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1.
Mol Pharmacol ; 75(3): 637-47, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19047484

RESUMO

We show here that the rat vasopressin V(1b) receptor simultaneously activates both the G(q/11)-inositol phosphate (IP) and G(s)-cAMP pathways when transiently expressed in Chinese hamster ovary, human embryonic kidney (HEK) 293, and COS-7 cells and stimulated with arginine-vasopressin. Higher concentrations of the hormone, however, were needed to trigger the cAMP pathway. The nonmammalian analog arginine-vasotocin and the selective V(1b) agonist d[Cha(4)]vasopressin also activated the cAMP and IP pathways, although d[Cha(4)]-vasopressin elicited the two responses with equivalent potencies. We determined that the V(1b) receptor is present as a homodimer at the plasma membrane. Treatment of V(1b)-transfected HEK-293 cells with methyl-beta-cyclodextrin, a drug known to dissociate cholesterol-rich domains of the plasma membrane, shifted the EC(50) of the vasopressin-induced cAMP accumulation to lower concentrations and, remarkably, increased the hormone efficacy related to the activation of this second messenger system. In parallel, the vasopressin-mediated activation of the IP pathway was slightly reduced without modification of its EC(50). These results suggest that, as with many other G protein-coupled receptors, when transfected in heterologous cell systems, the V(1b) receptor forms dimers that signal differentially through the G(q/11) and G(s) proteins depending on the nature of the ligand as well as on its localization within specialized compartments of the plasma membrane. The present study thus illustrates how signal transduction associated with the activation of a G protein-coupled receptor can be versatile and highly dependent on both the cell context and the chemical nature of the extracellular signaling messenger.


Assuntos
Compartimento Celular/fisiologia , Microdomínios da Membrana/metabolismo , Receptores de Vasopressinas/metabolismo , Animais , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , AMP Cíclico/fisiologia , Dimerização , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/fisiologia , Humanos , Microdomínios da Membrana/química , Ligação Proteica/fisiologia , Ratos , Receptores de Vasopressinas/química , Receptores de Vasopressinas/fisiologia , Transdução de Sinais/fisiologia
2.
Nat Struct Mol Biol ; 11(8): 706-13, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15235591

RESUMO

Membrane receptors, key components in signal transduction, often function as dimers. These include some G protein-coupled receptors such as metabotropic glutamate (mGlu) receptors that have large extracellular domains (ECDs) where agonists bind. How agonist binding in dimeric ECDs activates the effector domains remains largely unknown. The structure of the dimeric ECDs of mGlu(1) solved in the presence of agonist revealed two specific conformations in which either one or both protomers are in an agonist-stabilized closed form. Here we examined whether both conformations correspond to an active form of the full-length receptor. Using a system that allows the formation of dimers made of a wild-type and a mutant subunit, we show that the closure of one ECD per dimer is sufficient to activate the receptor, but the closure of both ECDs is required for full activity.


Assuntos
Dimerização , Receptores de Glutamato Metabotrópico/química , Sítios de Ligação , Cálcio/metabolismo , Linhagem Celular , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Transferência Ressonante de Energia de Fluorescência , Humanos , Fosfatos de Inositol/química , Microscopia de Fluorescência , Modelos Biológicos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Receptores de GABA-B/química , Transdução de Sinais , Fatores de Tempo , Transfecção
3.
J Med Chem ; 50(20): 4976-85, 2007 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-17850055

RESUMO

A series of fluorescent ligands designed for vasopressin and oxytocin G protein-coupled receptors was synthesized and characterized to develop fluorescence polarization or homogeneous time-resolved fluorescence (HTRF) binding assays. These ligands, labeled with europium pyridine-bis-bipyridine cryptate or with Alexa 488,546,647 selectively bound to the vasopressin V1a and oxytocin receptors with high affinities and exhibited antagonistic properties. The affinities of several unlabeled ligands determined by our homogeneous assays on membrane preparations or on intact cells into 96- and 384-well plate formats were similar to those determined by usual radioligand binding methods. Compared to other binding assays, the polarization and HTRF binding assays are nonradiaoactive, therefore safer to perform, yet very sensitive and homogeneous, therefore easier and faster to automate. These methods are thus suitable for efficient drug high-throughput screening procedures and can easily be applied to other G protein-coupled receptor models.


Assuntos
Antagonistas dos Receptores de Hormônios Antidiuréticos , Corantes Fluorescentes/química , Oligopeptídeos/síntese química , Receptores de Ocitocina/antagonistas & inibidores , Animais , Ligação Competitiva , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , AMP Cíclico/análogos & derivados , AMP Cíclico/química , Fluoresceínas/química , Polarização de Fluorescência , Transferência Ressonante de Energia de Fluorescência , Ligantes , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Compostos Organometálicos/química , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Compostos de Quinolínio/química , Ensaio Radioligante , Receptores de Ocitocina/agonistas , Receptores de Vasopressinas/agonistas
4.
J Biol Chem ; 283(44): 30225-34, 2008 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-18725411

RESUMO

In its native form, the chemokine CX3CL1 is a firmly adhesive molecule promoting leukocyte adhesion and migration and hence involved, along with its unique receptor CX3CR1, in various inflammatory processes. Here we investigated the role of molecular aggregation in the CX3CL1 adhesiveness. Assays of bioluminescence resonance energy transfer (BRET) and homogeneous time-resolved fluorescence (HTRF) in transfected cell lines and in primary cells showed specific signals indicative of CX3CL1 clustering. Truncation experiments showed that the transmembrane domain played a central role in this aggregation. A chimera with mutations of the 12 central transmembrane domain residues had significantly reduced BRET signals and characteristics of a non-clustering molecule. This mutant was weakly adhesive according to flow and dual pipette adhesion assays and was less glycosylated than CX3CL1, although, as we demonstrated, loss of glycosylation did not affect the CX3CL1 adhesive potency. We postulate that cell surfaces express CX3CL1 as a constitutive oligomer and that this oligomerization is essential for its adhesive potency. Inhibition of CX3CL1 self-assembly could limit the recruitment of CX3CR1-positive cells and may be a new pathway for anti-inflammatory therapies.


Assuntos
Quimiocina CX3CL1/metabolismo , Mutação , Sequência de Aminoácidos , Anti-Inflamatórios/farmacologia , Adesão Celular , Linhagem Celular , Deleção de Genes , Glicosilação , Humanos , Inflamação , Modelos Biológicos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Transdução de Sinais
5.
Mol Pharmacol ; 71(5): 1329-40, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17267663

RESUMO

G protein-coupled receptors transmit extracellular signals into the cells by activating heterotrimeric G proteins, a process that is often followed by receptor desensitization. Monitoring such a process in real time and in living cells will help better understand how G protein activation occurs. Energy transfer-based approaches [fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET)] were recently shown to be powerful methods to monitor the G protein-coupled receptors (GPCRs)-G protein association in living cells. Here, we used a BRET technique to monitor the coupling between the protease-activated receptor 1 (PAR1) and Galpha(i1) protein. A specific constitutive BRET signal can be measured between nonactivated PAR1 and the Galpha(i1) protein expressed at a physiological level. This signal is insensitive to pertussis toxin (PTX) and probably reflects the preassembly of these two proteins. The BRET signal rapidly increases upon receptor activation in a PTX-sensitive manner. The BRET signal then returns to the basal level after few minutes. The desensitization of the BRET signal is concomitant with beta-arrestin-1 recruitment to the receptor, consistent with the known rapid desensitization of PARs. The agonist-induced BRET increase was dependent on the insertion site of fluorophores in proteins. Taken together, our results show that BRET between GPCRs and Galpha proteins can be used to monitor the receptor activation in real time and in living cells. Our data also revealed that PAR1 can be part of a preassembled complex with Galpha(i1) protein, resulting either from a direct interaction between these partners or from their colocalization in specific microdomains, and that receptor activation probably results in rearrangements within such complexes.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/agonistas , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Proteínas Luminescentes/metabolismo , Receptor PAR-1/agonistas , Receptor PAR-1/metabolismo , Trombina/farmacologia , Sequência de Aminoácidos , Animais , Arrestinas/metabolismo , Células COS , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Humanos , Cinética , Dados de Sequência Molecular , Toxina Pertussis/farmacologia , Estrutura Secundária de Proteína/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Receptor PAR-1/química , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de Fluorescência , beta-Arrestina 1 , beta-Arrestinas
6.
Anal Biochem ; 358(1): 126-35, 2006 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16965760

RESUMO

Phospholipase C beta (PLC-beta)-coupled G protein-coupled receptor (GPCR) activities traditionally are assessed by measuring Ca2+ triggered by D-myo-inositol 1,4,5-trisphosphate (IP3), a PLC-beta hydrolysis product, or by measuring the production of inositol phosphate using cumbersome radioactive assays. A specific detection of IP3 production was also established using IP3 binding proteins. The short lifetime of IP3 makes this detection very challenging in measuring GPCR responses. Indeed, this IP3 rapidly enters the metabolic inositol phosphate cascade. It has been known for decades that lithium chloride (LiCl) leads to D-myo-inositol 1-phosphate accumulation on GPCR activation by inhibiting inositol monophosphatase, the final enzyme of the IP3 metabolic cascade. We show here that IP1 can be used as a surrogate of IP3 to monitor GPCR activation. We developed a novel homogeneous time-resolved fluorescence (HTRF) assay that correlates perfectly with existing methods and is easily amenable to high-throughput screening. The IP-One assay was validated on various GPCR models. It has the advantage over the traditional Ca2+ assay of allowing the measurement of inverse agonist activity as well as the analysis of PLC-beta activity in any nontransfected primary cultures. Finally, the high assay specificity for D-myo-inositol 1 monophosphate (IP1(1)) opens new possibilities in developing selective assays to study the functional roles of the various isoforms of inositol phosphates.


Assuntos
Inositol 1,4,5-Trifosfato , Fosfatos de Inositol , Receptores Acoplados a Proteínas G/metabolismo , Animais , Células CHO , Cricetinae , Humanos , Inositol 1,4,5-Trifosfato/química , Fosfatos de Inositol/química , Fosfolipases Tipo C/metabolismo
7.
Anal Biochem ; 329(2): 253-62, 2004 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-15158484

RESUMO

Direct or indirect interactions between membrane proteins at the cell surface play a central role in numerous cell processes, including possible synergistic effects between different types of receptors. Here we describe a method and tools to analyze membrane protein-protein interaction at the surface of living cells. This technology is based on the use of specific antibodies directed against each partner and labeled either with europium cryptate or with Alexa Fluor 647. This allows the measurement of a fluorescence resonance energy transfer (FRET) signal in a time-resolved manner if both antibodies are in close proximity. This approach is here validated using the heterodimeric gamma-aminobutyrate B receptor as a model. We show that after washing out the unbound antibodies, the time-resolved FRET signal can be measured together with the expression level of both partners via the quantification of the donor and the acceptor fluorophores bound to the cells. Thanks to the high sensitivity of this method and to the low concentration of antibodies required, we show that the signal can also be measured directly after the incubation period without washing out the unbound antibody (homogeneous time-resolved FRET). As such, this method is highly sensitive, reproducible, and compatible with the development of high-throughput screening protocols.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Mapeamento de Interação de Proteínas/métodos , Receptores de GABA-B/metabolismo , Anticorpos/imunologia , Humanos , Receptores de GABA-B/imunologia , Fatores de Tempo
8.
J Biol Chem ; 279(16): 15824-30, 2004 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-14736871

RESUMO

The gamma-aminobutyric acid type B (GABAB) receptor is an allosteric complex made of two subunits, GABAB1 (GB1) and GABAB2 (GB2). Both subunits are composed of an extracellular Venus flytrap domain (VFT) and a heptahelical domain (HD). GB1 binds GABA, and GB2 plays a major role in G-protein activation as well as in the high agonist affinity state of GB1. How agonist affinity in GB1 is regulated in the receptor remains unknown. Here, we demonstrate that GB2 VFT is a major molecular determinant involved in this control. We show that isolated versions of GB1 and GB2 VFTs in the absence of the HD and C-terminal tail can form hetero-oligomers as shown by time-resolved fluorescence resonance energy transfer (based on HTRF technology). GB2 VFT and its association with GB1 VFT controlled agonist affinity in GB1 in two ways. First, GB2 VFT exerted a direct action on GB1 VFT, as it slightly increased agonist affinity in isolated GB1 VFT. Second and most importantly, GB2 VFT prevented inhibitory interaction between the two main domains (VFT and HD) of GB1. According to this model, we propose that GB1 HD prevents the possible natural closure of GB1 VFT. In contrast, GB2 VFT facilitates this closure. Finally, such inhibitory contacts between HD and VFT in GB1 could be similar to those important to maintain the inactive state of the receptor.


Assuntos
Receptores de GABA-B/química , Regulação Alostérica , Sítios de Ligação , Linhagem Celular , Agonistas GABAérgicos/farmacologia , Agonistas dos Receptores de GABA-B , Humanos , Modelos Moleculares , Subunidades Proteicas , Transdução de Sinais , Relação Estrutura-Atividade
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