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1.
Inorg Chem ; 59(2): 1496-1512, 2020 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-31913029

RESUMO

We report a detailed characterization of Eu3+ and Tb3+ complexes derived from a tripyridinophane macrocycle bearing three acetate side arms (H3tpptac). Tpptac3- displays an overall basicity (∑ log KiH) of 24.5, provides the formation of mononuclear ML species, and shows a good binding affinity for Ln3+ (log KLnL = 17.5-18.7). These complexes are also thermodynamically stable at physiological pH (pEu = 18.6, pTb = 18.0). It should be noted that the pGd value of Gd-tpptac (18.4) is only slightly lower than that of commercially available MRI contrast agents such as Gd-dota (pGd = 19.2). Moreover, a very good selectivity for these ions over the endogenous cations (log KCuL = 14.4, log KZnL = 12.9, and log KCaL = 9.3) is observed. The X-ray structure of the terbium complex shows the metal coordinated by the nine N6O3 donor set of the ligand and one inner-sphere water molecule. DFT calculations result in two Eu-tpptac structures with similar bond energies (ΔE = 0.145 eV): one structure in which the water is coordinated to the metal ion and one structure in which the water molecule is farther away from the ion, bound to the ligand with an OH-π bond. By detailed luminescence experiments, we demonstrate that the europium complex in aqueous solution presents a hydration equilibrium between nine-coordinate, dehydrated [Eu-tpptac]0 and ten-coordinate, monohydrated [Eu-tpptac(H2O)]0 species. A similar trend is observed for the terbium complex. Despite the presence of this hydration equilibrium, the H3tpptac ligand sensitizes Eu3+ and Tb3+ luminescence efficiently in buffered water at physiological pH. Particularly, the terbium complex displays a long excited-state lifetime of 2.24 ms and an overall quantum yield of 33% with a brightness of 3600 M-1 cm-1. Such features of Ln3+ complexes of H3tpptac indicate that this platform appears to be particularly appealing for the further development of luminescent lanthanide labels.

2.
Anal Bioanal Chem ; 412(1): 73-80, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31776644

RESUMO

Although water has been extensively studied, not all of its unique properties have been fully understood. There is still controversy about the temperature at which hydrogen bonds are broken or weakened, producing the anomalous temperature dependence of many water properties. Different temperatures between 23 and 48 °C have been reported, but no study has scrutinized the reasons for this discrepancy. We suggest the determining role of pH in the alteration of the water anomaly temperature. We employed a luminescent europium trisbipyridine cryptate, which is highly sensitive to changes in the arrangement of water molecules and whose luminescence intensity and lifetime are not significantly influenced by variations over a broad pH range. Our results revealed an increase of the crossover temperature from circa 35 °C at pH 3.5 to circa 45 °C at pH 7 to 9, which explains the discrepancies of previous studies. The pH dependence of water anomaly temperature is an important property for a better understanding of water and water-based systems and applications.

3.
Angew Chem Int Ed Engl ; 57(41): 13686-13690, 2018 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-30084526

RESUMO

Fluorescence barcoding based on nanoparticles provides many advantages for multiparameter imaging. However, creating different concentration-independent codes without mixing various nanoparticles and by using single-wavelength excitation and emission for multiplexed cellular imaging is extremely challenging. Herein, we report the development of quantum dots (QDs) with two different SiO2 shell thicknesses (6 and 12 nm) that are coated with two different lanthanide complexes (Tb and Eu). FRET from the Tb or Eu donors to the QD acceptors resulted in four distinct photoluminescence (PL) decays, which were encoded by simple time-gated (TG) PL intensity detection in three individual temporal detection windows. The well-defined single-nanoparticle codes were used for live cell imaging and a one-measurement distinction of four different cells in a single field of view. This single-color barcoding strategy opens new opportunities for multiplexed labeling and tracking of cells.


Assuntos
Európio/química , Transferência Ressonante de Energia de Fluorescência/métodos , Nanopartículas , Pontos Quânticos , Térbio/química
4.
Sci Rep ; 8(1): 10414, 2018 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-29991736

RESUMO

G protein coupled receptors (GPCRs) play essential roles in intercellular communication. Although reported two decades ago, the assembly of GPCRs into dimer and larger oligomers in their native environment is still a matter of intense debate. Here, using number and brightness analysis of fluorescently labeled receptors in cultured hippocampal neurons, we confirm that the metabotropic glutamate receptor type 2 (mGlu2) is a homodimer at expression levels in the physiological range, while heterodimeric GABAB receptors form larger complexes. Surprisingly, we observed the formation of larger mGlu2 oligomers upon both activation and inhibition of the receptor. Stabilizing the receptor in its inactive conformation using biochemical constraints also led to the observation of oligomers. Following our recent observation that mGlu receptors are in constant and rapid equilibrium between several states under basal conditions, we propose that this structural heterogeneity limits receptor oligomerization. Such assemblies are expected to stabilize either the active or the inactive state of the receptor.


Assuntos
Neurônios/química , Conformação Proteica , Receptores Acoplados a Proteínas-G/química , Receptores de GABA-B/química , Hipocampo/química , Hipocampo/metabolismo , Humanos , Neurônios/metabolismo , Multimerização Proteica/genética , Receptores Acoplados a Proteínas-G/genética , Receptores de GABA-B/metabolismo , Transdução de Sinais
5.
Elife ; 62017 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-28661401

RESUMO

Metabotropic glutamate receptors (mGluRs) are mandatory dimers playing important roles in regulating CNS function. Although assumed to form exclusive homodimers, 16 possible heterodimeric mGluRs have been proposed but their existence in native cells remains elusive. Here, we set up two assays to specifically identify the pharmacological properties of rat mGlu heterodimers composed of mGlu2 and 4 subunits. We used either a heterodimer-specific conformational LRET-based biosensor or a system that guarantees the cell surface targeting of the heterodimer only. We identified mGlu2-4 specific pharmacological fingerprints that were also observed in a neuronal cell line and in lateral perforant path terminals naturally expressing mGlu2 and mGlu4. These results bring strong evidence for the existence of mGlu2-4 heterodimers in native cells. In addition to reporting a general approach to characterize heterodimeric mGluRs, our study opens new avenues to understanding the pathophysiological roles of mGlu heterodimers.


Assuntos
Compostos Bicíclicos com Pontes/farmacologia , Embrião de Mamíferos/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Multimerização Proteica/efeitos dos fármacos , Receptores de Glutamato Metabotrópico/química , Animais , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/farmacologia , Células HEK293 , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Humanos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de Glutamato Metabotrópico/metabolismo
6.
Cell Chem Biol ; 24(3): 360-370, 2017 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-28286129

RESUMO

The main inhibitory neurotransmitter, γ-aminobutyric acid (GABA), modulates many synapses by activating the G protein-coupled receptor GABAB, which is a target for various therapeutic applications. It is an obligatory heterodimer made of GB1 and GB2 that can be regulated by positive allosteric modulators (PAMs). The molecular mechanism of activation of the GABAB receptor remains poorly understood. Here, we have developed FRET-based conformational GABAB sensors compatible with high-throughput screening. We identified conformational changes occurring within the extracellular and transmembrane domains upon receptor activation, which are smaller than those observed in the related metabotropic glutamate receptors. These sensors also allow discrimination between agonists of different efficacies and between PAMs that have different modes of action, which has not always been possible using conventional functional assays. Our study brings important new information on the activation mechanism of the GABAB receptor and should facilitate the screening and identification of new chemicals targeting this receptor.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Receptores de GABA-B/metabolismo , Regulação Alostérica , Cálcio/análise , Cálcio/metabolismo , Agonistas dos Receptores de GABA-B/química , Agonistas dos Receptores de GABA-B/metabolismo , Antagonistas de Receptores de GABA-B/química , Antagonistas de Receptores de GABA-B/metabolismo , Células HEK293 , Humanos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Receptores de GABA-B/química , Ácido gama-Aminobutírico/química , Ácido gama-Aminobutírico/metabolismo
7.
Nat Chem Biol ; 13(4): 372-380, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28135236

RESUMO

Cell surface receptors represent a vast majority of drug targets. Efforts have been conducted to develop biosensors reporting their conformational changes in live cells for pharmacological and functional studies. Although Förster resonance energy transfer (FRET) appears to be an ideal approach, its use is limited by the low signal-to-noise ratio. Here we report a toolbox composed of a combination of labeling technologies, specific fluorophores compatible with time-resolved FRET and a novel method to quantify signals. This approach enables the development of receptor biosensors with a large signal-to-noise ratio. We illustrate the usefulness of this toolbox through the development of biosensors for various G-protein-coupled receptors and receptor tyrosine kinases. These receptors include mGlu, GABAB, LH, PTH, EGF and insulin receptors among others. These biosensors can be used for high-throughput studies and also revealed new information on the activation process of these receptors in their cellular environment.


Assuntos
Técnicas Biossensoriais , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Células HEK293 , Humanos , Ratos
8.
Artigo em Inglês | MEDLINE | ID: mdl-26617570

RESUMO

Although G protein-coupled receptor (GPCR) internalization has long been considered as a major aspect of the desensitization process that tunes ligand responsiveness, internalization is also involved in receptor resensitization and signaling, as well as the ligand scavenging function of some atypical receptors. Internalization thus contributes to the diversity of GPCR-dependent signaling, and its dynamics and quantification in living cells has generated considerable interest. We developed a robust and sensitive assay to follow and quantify ligand-induced and constitutive-induced GPCR internalization but also receptor recycling in living cells. This assay is based on diffusion-enhanced resonance energy transfer (DERET) between cell surface GPCRs labeled with a luminescent terbium cryptate donor and a fluorescein acceptor present in the culture medium. GPCR internalization results in a quantifiable reduction of energy transfer. This method yields a high signal-to-noise ratio due to time-resolved measurements. For various GPCRs belonging to different classes, we demonstrated that constitutive and ligand-induced internalization could be monitored as a function of time and ligand concentration, thus allowing accurate quantitative determination of kinetics of receptor internalization but also half-maximal effective or inhibitory concentrations of compounds. In addition to its selectivity and sensitivity, we provided evidence that DERET-based internalization assay is particularly suitable for characterizing biased ligands. Furthermore, the determination of a Z'-factor value of 0.45 indicates the quality and suitability of DERET-based internalization assay for high-throughput screening (HTS) of compounds that may modulate GPCRs internalization.

9.
FASEB J ; 29(6): 2235-46, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25690655

RESUMO

Identifying the interacting partners and the dynamics of the molecular networks constitutes the key point in understanding cellular processes. Different methods often based on energy transfer strategies have been developed to examine the molecular dynamics of protein complexes. However, these methods suffer a couple of drawbacks: a single complex can be studied at a time, and its localization and tracking cannot generally be investigated. Here, we report a multicolor time-resolved Förster resonance energy transfer microscopy method that allows the identification of up to 3 different complexes simultaneously, their localization in cells, and their tracking after activation. Using this technique, we studied GPCR oligomerization and internalization in human embryonic kidney 293 cells. We definitively show that receptors can internalize as oligomers and that receptor coexpression deeply impacts oligomer internalization processes.


Assuntos
Endocitose , Transferência Ressonante de Energia de Fluorescência/métodos , Microscopia de Fluorescência/métodos , Multimerização Proteica , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Transferência Ressonante de Energia de Fluorescência/instrumentação , Células HEK293 , Humanos , Microscopia de Fluorescência/instrumentação , Receptores de Vasopressinas/agonistas , Receptores de Vasopressinas/química , Receptores de Vasopressinas/metabolismo , Reprodutibilidade dos Testes , Imagem com Lapso de Tempo/instrumentação , Imagem com Lapso de Tempo/métodos
10.
Methods Mol Biol ; 1272: 23-36, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25563174

RESUMO

Screening chemical libraries to find specific drugs for G protein-coupled receptors is still of major interest. Indeed, because of their major roles in all physiological functions, G protein-coupled receptors remain major targets for drug development programs. Currently, interest in GPCRs as drug targets has been boosted by the discovery of biased ligands, thus allowing the development of drugs not only specific for one target but also for the specific signaling cascade expected to have the therapeutic effect. Such molecules are then expected to display fewer side effects. To reach such a goal, there is much interest in novel, efficient, simple, and direct screening assays that may help identify any drugs interacting with the target, these being then analyzed for their biased activity. Here, we present an efficient strategy to screen ligands on their binding properties. The method described is based on time-resolved FRET between a receptor and a ligand. This method has already been used to develop new assays called Tag-lite(®) binding assays for numerous G protein-coupled receptors, proving its broad application and its power.


Assuntos
Ensaios de Triagem em Larga Escala , Receptores Acoplados a Proteínas-G/agonistas , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Coloração e Rotulagem/métodos , Sítios de Ligação , Complexos de Coordenação/química , Desenho de Fármacos , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Expressão Gênica , Guanidinas/química , Células HEK293 , Humanos , Cinética , Ligantes , O(6)-Metilguanina-DNA Metiltransferase/química , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Ligação Proteica , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/química , Térbio/química
11.
Dalton Trans ; 44(11): 4791-803, 2015 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-25341077

RESUMO

The development of the brightest luminescent europium(iii) complexes is traced, including analysis of the C3-symmetric core complex based on a functionalized triazacyclononane and identification of the most suitable strongly absorbing chromophore. Strategies for the synthesis of the complexes, including enantiopure analogues, are outlined and opportunities for applications in time-resolved microscopy and spectral imaging emphasised. Practicable examples are introduced, including selective organelle staining for cellular optical imaging at 65 nm resolution and the development of new bioassays using time-resolved FRET methods.


Assuntos
Bioensaio/métodos , Corantes/síntese química , Európio/química , Imagem Óptica/métodos , Compostos Organometálicos/síntese química , Animais , Técnicas de Química Sintética , Corantes/química , Corantes/metabolismo , Humanos , Compostos Organometálicos/química , Compostos Organometálicos/metabolismo
12.
Nat Commun ; 5: 5206, 2014 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-25323157

RESUMO

Efficient cell-to-cell communication relies on the accurate signalling of cell surface receptors. Understanding the molecular bases of their activation requires the characterization of the dynamic equilibrium between active and resting states. Here, we monitor, using single-molecule Förster resonance energy transfer, the kinetics of the reorientation of the extracellular ligand-binding domain of the metabotropic glutamate receptor (mGluR), a class C G-protein-coupled receptor. We demonstrate that most receptors oscillate between a resting- and an active-conformation on a sub-millisecond timescale. Interestingly, we demonstrate that differences in agonist efficacies stem from differing abilities to shift the conformational equilibrium towards the fully active state, rather than from the stabilization of alternative static conformations, which further highlights the dynamic nature of mGluRs and revises our understanding of receptor activation and allosteric modulation.


Assuntos
Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/metabolismo , Sítio Alostérico , Domínio Catalítico , Comunicação Celular , Transferência Ressonante de Energia de Fluorescência , Guanidinas/química , Células HEK293 , Humanos , Cinética , Ligantes , Conformação Molecular , Mutação , Fótons , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Transdução de Sinais
13.
Angew Chem Int Ed Engl ; 53(40): 10718-22, 2014 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-25115848

RESUMO

Luminescent europium complexes are used in a broad range of applications as a result of their particular emissive properties. The synthesis and application of bright, highly water-soluble, and negatively charged sulfonic- or carboxylic acid derivatives of para-substituted aryl-alkynyl triazacyclononane complexes are described. Introduction of the charged solubilizing moieties suppresses cellular uptake or adsorption to living cells making them applicable for labeling and performing assays on membrane receptors. These europium complexes are applied to monitor fluorescent ligand binding on cell-surface proteins with time-resolved Förster resonance energy transfer (TR-FRET) assays in plate-based format and using TR-FRET microscopy.


Assuntos
Compostos Aza/análise , Complexos de Coordenação/análise , Európio/análise , Transferência Ressonante de Energia de Fluorescência/métodos , Substâncias Luminescentes/análise , Microscopia/métodos , Piperidinas/análise , Receptores Acoplados a Proteínas-G/metabolismo , Compostos Aza/metabolismo , Complexos de Coordenação/metabolismo , Európio/metabolismo , Células HEK293 , Humanos , Ligantes , Substâncias Luminescentes/metabolismo , Piperidinas/metabolismo , Ligação Proteica , Receptores Acoplados a Proteínas-G/análise , Solubilidade , Água/química
14.
Chemistry ; 20(28): 8636-46, 2014 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-24938657

RESUMO

A series of europium and terbium complexes based on a functionalized triazacyclononane carboxylate or phosphinate macrocyclic ligand is described. The influence of the anionic group, that is, carboxylate, methylphosphinate, or phenylphosphinate, on the photophysical properties was studied and rationalized on the basis of DFT calculated structures. The nature, number, and position of electron-donating or electron-withdrawing aryl substituents were varied systematically within the same phenylethynyl scaffold in order to optimize the brightness of the corresponding europium complexes and investigate their two-photon absorption properties. Finally, the europium complexes were examined in cell-imaging applications, and selected terbium complexes were studied as potential oxygen sensors.


Assuntos
Alquinos/química , Compostos Aza/química , Európio/química , Compostos Organometálicos/química , Piperidinas/química , Térbio/química , Ligantes , Estrutura Molecular
15.
Inorg Chem ; 53(4): 1854-66, 2014 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-24392868

RESUMO

The design and application of luminescent lanthanide cryptates for sensing biological interactions is highlighted through the review of the work performed in our laboratory and with academic collaborations. The path from the initial applications probing biochemical interaction in vitro to "state-of-the-art" cellular assays toward clinical applications using homogeneous time-resolved fluorescence technology is described. An overview of the luminescent lanthanide macrocyclic compounds developed at Cisbio in the recent past is given with an emphasis on specific constraints required by specific applications. Recent assays for drug-discovery and diagnostic purposes using both antibody-based and suicide-enzyme-based technology are illustrated. New perspectives in the field of molecular medicine and time-resolved microscopy are discussed.


Assuntos
Éteres de Coroa/química , Descoberta de Drogas , Elementos da Série dos Lantanídeos/química , Substâncias Luminescentes/química , Medicina Molecular/tendências , Humanos
16.
Proc Natl Acad Sci U S A ; 110(15): E1416-25, 2013 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-23487753

RESUMO

In multimeric cell-surface receptors, the conformational changes of the extracellular ligand-binding domains (ECDs) associated with receptor activation remain largely unknown. This is the case for the dimeric metabotropic glutamate receptors even though a number of ECD structures have been solved. Here, using an innovative approach based on cell-surface labeling and FRET, we demonstrate that a reorientation of the ECDs is associated with receptor and G-protein activation. Our approach helps identify partial agonists and highlights allosteric interactions between the effector and binding domains. Any approach expected to stabilize the active conformation of the effector domain increased the agonist potency in stabilizing the active ECDs conformation. These data provide key information on the structural dynamics and drug action at metabotropic glutamate receptors and validate an approach for tackling such analysis on other receptors.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Ácido Glutâmico/química , Receptores de Glutamato Metabotrópico/química , Sítio Alostérico , Animais , Cálcio/metabolismo , Membrana Celular/metabolismo , Cristalografia por Raios X , Análise Mutacional de DNA , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Ligantes , Mutação , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Ratos
17.
Chem Commun (Camb) ; 49(16): 1600-2, 2013 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-23336102

RESUMO

The synthesis, structure and photophysical properties of a series of highly emissive europium complexes is reported. Certain complexes enter mammalian cells by macropinocytosis and stain the mitochondria selectively, allowing observation of the Eu emission in cellulo by time-gated spectral imaging.


Assuntos
Európio/análise , Európio/química , Mitocôndrias/química , Amilorida/farmacologia , Androstadienos/farmacologia , Animais , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Moleculares , Estrutura Molecular , Células NIH 3T3 , Compostos Organometálicos/análise , Compostos Organometálicos/antagonistas & inibidores , Compostos Organometálicos/química , Temperatura , Wortmanina
18.
Prog Mol Biol Transl Sci ; 113: 275-312, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23244793

RESUMO

High-throughput screening requires easy-to-monitor, rapid, robust, reliable, and miniaturized methods to test thousands of compounds on a target in a short period, in order to find active drugs. Only a few methods have been proved to fulfill all these requirements. New screening approaches based on fluorescence and especially on the principle of resonance energy transfer are being developed to study one of the main targets in the pharmaceutical industry, namely, the G protein-coupled receptors (GPCRs). Two types of approaches are clearly defined: generic approaches that are immediately applicable to a lot of targets such as second messenger kits or kinase kits; target-specific approaches that sense the receptor itself such as fluorescent ligands or fluorescent partners. This chapter focuses on sensors and approaches using the time-resolved Förster resonance energy transfer and homogeneous time-resolved fluorescence principle, their use, and their prospective applications for screening drugs acting on GPCRs.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Receptores Acoplados a Proteínas-G/química , Animais , Anticorpos/química , Ligação Competitiva , Técnicas Biossensoriais , Membrana Celular/metabolismo , Química Farmacêutica/métodos , Desenho de Fármacos , Corantes Fluorescentes/química , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Ligantes , Ligação Proteica , Transdução de Sinais
19.
Proc Natl Acad Sci U S A ; 109(17): 6733-8, 2012 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-22493271

RESUMO

G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters, representing the largest group of therapeutic targets. Recent studies show that some GPCRs signal through both G protein and arrestin pathways in a ligand-specific manner. Ligands that direct signaling through a specific pathway are known as biased ligands. The arginine-vasopressin type 2 receptor (V2R), a prototypical peptide-activated GPCR, is an ideal model system to investigate the structural basis of biased signaling. Although the native hormone arginine-vasopressin leads to activation of both the stimulatory G protein (Gs) for the adenylyl cyclase and arrestin pathways, synthetic ligands exhibit highly biased signaling through either Gs alone or arrestin alone. We used purified V2R stabilized in neutral amphipols and developed fluorescence-based assays to investigate the structural basis of biased signaling for the V2R. Our studies demonstrate that the Gs-biased agonist stabilizes a conformation that is distinct from that stabilized by the arrestin-biased agonists. This study provides unique insights into the structural mechanisms of GPCR activation by biased ligands that may be relevant to the design of pathway-biased drugs.


Assuntos
Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais , Espectrometria de Fluorescência/métodos , Ligantes , Conformação Proteica , Receptores Acoplados a Proteínas-G/química
20.
Mol Pharmacol ; 80(6): 1033-46, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21880827

RESUMO

Molecular evolution and chemical genetics have been applied to generate functional pairings of mutated G protein-coupled receptors (GPCRs) and nonendogenous ligands. These mutant receptors, referred to as receptors activated solely by synthetic ligands (RASSLs) or designer receptors exclusively activated by designer drugs (DREADDs), have huge potential to define physiological roles of GPCRs and to validate receptors in animal models as therapeutic targets to treat human disease. However, appreciation of ligand bias and functional selectivity of different ligands at the same receptor suggests that RASSLs may signal differently than wild-type receptors activated by endogenous agonists. We assessed this by generating forms of wild-type human M(3) muscarinic receptor and a RASSL variant that responds selectively to clozapine N-oxide. Although the RASSL receptor had reduced affinity for muscarinic antagonists, including atropine, stimulation with clozapine N-oxide produced effects very similar to those generated by acetylcholine at the wild-type M(3)-receptor. Such effects included the relative movement of the third intracellular loop and C-terminal tail of intramolecular fluorescence resonance energy transfer sensors and the ability of the wild type and evolved mutant to regulate extracellular signal-regulated kinase 1/2 phosphorylation. Each form interacted similarly with ß-arrestin 2 and was internalized from the cell surface in response to the appropriate ligand. Furthermore, the pattern of phosphorylation of specific serine residues within the evolved receptor in response to clozapine N-oxide was very similar to that produced by acetylcholine at the wild type. Such results provide confidence that, at least for the M(3) muscarinic receptor, results obtained after transgenic expression of this RASSL are likely to mirror the actions of acetylcholine at the wild type receptor.


Assuntos
Mutagênese Sítio-Dirigida/normas , Receptor Muscarínico M3/fisiologia , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Opioides kappa/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Células HEK293 , Humanos , Ligantes , Mutagênese Sítio-Dirigida/métodos , Receptor Muscarínico M3/química , Receptores Opioides kappa/genética , Proteínas Recombinantes de Fusão/genética , Reprodutibilidade dos Testes
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