(1): 8779, 2020 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-32471984

RESUMO

A comprehensive understanding of signalling downstream of GPCRs requires a broad approach to capture novel signalling modalities in addition to established pathways. Here, using an array of sixteen validated BRET-based biosensors, we analyzed the ability of seven different ß-adrenergic ligands to engage five distinct signalling pathways downstream of the ß1-adrenergic receptor (ß1AR). In addition to generating signalling signatures and capturing functional selectivity for the different ligands toward these pathways, we also revealed coupling to signalling pathways that have not previously been ascribed to the ßAR. These include coupling to Gz and G12 pathways. The signalling cascade linking the ß1AR to calcium mobilization was also characterized using a combination of BRET-based biosensors and CRISPR-engineered HEK 293 cells lacking the Gαs subunit or with pharmacological or genetically engineered pathway inhibitors. We show that both Gs and G12 are required for the full calcium response. Our work highlights the power of combining signal profiling with genome editing approaches to capture the full complement of GPCR signalling activities in a given cell type and to probe their underlying mechanisms.

3.
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
4.
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
5.
iScience ; 14: 47-57, 2019 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-30925410

RESUMO

G protein-coupled receptors are key signaling molecules and major targets for pharmaceuticals. The concept of ligand-dependent biased signaling raises the possibility of developing drugs with improved efficacy and safety profiles, yet translating this concept to native tissues remains a major challenge. Whether drug activity profiling in recombinant cell-based assays, traditionally used for drug discovery, has any relevance to physiology is unknown. Here we focused on the mu opioid receptor, the unrivalled target for pain treatment and also the key driver for the current opioid crisis. We selected a set of clinical and novel mu agonists, and profiled their activities in transfected cell assays using advanced biosensors and in native neurons from knock-in mice expressing traceable receptors endogenously. Our data identify Gi-biased agonists, including buprenorphine, and further show highly correlated drug activities in the two otherwise very distinct experimental systems, supporting in vivo translatability of biased signaling for mu opioid drugs.

6.
Sci Signal ; 11(559)2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30514808

RESUMO

G protein-coupled receptors (GPCRs) are important therapeutic targets that exhibit functional selectivity (biased signaling), in which different ligands or receptor variants elicit distinct downstream signaling. Understanding all the signaling events and biases that contribute to both the beneficial and adverse effects of GPCR stimulation by given ligands is important for drug discovery. Here, we report the design, validation, and use of pathway-selective bioluminescence resonance energy transfer (BRET) biosensors that monitor the engagement and activation of signaling effectors downstream of G proteins, including protein kinase C (PKC), phospholipase C (PLC), p63RhoGEF, and Rho. Combined with G protein and ß-arrestin BRET biosensors, our sensors enabled real-time monitoring of GPCR signaling at different levels in downstream pathways in both native and engineered cells. Profiling of the responses to 14 angiotensin II (AngII) type 1 receptor (AT1R) ligands enabled the clustering of compounds into different subfamilies of biased ligands and showed that, in addition to the previously reported functional selectivity between Gαq and ß-arrestin, there are also biases among G protein subtypes. We also demonstrated that biases observed at the receptor and G protein levels propagated to downstream signaling pathways and that these biases could occur through the engagement of different G proteins to activate a common effector. We also used these tools to determine how naturally occurring AT1R variants affected signaling bias. This suite of BRET biosensors provides a useful resource for fingerprinting biased ligands and mutant receptors and for dissecting functional selectivity at various levels of GPCR signaling.


Assuntos
Angiotensina II/análogos & derivados , Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Técnicas Biossensoriais/métodos , Proteínas de Ligação ao GTP/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , beta-Arrestinas/metabolismo , Sequência de Aminoácidos , Angiotensina II/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Transferência de Energia , Células HEK293 , Humanos , Ligantes , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Ratos , Receptor Tipo 1 de Angiotensina/genética , Transdução de Sinais
7.
Sci Signal ; 11(559)2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30514810

RESUMO

Frizzleds (FZDs) are a group of seven transmembrane-spanning (7TM) receptors that belong to class F of the G protein-coupled receptor (GPCR) superfamily. FZDs bind WNT proteins to stimulate diverse signaling cascades involved in embryonic development, stem cell regulation, and adult tissue homeostasis. Frizzled 5 (FZD5) is one of the most studied class F GPCRs that promote the functional inactivation of the ß-catenin destruction complex in response to WNTs. However, whether FZDs function as prototypical GPCRs has been heavily debated and, in particular, FZD5 has not been shown to activate heterotrimeric G proteins. Here, we show that FZD5 exhibited a conformational change after the addition of WNT-5A, which is reminiscent of class A and class B GPCR activation. In addition, we performed several live-cell imaging and spectrometric-based approaches, such as dual-color fluorescence recovery after photobleaching (dcFRAP) and resonance energy transfer (RET)-based assays that demonstrated that FZD5 activated Gαq and its downstream effectors upon stimulation with WNT-5A. Together, these findings suggest that FZD5 is a 7TM receptor with a bona fide GPCR activation profile and suggest novel targets for drug discovery in WNT-FZD signaling.


Assuntos
Proliferação de Células , Receptores Frizzled/metabolismo , Neoplasias Pancreáticas/patologia , Proteína Wnt-5a/metabolismo , Cálcio/metabolismo , Diglicerídeos/metabolismo , Receptores Frizzled/química , Células HEK293 , Humanos , Neoplasias Pancreáticas/metabolismo , Conformação Proteica , Proteína Quinase C/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Proteína Wnt-5a/química
8.
Sci Signal ; 11(545)2018 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-30154102

RESUMO

Melatonin is produced during the night and regulates sleep and circadian rhythms. Loss-of-function variants in MTNR1B, which encodes the melatonin receptor MT2, a G protein-coupled receptor (GPCR), are associated with an increased risk of type 2 diabetes (T2D). To identify specific T2D-associated signaling pathway(s), we profiled the signaling output of 40 MT2 variants by monitoring spontaneous (ligand-independent) and melatonin-induced activation of multiple signaling effectors. Genetic association analysis showed that defects in the melatonin-induced activation of Gαi1 and Gαz proteins and in spontaneous ß-arrestin2 recruitment to MT2 were the most statistically significantly associated with an increased T2D risk. Computational variant impact prediction by in silico evolutionary lineage analysis strongly correlated with the measured phenotypic effect of each variant, providing a predictive tool for future studies on GPCR variants. Together, this large-scale functional study provides an operational framework for the postgenomic analysis of the multiple GPCR variants present in the human population. The association of T2D risk with signaling pathway-specific defects opens avenues for pathway-specific personalized therapeutic intervention and reveals the potential relevance of MT2 function during the day, when melatonin is undetectable, but spontaneous activity of the receptor occurs.


Assuntos
Diabetes Mellitus Tipo 2/genética , Variação Genética , Receptor MT2 de Melatonina/genética , Transdução de Sinais/genética , Antioxidantes/farmacologia , Diabetes Mellitus Tipo 2/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HEK293 , Humanos , Melatonina/farmacologia , Fosforilação/efeitos dos fármacos , Receptor MT2 de Melatonina/metabolismo , Transdução de Sinais/efeitos dos fármacos , beta-Arrestina 2/genética , beta-Arrestina 2/metabolismo
9.
Brain Struct Funct ; 223(3): 1275-1296, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29110094

RESUMO

GPR88 is an orphan G-protein coupled receptor originally characterized as a striatal-enriched transcript and is a potential target for neuropsychiatric disorders. At present, gene knockout studies in the mouse have essentially focused on striatal-related functions and a comprehensive knowledge of GPR88 protein distribution and function in the brain is still lacking. Here, we first created Gpr88-Venus knock-in mice expressing a functional fluorescent receptor to fine-map GPR88 localization in the brain. The receptor protein was detected in neuronal soma, fibers and primary cilia depending on the brain region, and remarkably, whole-brain mapping revealed a yet unreported layer-4 cortical lamination pattern specifically in sensory processing areas. The unique GPR88 barrel pattern in L4 of the somatosensory cortex appeared 3 days after birth and persisted into adulthood, suggesting a potential function for GPR88 in sensory integration. We next examined Gpr88 knockout mice for cortical structure and behavioral responses in sensory tasks. Magnetic resonance imaging of live mice revealed abnormally high fractional anisotropy, predominant in somatosensory cortex and caudate putamen, indicating significant microstructural alterations in these GPR88-enriched areas. Further, behavioral analysis showed delayed responses in somatosensory-, visual- and olfactory-dependent tasks, demonstrating a role for GPR88 in the integration rather than perception of sensory stimuli. In conclusion, our data show for the first time a prominent role for GPR88 in multisensory processing. Because sensory integration is disrupted in many psychiatric diseases, our study definitely positions GPR88 as a target to treat mental disorders perhaps via activity on cortical sensory networks.


Assuntos
Proteínas de Bactérias/metabolismo , Mapeamento Encefálico , Encéfalo/metabolismo , Proteínas Luminescentes/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Fatores de Ribosilação do ADP/metabolismo , Animais , Proteínas de Bactérias/genética , Encéfalo/citologia , Encéfalo/diagnóstico por imagem , Proteínas de Transporte/metabolismo , Células Cultivadas , Discriminação Psicológica/fisiologia , Feminino , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacocinética , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Imagem por Ressonância Magnética , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Nucleares/metabolismo , Odorantes , Fosfopiruvato Hidratase/metabolismo , Desempenho Psicomotor/fisiologia , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas-G/genética , Reconhecimento Psicológico/fisiologia , Transfecção
10.
Nat Commun ; 8(1): 2169, 2017 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-29255305

RESUMO

Functional selectivity of G-protein-coupled receptors is believed to originate from ligand-specific conformations that activate only subsets of signaling effectors. In this study, to identify molecular motifs playing important roles in transducing ligand binding into distinct signaling responses, we combined in silico evolutionary lineage analysis and structure-guided site-directed mutagenesis with large-scale functional signaling characterization and non-negative matrix factorization clustering of signaling profiles. Clustering based on the signaling profiles of 28 variants of the ß2-adrenergic receptor reveals three clearly distinct phenotypical clusters, showing selective impairments of either the Gi or ßarrestin/endocytosis pathways with no effect on Gs activation. Robustness of the results is confirmed using simulation-based error propagation. The structural changes resulting from functionally biasing mutations centered around the DRY, NPxxY, and PIF motifs, selectively linking these micro-switches to unique signaling profiles. Our data identify different receptor regions that are important for the stabilization of distinct conformations underlying functional selectivity.


Assuntos
Evolução Molecular , Mutação , Receptores Adrenérgicos beta 2/genética , Transdução de Sinais/genética , Agonistas Adrenérgicos beta/farmacologia , Sequência de Bases , Análise por Conglomerados , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Isoproterenol/farmacologia , Modelos Moleculares , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais/efeitos dos fármacos
11.
Mol Pharmacol ; 91(5): 533-544, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28280061

RESUMO

The ß2 adrenergic receptor (ß2AR) increases intracellular Ca2+ in a variety of cell types. By combining pharmacological and genetic manipulations, we reveal a novel mechanism through which the ß2AR promotes Ca2+ mobilization (pEC50 = 7.32 ± 0.10) in nonexcitable human embryonic kidney (HEK)293S cells. Downregulation of Gs with sustained cholera toxin pretreatment and the use of Gs-null HEK293 (∆Gs-HEK293) cells generated using the clustered regularly interspaced short palindromic repeat-associated protein-9 nuclease (CRISPR/Cas9) system, combined with pharmacological modulation of cAMP formation, revealed a Gs-dependent but cAMP-independent increase in intracellular Ca2+ following ß2AR stimulation. The increase in cytoplasmic Ca2+ was inhibited by P2Y purinergic receptor antagonists as well as a dominant-negative mutant form of Gq, a Gq-selective inhibitor, and an inositol 1,4,5-trisphosphate (IP3) receptor antagonist, suggesting a role for this Gq-coupled receptor family downstream of the ß2AR activation. Consistent with this mechanism, ß2AR stimulation promoted the extracellular release of ATP, and pretreatment with apyrase inhibited the ß2AR-promoted Ca2+ mobilization. Together, these data support a model whereby the ß2AR stimulates a Gs-dependent release of ATP, which transactivates Gq-coupled P2Y receptors through an inside-out mechanism, leading to a Gq- and IP3-dependent Ca2+ mobilization from intracellular stores. Given that ß2AR and P2Y receptors are coexpressed in various tissues, this novel signaling paradigm could be physiologically important and have therapeutic implications. In addition, this study reports the generation and validation of HEK293 cells deleted of Gs using the CRISPR/Cas9 genome editing technology that will undoubtedly be powerful tools to study Gs-dependent signaling.


Assuntos
Cálcio/metabolismo , Receptores Purinérgicos P2Y/metabolismo , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/metabolismo , Toxina da Cólera/farmacologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Proteínas de Ligação ao GTP/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2Y/genética , Transdução de Sinais , Ativação Transcricional
12.
Nat Commun ; 7: 12178, 2016 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-27397672

RESUMO

Endocytosis and intracellular trafficking of receptors are pivotal to maintain physiological functions and drug action; however, robust quantitative approaches are lacking to study such processes in live cells. Here we present new bioluminescence resonance energy transfer (BRET) sensors to quantitatively monitor G protein-coupled receptors (GPCRs) and ß-arrestin trafficking. These sensors are based on bystander BRET and use the naturally interacting chromophores luciferase (RLuc) and green fluorescent protein (rGFP) from Renilla. The versatility and robustness of this approach are exemplified by anchoring rGFP at the plasma membrane or in endosomes to generate high dynamic spectrometric BRET signals on ligand-promoted recruitment or sequestration of RLuc-tagged proteins to, or from, specific cell compartments, as well as sensitive subcellular BRET imaging for protein translocation visualization. These sensors are scalable to high-throughput formats and allow quantitative pharmacological studies of GPCR trafficking in real time, in live cells, revealing ligand-dependent biased trafficking of receptor/ß-arrestin complexes.


Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Membrana Celular/metabolismo , Endossomos/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , beta-Arrestinas/metabolismo , Proteínas de Fluorescência Verde , Células HEK293 , Humanos , Luciferases , Transporte Proteico
13.
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
14.
Methods Cell Biol ; 132: 319-37, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26928550

RESUMO

The use of biosensors either individually or as part of panels has now become a common technique to capturing signaling events in living cells. Such biosensors have become particularly important for probing biased signaling and allostery in G protein-coupled receptor drug screening efforts. However, assumptions about the portability of such biosensors between cell types may lead to misinterpretation of drug effects on specific signaling pathways in a given cellular context. Further, the output of a particular biosensor may be different depending on where it is localized in a cell. Here, we discuss strategies to mitigate these concerns which should feed into future biosensor design and usage.


Assuntos
Técnicas Biossensoriais , Transdução de Sinais , Núcleo Celular/enzimologia , Ativação Enzimática , Fator de Crescimento Epidérmico/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Células HeLa , Humanos , Sistema de Sinalização das MAP Quinases , Receptores Acoplados a Proteínas-G/metabolismo , Acetato de Tetradecanoilforbol/farmacologia
15.
Methods ; 92: 5-10, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-25890247

RESUMO

There has been a growing appreciation that G protein-coupled receptor (GPCR) functional selectivity (viz. biased signaling), in particular between G protein- and ß-arrestin-dependent signaling, can be achieved with specific ligands, and that such directed signaling represents a promising avenue for improving drug efficacy and therapy. Thus, for any given GPCRs it is important to define means to pharmacologically characterize and classify drugs for their propensity to bias signaling. Here we describe an experimental protocol and step-by-step approach to assess functional selectivity between Gαq and ß-arrestin-dependent responses using the prototypical angiotensin II (AngII) type 1 receptor (AT1R) expressed in HEK 293 cells. The protocol describes the expression of Bioluminescence Resonance Energy Transfer (BRET) sensors for either Gαq or ß-arrestin with AT1R, and the use of the operational model of pharmacological agonism to quantify ligand bias. Such methods are equally applicable to other GPCRs and their downstream signaling effectors.


Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Técnicas Biossensoriais/métodos , Receptor Tipo 1 de Angiotensina/análise , Receptor Tipo 1 de Angiotensina/metabolismo , Transdução de Sinais/fisiologia , Angiotensina II/metabolismo , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Ligantes
16.
Biophys J ; 99(12): 4037-46, 2010 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-21156147

RESUMO

Bioluminescence resonance energy transfer (BRET) is increasingly being used to monitor protein-protein interactions and cellular events in cells. However, the ability to monitor multiple events simultaneously is limited by the spectral properties of the existing BRET partners. Taking advantage of newly developed Renilla luciferases and blue-shifted fluorescent proteins (FPs), we explored the possibility of creating novel BRET configurations using a single luciferase substrate and distinct FPs. Three new (to our knowledge) BRET assays leading to distinct color bioluminescence emission were generated and validated. The spectral properties of two of the FPs used (enhanced blue (EB) FP2 and mAmetrine) and the selection of appropriate detection filters permitted the concomitant detection of two independent BRET signals, without cross-interference, in the same cells after addition of a unique substrate for Renilla luciferase-II, coelentrazine-400a. Using individual BRET-based biosensors to monitor the interaction between G-protein-coupled receptors and G-protein subunits or activation of different G-proteins along with the production of a second messenger, we established the proof of principle that two new BRET configurations can be multiplexed to simultaneously monitor two dependent or independent cellular events. The development of this new multiplexed BRET configuration opens the way for concomitant monitoring of various independent biological processes in living cells.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas Luminescentes/metabolismo , Cor , AMP Cíclico/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Receptores Acoplados a Proteínas-G/metabolismo
17.
J Pharmacol Exp Ther ; 335(3): 520-32, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20826565

RESUMO

Heterozygous null mutations in the melanocortin-4 receptor (MC4R) cause early-onset obesity in humans, indicating that metabolic homeostasis is sensitive to quantitative variation in MC4R function. Most of the obesity-causing MC4R mutations functionally characterized so far lead to intracellular retention of receptors by the cell's quality control system. Thus, recovering cell surface expression of mutant MC4Rs could have a beneficial therapeutic value. We tested a pharmacological chaperone approach to restore cell surface expression and function of 10 different mutant forms of human melanocortin-4 receptor found in obese patients. Five cell-permeant MC4R-selective ligands were tested and displayed pharmacological chaperone activities, restoring cell surface targeting and function of the receptors with distinct efficacy profiles for the different mutations. Such mutation-specific efficacies suggested a structure-activity relationship between compounds and mutant receptor conformations that may open a path toward personalized therapy. In addition, one of the five pharmacological chaperones restored function to most of the mutant receptors tested. Combined with its ability to reach the central nervous system and its selectivity for the MC4R, this pharmacological chaperone may represent a candidate for the development of a targeted therapy suitable for a large subset of patients with MC4R-deficient obesity.


Assuntos
Mutação de Sentido Incorreto/fisiologia , Obesidade/tratamento farmacológico , Obesidade/genética , Dobramento de Proteína/efeitos dos fármacos , Receptor Tipo 4 de Melanocortina/antagonistas & inibidores , Idade de Início , Animais , Sítios de Ligação , Encéfalo/metabolismo , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Endocitose/efeitos dos fármacos , Células HEK293 , Humanos , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estrutura Molecular , Preparações Farmacêuticas/sangue , Preparações Farmacêuticas/química , Preparações Farmacêuticas/metabolismo , Farmacocinética , Transporte Proteico/efeitos dos fármacos , Receptor Tipo 4 de Melanocortina/agonistas , Receptor Tipo 4 de Melanocortina/química , Receptor Tipo 4 de Melanocortina/genética , Receptor Tipo 4 de Melanocortina/metabolismo , Transfecção , alfa-MSH/análogos & derivados , alfa-MSH/farmacologia
18.
J Biol Chem ; 285(33): 25624-36, 2010 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-20551320

RESUMO

The prostaglandin F2alpha (PGF2alpha) receptor (FP) is a key regulator of parturition and a target for pharmacological management of preterm labor. However, an incomplete understanding of signaling pathways regulating myometrial contraction hinders the development of improved therapeutics. Here we used a peptidomimetic inhibitor of parturition in mice, PDC113.824, whose structure was based on the NH(2)-terminal region of the second extracellular loop of FP receptor, to gain mechanistic insight underlying FP receptor-mediated cell responses in the context of parturition. We show that PDC113.824 not only delayed normal parturition in mice but also that it inhibited both PGF2alpha- and lipopolysaccharide-induced preterm labor. PDC113.824 inhibited PGF2alpha-mediated, G(alpha)(12)-dependent activation of the Rho/ROCK signaling pathways, actin remodeling, and contraction of human myometrial cells likely by acting as a non-competitive, allosteric modulator of PGF2alpha binding. In contrast to its negative allosteric modulating effects on Rho/ROCK signaling, PDC113.824 acted as a positive allosteric modulator on PGF2alpha-mediated protein kinase C and ERK1/2 signaling. This bias in receptor-dependent signaling was explained by an increase in FP receptor coupling to G(alpha)(q), at the expense of coupling to G(alpha)(12). Our findings regarding the allosteric and biased nature of PDC113.824 offer new mechanistic insights into FP receptor signaling relevant to parturition and suggest novel therapeutic opportunities for the development of new tocolytic drugs.


Assuntos
Dinoprosta/metabolismo , Parto/efeitos dos fármacos , Peptídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Quinases Associadas a rho/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Linhagem Celular , Feminino , Imunofluorescência , Humanos , Camundongos , Trabalho de Parto Prematuro/induzido quimicamente , Trabalho de Parto Prematuro/tratamento farmacológico , Peptídeos/síntese química , Peptídeos/uso terapêutico , Gravidez , Proteína Quinase C/metabolismo
19.
J Pharmacol Exp Ther ; 331(1): 297-307, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19584306

RESUMO

Classically, the prostaglandin E(2) (PGE(2)) receptor EP(4) has been classified as coupling to the Galpha(s) subunit, leading to intracellular cAMP increases. However, EP(4) signaling has been revealed to be more complex and also involves coupling to pertussis toxin-sensitive Galpha(i) proteins and beta-arrestin-mediated effects. There are now many examples of selective activation of independent pathways by G protein-coupled receptor (GPCR) ligands, a concept referred to as functional selectivity. Because most EP(4) ligands had thus far only been functionally characterized by their ability to stimulate cAMP production, we systematically determined the potencies and efficacies of a panel of EP(4) ligands for activation of Galpha(s), Galpha(i), and beta-arrestin relative to the endogenous ligand PGE(2). For this purpose, we adapted three bioluminescence resonance energy transfer (BRET) assays to evaluate the respective pathways in living cells. Our results suggest considerable functional selectivity among the tested, structurally related agonists. PGE(2) was the most selective in activating Galpha(s), whereas PGF(2alpha) and PGE(1) alcohol were the most biased for activating Galpha(i1) and beta-arrestin, respectively. We observed reversal in order of potencies between beta-arrestin 2 and Galpha(i1) functional assays comparing PGE(1) alcohol and either PGF(2alpha), PGD(2), or 7-[(1R,2R)-2-[(E,3R)-3-hydroxy-4-(phenoxy)but-1-enyl]-5-oxocyclopentyl]heptanoic acid (M&B28767). Most ligands were full agonists for the three pathways tested. Our results have implications for the use of PGE(2) analogs in experimental and possibly clinical settings, because their activity spectra on EP(4) differ from that of the native agonist. The BRET-based methodology used for this first systematic assessment of a set of EP(4) agonists should be applicable for the study of other GPCRs.


Assuntos
Dinoprostona/metabolismo , Receptores de Prostaglandina E/metabolismo , Animais , Arrestinas/metabolismo , Arrestinas/fisiologia , Linhagem Celular , Dinoprostona/fisiologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/fisiologia , Humanos , Insetos , Ligantes , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Receptores de Prostaglandina E/fisiologia , Receptores de Prostaglandina E Subtipo EP4 , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , beta-Arrestina 2 , beta-Arrestinas
20.
J Immunol ; 183(4): 2747-57, 2009 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-19620302

RESUMO

Certain proteins, including receptors and signaling molecules, are known to be enriched in caveolae and lipid rafts. Caveolin-1, the major structural protein of caveolae, specifically interacts with many signaling molecules and, thus, caveolae and lipid rafts are often seen as preassembled signaling platforms. A potential binding site for caveolin-1 is present in the platelet-activating factor receptor (PAFR) sequence, and many downstream signaling components of PAFR activation preferentially localize in caveolae. The aim of this study was to investigate whether the PAFR was localized in caveolae/lipid raft domains and, if so, what would be the significance of such localization for PAFR signaling. In this study, we demonstrate that PAFR localizes within membrane microdomains, in close proximity to caveolin-1 in living cells, with potential interaction through a caveolin-1-binding sequence in the PAFR C terminus. Caveolin-1, however, is not essential for PAFR localization in lipid rafts. Disruption of caveolae/lipid rafts with methyl-beta-cyclodextrin markedly reduced PAF-triggered inositol phosphate production and cytosolic calcium flux, suggesting that PAFR signaling through the Galphaq protein was critically dependent on integrity of lipid rafts and/or caveolae. Interestingly, whereas in caveolin-1-expressing cells lipid raft disruption markedly decreased PAFR-mediated activation of the ERK/MAPK pathway, in cells lacking caveolae, such as leukocytes, lipid raft disruption had either the same inhibitory effect (Ramos B cells) or no effect (monocytes) on PAFR capacity to signal through the ERK/MAPK pathway. In conclusion, PAFR appears to localize within caveolae or lipid rafts in different cell types, and this location may be important for specific signaling events.


Assuntos
Sinalização do Cálcio , Cavéolas/metabolismo , Caveolina 1/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator de Ativação de Plaquetas/fisiologia , Animais , Sítios de Ligação/imunologia , Células CHO , Sinalização do Cálcio/imunologia , Cavéolas/enzimologia , Cavéolas/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Ativação Enzimática/imunologia , Humanos , Microdomínios da Membrana/imunologia , Microdomínios da Membrana/metabolismo , Fragmentos de Peptídeos/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/fisiologia , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Acoplados a Proteínas-G/fisiologia
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