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1.
Proc Natl Acad Sci U S A ; 121(30): e2404000121, 2024 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-39008676

RESUMO

Atypical Chemokine Receptor 3 (ACKR3) belongs to the G protein-coupled receptor family but it does not signal through G proteins. The structural properties that govern the functional selectivity and the conformational dynamics of ACKR3 activation are poorly understood. Here, we combined hydrogen/deuterium exchange mass spectrometry, site-directed mutagenesis, and molecular dynamics simulations to examine the binding mode and mechanism of action of ACKR3 ligands of different efficacies. Our results show that activation or inhibition of ACKR3 is governed by intracellular conformational changes of helix 6, intracellular loop 2, and helix 7, while the DRY motif becomes protected during both processes. Moreover, we identified the binding sites and the allosteric modulation of ACKR3 upon ß-arrestin 1 binding. In summary, this study highlights the structure-function relationship of small ligands, the binding mode of ß-arrestin 1, the activation dynamics, and the atypical dynamic features in ACKR3 that may contribute to its inability to activate G proteins.


Assuntos
Simulação de Dinâmica Molecular , Ligação Proteica , Receptores CXCR , Humanos , Receptores CXCR/metabolismo , Receptores CXCR/genética , Sítios de Ligação , Conformação Proteica , beta-Arrestina 1/metabolismo , beta-Arrestina 1/genética , Ligantes , Células HEK293 , Mutagênese Sítio-Dirigida , Regulação Alostérica , Relação Estrutura-Atividade
2.
Mol Psychiatry ; 28(5): 1960-1969, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-36604603

RESUMO

Increasing evidence supports a relationship between lipid metabolism and mental health. In particular, the biostatus of polyunsaturated fatty acids (PUFAs) correlates with some symptoms of psychiatric disorders, as well as the efficacy of pharmacological treatments. Recent findings highlight a direct association between brain PUFA levels and dopamine transmission, a major neuromodulatory system implicated in the etiology of psychiatric symptoms. However, the mechanisms underlying this relationship are still unknown. Here we demonstrate that membrane enrichment in the n-3 PUFA docosahexaenoic acid (DHA), potentiates ligand binding to the dopamine D2 receptor (D2R), suggesting that DHA acts as an allosteric modulator of this receptor. Molecular dynamics simulations confirm that DHA has a high preference for interaction with the D2R and show that membrane unsaturation selectively enhances the conformational dynamics of the receptor around its second intracellular loop. We find that membrane unsaturation spares G protein activity but potentiates the recruitment of ß-arrestin in cells. Furthermore, in vivo n-3 PUFA deficiency blunts the behavioral effects of two D2R ligands, quinpirole and aripiprazole. These results highlight the importance of membrane unsaturation for D2R activity and provide a putative mechanism for the ability of PUFAs to enhance antipsychotic efficacy.

3.
Proc Natl Acad Sci U S A ; 118(42)2021 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-34663701

RESUMO

Atypical chemokine receptor 1 (ACKR1) is a G protein-coupled receptor (GPCR) targeted by Staphylococcus aureus bicomponent pore-forming leukotoxins to promote bacterial growth and immune evasion. Here, we have developed an integrative molecular pharmacology and structural biology approach in order to characterize the effect of leukotoxins HlgA and HlgB on ACKR1 structure and function. Interestingly, using cell-based assays and native mass spectrometry, we found that both components HlgA and HlgB compete with endogenous chemokines through a direct binding with the extracellular domain of ACKR1. Unexpectedly, hydrogen/deuterium exchange mass spectrometry analysis revealed that toxin binding allosterically modulates the intracellular G protein-binding domain of the receptor, resulting in dissociation and/or changes in the architecture of ACKR1-Gαi1 protein complexes observed in living cells. Altogether, our study brings important molecular insights into the initial steps of leukotoxins targeting a host GPCR.


Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Staphylococcus aureus/fisiologia , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Dimerização , Sistema do Grupo Sanguíneo Duffy/isolamento & purificação , Sistema do Grupo Sanguíneo Duffy/metabolismo , Exotoxinas/metabolismo , Humanos , Espectrometria de Massas/métodos , Ligação Proteica , Receptores de Superfície Celular/isolamento & purificação , Receptores de Superfície Celular/metabolismo , Células Sf9
4.
J Lipid Res ; 62: 100059, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33647276

RESUMO

Cholesterol is a major component of mammalian plasma membranes that not only affects the physical properties of the lipid bilayer but also is the function of many membrane proteins including G protein-coupled receptors. The oxytocin receptor (OXTR) is involved in parturition and lactation of mammals and in their emotional and social behaviors. Cholesterol acts on OXTR as an allosteric modulator inducing a high-affinity state for orthosteric ligands through a molecular mechanism that has yet to be determined. Using the ion channel-coupled receptor technology, we developed a functional assay of cholesterol modulation of G protein-coupled receptors that is independent of intracellular signaling pathways and operational in living cells. Using this assay, we discovered a stable binding of cholesterol molecules to the receptor when it adopts an orthosteric ligand-bound state. This stable interaction preserves the cholesterol-dependent activity of the receptor in cholesterol-depleted membranes. This mechanism was confirmed using time-resolved FRET experiments on WT OXTR expressed in CHO cells. Consequently, a positive cross-regulation sequentially occurs in OXTR between cholesterol and orthosteric ligands.


Assuntos
Receptores Acoplados a Proteínas G
5.
Traffic ; 19(1): 58-82, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29044966

RESUMO

The signaling pathway of G protein-coupled receptors is strongly linked to their trafficking profile. Little is known about the molecular mechanisms involved in the vasopressin receptor V1b subtype (V1b R) trafficking and its impact on receptor signaling and regulation. For this purpose, we investigated the role of ß-arrestins in receptor desensitization, internalization and recycling and attempted to dissect the V1b R-mediated MAP kinase pathway. Using MEF cells Knocked-out for ß-arrestins 1 and 2, we demonstrated that both ß-arrestins 1 and 2 play a fundamental role in internalization and recycling of V1b R with a rapid and transient V1b R-ß-arrestin interaction in contrast to a slow and long-lasting ß-arrestin recruitment of the V2 vasopressin receptor subtype (V2 R). Using V1b R-V2 R chimeras and V1b R C-terminus truncations, we demonstrated the critical role of the V1b R C-terminus in its interaction with ß-arrestins thereby regulating the receptor internalization and recycling kinetics in a phosphorylation-independent manner. In parallel, V1b R MAP kinase activation was dependent on arrestins and Src-kinase but independent on G proteins. Interestingly, Src interacted with hV1b R at basal state and dissociated when receptor internalization occurred. Altogether, our data describe for the first time the trafficking profile and MAP kinase pathway of V1b R involving both arrestins and Src kinase family.


Assuntos
Sistema de Sinalização das MAP Quinases , Receptores de Vasopressinas/metabolismo , beta-Arrestinas/metabolismo , Animais , Sítios de Ligação , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Camundongos , Ligação Proteica , Transporte Proteico , beta-Arrestinas/química , Quinases da Família src/metabolismo
6.
Mol Pharmacol ; 96(6): 778-793, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31092552

RESUMO

G protein-coupled receptors (GPCRs) are regulated by complex molecular mechanisms, both in physiologic and pathologic conditions, and their signaling can be intricate. Many factors influence their signaling behavior, including the type of ligand that activates the GPCR, the presence of interacting partners, the kinetics involved, or their location. The two CXC-type chemokine receptors, CXC chemokine receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3), both members of the GPCR superfamily, are important and established therapeutic targets in relation to cancer, human immunodeficiency virus infection, and inflammatory diseases. Therefore, it is crucial to understand how the signaling of these receptors works to be able to specifically target them. In this review, we discuss how the signaling pathways activated by CXCR4 and ACKR3 can vary in different situations. G protein signaling of CXCR4 depends on the cellular context, and discrepancies exist depending on the cell lines used. ACKR3, as an atypical chemokine receptor, is generally reported to not activate G proteins but can broaden its signaling spectrum upon heteromerization with other receptors, such as CXCR4, endothelial growth factor receptor, or the α 1-adrenergic receptor (α 1-AR). Also, CXCR4 forms heteromers with CC chemokine receptor (CCR) 2, CCR5, the Na+/H+ exchanger regulatory factor 1, CXCR3, α 1-AR, and the opioid receptors, which results in differential signaling from that of the monomeric subunits. In addition, CXCR4 is present on membrane rafts but can go into the nucleus during cancer progression, probably acquiring different signaling properties. In this review, we also provide an overview of the currently known critical amino acids involved in CXCR4 and ACKR3 signaling.


Assuntos
Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Receptores CXCR/genética , Receptores CXCR/metabolismo , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Humanos
8.
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
9.
EMBO J ; 30(12): 2336-49, 2011 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-21552208

RESUMO

G protein-coupled receptors (GPCRs) have key roles in cell-cell communication. Recent data suggest that these receptors can form large complexes, a possibility expected to expand the complexity of this regulatory system. Among the brain GPCRs, the heterodimeric GABA(B) receptor is one of the most abundant, being distributed in most brain regions, on either pre- or post-synaptic elements. Here, using specific antibodies labelled with time-resolved FRET compatible fluorophores, we provide evidence that the heterodimeric GABA(B) receptor can form higher-ordered oligomers in the brain, as suggested by the close proximity of the GABA(B1) subunits. Destabilizing the oligomers using a competitor or a GABA(B1) mutant revealed different G protein coupling efficiencies depending on the oligomeric state of the receptor. By examining, in heterologous system, the G protein coupling properties of such GABA(B) receptor oligomers composed of a wild-type and a non-functional mutant heterodimer, we provide evidence for a negative functional cooperativity between the GABA(B) heterodimers.


Assuntos
Receptores de GABA-B/química , Transdução de Sinais/fisiologia , Regulação Alostérica/genética , Animais , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Mutagênese Sítio-Dirigida , Isoformas de Proteínas/química , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genética , Multimerização Proteica/genética , Estabilidade Proteica , Receptores de GABA-B/deficiência , Receptores de GABA-B/genética , Transdução de Sinais/genética
10.
Anal Biochem ; 484: 105-12, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-25998104

RESUMO

Ligand-gated ion channels (LGICs) are considered as attractive protein targets in the search for new therapeutic agents. Nowadays, this strategy involves the capability to screen large chemical libraries. We present a new Tag-lite ligand binding assay targeting LGICs on living cells. This technology combines the use of suicide enzyme tags fused to channels of interest with homogeneous time-resolved fluorescence (HTRF) as the detection readout. Using the 5-HT3 receptor as system model, we showed that the pharmacology of the HALO-5HT3 receptor was identical to that of the native receptor. After validation of the assay by using 5-HT3 agonists and antagonists of reference, a pilot screen enabled us to identify azelastine, a well-known histamine H1 antagonist, as a potent 5-HT3 antagonist. This interesting result was confirmed with electrophysiological experiments. The method described here is easy to implement and could be applicable for other LGICs, opening new ways for the screening of chemical libraries.


Assuntos
Bioensaio/métodos , Receptores 5-HT3 de Serotonina/metabolismo , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Células HEK293 , Humanos , Miniaturização , Receptores 5-HT3 de Serotonina/química
11.
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
12.
Biochem Soc Trans ; 41(1): 148-53, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23356275

RESUMO

Fluorescent ligands for GPCRs (G-protein-coupled receptors) have been synthesized for a long time but their use was usually restricted to receptor localization in the cell by fluorescent imaging microscopy. During the last two decades, the emergence of new fluorescence-based strategies and the concomitant development of fluorescent measurement apparatus have dramatically widened the use of fluorescent ligands. Among the various strategies, TR (time-resolved)-FRET (fluorescence resonance energy transfer) approaches exhibit an interesting potential to study GPCR interactions with various partners. We have derived various sets of ligands that target different GPCRs with fluorophores, which are compatible with TR-FRET strategies. Fluorescent ligands labelled either with a fluorescent donor (such as europium or terbium cryptate) or with a fluorescent acceptor (such as fluorescein, dy647 or Alexa Fluor® 647), for example, kept high affinities for their cognate receptors. These ligands turn out to be interesting tools to develop FRET-based binding assays. We also used these fluorescent ligands to analyse GPCR oligomerization by measuring FRET between ligands bound to receptor dimers. In contrast with FRET strategies, on the basis of receptor labelling, the ligand-based approach we developed is fully compatible with the study of wild-type receptors and therefore with receptors expressed in native tissues. Therefore, by using fluorescent analogues of oxytocin, we demonstrated the existence of oxytocin receptor dimers in the mammary gland of lactating rats.


Assuntos
Biopolímeros/metabolismo , Corantes Fluorescentes/química , Receptores Acoplados a Proteínas G/metabolismo , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/metabolismo , Ligantes , Ligação Proteica
13.
Nat Chem Biol ; 6(8): 587-94, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20622858

RESUMO

G protein-coupled receptor (GPCR) oligomers have been proposed to play critical roles in cell signaling, but confirmation of their existence in a native context remains elusive, as no direct interactions between receptors have been reported. To demonstrate their presence in native tissues, we developed a time-resolved FRET strategy that is based on receptor labeling with selective fluorescent ligands. Specific FRET signals were observed with four different receptors expressed in cell lines, consistent with their dimeric or oligomeric nature in these transfected cells. More notably, the comparison between FRET signals measured with sets of fluorescent agonists and antagonists was consistent with an asymmetric relationship of the two protomers in an activated GPCR dimer. Finally, we applied the strategy to native tissues and succeeded in demonstrating the presence of oxytocin receptor dimers and/or oligomers in mammary gland.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Oligopeptídeos/química , Receptores Acoplados a Proteínas G/metabolismo , Algoritmos , Animais , Antagonistas dos Receptores de Hormônios Antidiuréticos , Células COS , Linhagem Celular , Chlorocebus aethiops , Dimerização , Antagonistas dos Receptores de Dopamina D2 , Feminino , Corantes Fluorescentes , Ligantes , Glândulas Mamárias Animais/metabolismo , Modelos Moleculares , Oligopeptídeos/metabolismo , Ensaio Radioligante , Ratos , Receptores de Dopamina D2/agonistas , Receptores de Dopamina D2/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores de Ocitocina/agonistas , Receptores de Ocitocina/antagonistas & inibidores , Receptores de Ocitocina/metabolismo , Receptores de Vasopressinas/agonistas , Receptores de Vasopressinas/metabolismo
14.
Elife ; 112022 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-35311641

RESUMO

Staphylococcus aureus (SA) leukocidin ED (LukED) belongs to a family of bicomponent pore forming toxins that play important roles in SA immune evasion and nutrient acquisition. LukED targets specific G protein-coupled chemokine receptors to lyse human erythrocytes (red blood cells) and leukocytes (white blood cells). The first recognition step of receptors is critical for specific cell targeting and lysis. The structural and molecular bases for this mechanism are not well understood but could constitute essential information to guide antibiotic development. Here, we characterized the interaction of LukE with chemokine receptors ACKR1, CCR2, and CCR5 using a combination of structural, pharmacological, and computational approaches. First, crystal structures of LukE in complex with a small molecule mimicking sulfotyrosine side chain (p-cresyl sulfate) and with peptides containing sulfotyrosines issued from receptor sequences revealed the location of receptor sulfotyrosine binding sites in the toxins. Then, by combining previous and novel experimental data with protein docking, classical and accelerated weight histogram (AWH) molecular dynamics we propose models of the ACKR1-LukE and CCR5-LukE complexes. This work provides novel insights into chemokine receptor recognition by leukotoxins and suggests that the conserved sulfotyrosine binding pocket could be a target of choice for future drug development.


Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Humanos , Evasão da Resposta Imune , Leucocidinas/metabolismo , Receptores de Quimiocinas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Staphylococcus aureus/genética
15.
J Biol Chem ; 285(23): 17907-17, 2010 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-20375012

RESUMO

At synaptic boutons, metabotropic glutamate receptor 7 (mGlu7 receptor) serves as an autoreceptor, inhibiting glutamate release. In this response, mGlu7 receptor triggers pertussis toxin-sensitive G protein activation, reducing presynaptic Ca(2+) influx and the subsequent depolarization evoked release. Here we report that receptor coupling to signaling pathways that potentiate release can be seen following prolonged exposure of nerve terminals to the agonist l-(+)-phosphonobutyrate, l-AP4. This novel mGlu7 receptor response involves an increase in the release induced by the Ca(2+) ionophore ionomycin, suggesting a mechanism that is independent of Ca(2+) channel activity, but dependent on the downstream exocytotic release machinery. The mGlu7 receptor-mediated potentiation resists exposure to pertussis toxin, but is dependent on phospholipase C, and increased phosphatidylinositol (4,5)-bisphosphate hydrolysis. Furthermore, the potentiation of release does not depend on protein kinase C, although it is blocked by the diacylglycerol-binding site antagonist calphostin C. We also found that activation of mGlu7 receptors translocate the active zone protein essential for synaptic vesicle priming, munc13-1, from soluble to particulate fractions. We propose that the mGlu7 receptor can facilitate or inhibit glutamate release through multiple pathways, thereby exerting homeostatic control of presynaptic function.


Assuntos
Ácido Glutâmico/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Cálcio/química , Diglicerídeos/química , Hidrólise , Ionomicina/farmacologia , Ionóforos , Masculino , Modelos Biológicos , Fosfatidilinositol 4,5-Difosfato , Fosfatos de Fosfatidilinositol/química , Ratos , Ratos Wistar , Fosfolipases Tipo C/química
16.
J Biol Chem ; 285(9): 6337-47, 2010 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-20026606

RESUMO

Accumulating evidence indicates that G protein-coupled receptors can assemble as dimers/oligomers but the role of this phenomenon in G protein coupling and signaling is not yet clear. We have used the purified leukotriene B(4) receptor BLT2 as a model to investigate the capacity of receptor monomers and dimers to activate the adenylyl cyclase inhibitory G(i2) protein. For this, we overexpressed the recombinant receptor as inclusion bodies in the Escherichia coli prokaryotic system, using a human alpha(5) integrin as a fusion partner. This strategy allowed the BLT2 as well as several other G protein-coupled receptors from different families to be produced and purified in large amounts. The BLT2 receptor was then successfully refolded to its native state, as measured by high-affinity LTB(4) binding in the presence of the purified G protein G alpha(i2). The receptor dimer, in which the two protomers displayed a well defined parallel orientation as assessed by fluorescence resonance energy transfer, was then separated from the monomer. Using two methods of receptor-catalyzed guanosine 5'-3-O-(thio)triphosphate binding assay, we clearly demonstrated that monomeric BLT2 stimulates the purified G alpha(i2) beta(1) gamma(2) protein more efficiently than the dimer. These data suggest that assembly of two BLT2 protomers into a dimer results in the reduced ability to signal.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Receptores do Leucotrieno B4/fisiologia , Transferência Ressonante de Energia de Fluorescência , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/isolamento & purificação , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Humanos , Integrina alfaV , Ligação Proteica , Multimerização Proteica , Transdução de Sinais
17.
Nat Methods ; 5(6): 561-7, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18488035

RESUMO

Cell-surface proteins are important in cell-cell communication. They assemble into heterocomplexes that include different receptors and effectors. Elucidation and manipulation of such protein complexes offers new therapeutic possibilities. We describe a methodology combining time-resolved fluorescence resonance energy transfer (FRET) with snap-tag technology to quantitatively analyze protein-protein interactions at the surface of living cells, in a high throughput-compatible format. Using this approach, we examined whether G protein-coupled receptors (GPCRs) are monomers or assemble into dimers or larger oligomers--a matter of intense debate. We obtained evidence for the oligomeric state of both class A and class C GPCRs. We also observed different quaternary structure of GPCRs for the neurotransmitters glutamate and gamma-aminobutyric acid (GABA): whereas metabotropic glutamate receptors assembled into strict dimers, the GABA(B) receptors spontaneously formed dimers of heterodimers, offering a way to modulate G-protein coupling efficacy. This approach will be useful in systematic analysis of cell-surface protein interaction in living cells.


Assuntos
Biofísica/métodos , Membrana Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Mapeamento de Interação de Proteínas/métodos , Receptores Acoplados a Proteínas G/metabolismo , Animais , Células COS , Chlorocebus aethiops , Dimerização , Humanos , Modelos Biológicos , Estrutura Quaternária de Proteína , Receptores de GABA-B/química , Propriedades de Superfície , Ácido gama-Aminobutírico
18.
Nat Chem Biol ; 5(3): 131-4, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19219011

RESUMO

Receptor heteromers constitute a new area of research that is reshaping our thinking about biochemistry, cell biology, pharmacology and drug discovery. In this commentary, we recommend clear definitions that should facilitate both information exchange and research on this growing class of transmembrane signal transduction units and their complex properties. We also consider research questions underlying the proposed nomenclature, with recommendations for receptor heteromer identification in native tissues and their use as targets for drug development.


Assuntos
Receptores de Superfície Celular/efeitos dos fármacos , Transdução de Sinais , Desenho de Fármacos , Receptores de Superfície Celular/metabolismo
19.
Cells ; 10(3)2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799570

RESUMO

Background: The atypical chemokine receptor 3 (ACKR3) belongs to the superfamily of G protein-coupled receptors (GPCRs). Unlike classical GPCRs, this receptor does not activate G proteins in most cell types but recruits ß-arrestins upon activation. ACKR3 plays an important role in cancer and vascular diseases. As recruitment of ß-arrestins is triggered by phosphorylation of the C-terminal tail of GPCRs, we studied the role of different potential phosphorylation sites within the ACKR3 C-tail to further delineate the molecular mechanism of internalization and trafficking of this GPCR. Methods: We used various bioluminescence and fluorescence resonance energy transfer-based sensors and techniques in Human Embryonic Kidney (HEK) 293T cells expressing WT or phosphorylation site mutants of ACKR3 to measure CXCL12-induced recruitment of ß-arrestins and G-protein-coupled receptor kinases (GRKs), receptor internalization and trafficking. Results: Upon CXCL12 stimulation, ACKR3 recruits both ß-arrestin 1 and 2 with equivalent kinetic profiles. We identified interactions with GRK2, 3 and 5, with GRK2 and 3 being important for ß-arrestin recruitment. Upon activation, ACKR3 internalizes and recycles back to the cell membrane. We demonstrate that ß-arrestin recruitment to the receptor is mainly determined by a single cluster of phosphorylated residues on the C-tail of ACKR3, and that residue T352 and in part S355 are important residues for ß-arrestin1 recruitment. Phosphorylation of the C-tail appears essential for ligand-induced internalization and important for differential ß-arrestin recruitment. GRK2 and 3 play a key role in receptor internalization. Moreover, ACKR3 can still internalize when ß-arrestin recruitment is impaired or in the absence of ß-arrestins, using alternative internalization pathways. Our data indicate that distinct residues within the C-tail of ACKR3 differentially regulate CXCL12-induced ß-arrestin recruitment, ACKR3 trafficking and internalization.


Assuntos
Endocitose , Receptores CXCR/metabolismo , beta-Arrestina 1/metabolismo , beta-Arrestina 2/metabolismo , Técnicas Biossensoriais , Quimiocina CXCL12/farmacologia , Transferência Ressonante de Energia de Fluorescência , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Quinase 3 de Receptor Acoplado a Proteína G/metabolismo , Células HEK293 , Humanos , Cinética , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Receptores CXCR/agonistas , Receptores CXCR/genética , beta-Arrestina 1/genética , beta-Arrestina 2/genética
20.
J Am Soc Nephrol ; 20(10): 2190-203, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19729439

RESUMO

X-linked congenital nephrogenic diabetes insipidus (cNDI) results from inactivating mutations of the human arginine vasopressin (AVP) V2 receptor (hV(2)R). Most of these mutations lead to intracellular retention of the hV(2)R, preventing its interaction with AVP and thereby limiting water reabsorption and concentration of urine. Because the majority of cNDI-hV(2)Rs exhibit protein misfolding, molecular chaperones hold promise as therapeutic agents; therefore, we sought to identify pharmacochaperones for hV(2)R that also acted as agonists. Here, we describe high-affinity nonpeptide compounds that promoted maturation and membrane rescue of L44P, A294P, and R337X cNDI mutants and restored a functional AVP-dependent cAMP signal. Contrary to pharmacochaperone antagonists, these compounds directly activated a cAMP signal upon binding to several cNDI mutants. In addition, these molecules displayed original functionally selective properties (biased agonism) toward the hV(2)R, being unable to recruit arrestin, trigger receptor internalization, or stimulate mitogen-activated protein kinases. These characteristics make these hV(2)R agonist pharmacochaperones promising therapeutic candidates for cNDI.


Assuntos
Diabetes Insípido Nefrogênico/tratamento farmacológico , Chaperonas Moleculares/farmacologia , Receptores de Vasopressinas/agonistas , Arginina Vasopressina/metabolismo , Arrestina/antagonistas & inibidores , Arrestina/metabolismo , Células Cultivadas , AMP Cíclico/biossíntese , Glicosilação , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Chaperonas Moleculares/uso terapêutico , Receptores de Vasopressinas/fisiologia
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