RESUMO
The quaternary organization of rhodopsin-like G protein-coupled receptors in native tissues is unknown. To address this we generated mice in which the M1 muscarinic acetylcholine receptor was replaced with a C-terminally monomeric enhanced green fluorescent protein (mEGFP)-linked variant. Fluorescence imaging of brain slices demonstrated appropriate regional distribution, and using both anti-M1 and anti-green fluorescent protein antisera the expressed transgene was detected in both cortex and hippocampus only as the full-length polypeptide. M1-mEGFP was expressed at levels equal to the M1 receptor in wild-type mice and was expressed throughout cell bodies and projections in cultured neurons from these animals. Signaling and behavioral studies demonstrated M1-mEGFP was fully active. Application of fluorescence intensity fluctuation spectrometry to regions of interest within M1-mEGFP-expressing neurons quantified local levels of expression and showed the receptor was present as a mixture of monomers, dimers, and higher-order oligomeric complexes. Treatment with both an agonist and an antagonist ligand promoted monomerization of the M1-mEGFP receptor. The quaternary organization of a class A G protein-coupled receptor in situ was directly quantified in neurons in this study, which answers the much-debated question of the extent and potential ligand-induced regulation of basal quaternary organization of such a receptor in native tissue when present at endogenous expression levels.
Assuntos
Córtex Cerebral , Hipocampo , Receptor Muscarínico M1 , Animais , Córtex Cerebral/metabolismo , Proteínas de Fluorescência Verde , Hipocampo/metabolismo , Ligantes , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Imagem Óptica , Receptor Muscarínico M1/química , Receptor Muscarínico M1/genética , Receptor Muscarínico M1/metabolismoRESUMO
CXCR4, a member of the family of chemokine-activated G protein-coupled receptors, is widely expressed in immune response cells. It is involved in both cancer development and progression as well as viral infection, notably by HIV-1. A variety of methods, including structural information, have suggested that the receptor may exist as a dimer or an oligomer. However, the mechanistic details surrounding receptor oligomerization and its potential dynamic regulation remain unclear. Using both biochemical and biophysical means, we confirm that CXCR4 can exist as a mixture of monomers, dimers, and higher-order oligomers in cell membranes and show that oligomeric structure becomes more complex as receptor expression levels increase. Mutations of CXCR4 residues located at a putative dimerization interface result in monomerization of the receptor. Additionally, binding of the CXCR4 antagonist IT1t-a small drug-like isothiourea derivative-rapidly destabilizes the oligomeric structure, whereas AMD3100, another well-characterized CXCR4 antagonist, does not. Although a mutation that regulates constitutive activity of CXCR4 also results in monomerization of the receptor, binding of IT1t to this variant promotes receptor dimerization. These results provide novel insights into the basal organization of CXCR4 and how antagonist ligands of different chemotypes differentially regulate its oligomerization state.
Assuntos
Benzilaminas/farmacologia , Ciclamos/farmacologia , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Tioureia/farmacologia , Fármacos Anti-HIV/farmacologia , Células Cultivadas , Proteínas de Fluorescência Verde/metabolismo , Compostos Heterocíclicos/química , Compostos Heterocíclicos/farmacologia , Humanos , Ligantes , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Receptores CXCR4/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Transdução de SinaisRESUMO
Here, we introduce fluorescence intensity fluctuation spectrometry for determining the identity, abundance and stability of protein oligomers. This approach was tested on monomers and oligomers of known sizes and was used to uncover the oligomeric states of the epidermal growth factor receptor and the secretin receptor in the presence and absence of their agonist ligands. This method is fast and is scalable for high-throughput screening of drugs targeting protein-protein interactions.
Assuntos
Fluorescência , Processamento de Imagem Assistida por Computador/métodos , Multimerização Proteica , Receptores Acoplados a Proteínas G/metabolismo , Receptores dos Hormônios Gastrointestinais/metabolismo , Receptores ErbB/química , Receptores ErbB/metabolismo , Humanos , Ligantes , Microscopia Confocal , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Espectrometria de FluorescênciaRESUMO
Activation of the histamine-3 receptor (H3R) is involved in memory processes and cognitive action, while blocking H3R activation can slow the progression of neurological disorders, such as Alzheimer's disease, schizophrenia and narcolepsy. To date, however, no direct way to examine the activation of H3R has been utilized. Here, we describe a novel biosensor that can visualize the activation of H3R through an intramolecular fluorescence resonance energy transfer (FRET) signal. To achieve this, we constructed an intramolecular H3R FRET sensor with cyan fluorescent protein (CFP) attached at the C terminus and yellow fluorescent protein (YFP) inserted into the third intracellular loop. The sensor was found to internalize normally on agonist treatment. We measured FRET signals between the donor CFP and the acceptor YFP in living cells in real time, the results of which indicated that H3R agonist treatment (imetit or histamine) increases the FRET signal in a time- and concentration-dependent manner with Kon and Koff values consistent with published data and which maybe correlated with decreasing cAMP levels and the promotion of ERK1/2 phosphorylation. The FRET signal was inhibited by H3R antagonists, and the introduction of mutations at F419A, F423A, L426A and L427A, once again, the promotion of ERK1/2 phosphorylation, was diminished. Thus, we have built a H3R biosensor which can visualize the activation of receptor through real-time structure changes and which can obtain pharmacological kinetic data at the same time. The FRET signals may allow the sensor to become a useful tool for screening compounds and optimizing useful ligands.
Assuntos
Técnicas Biossensoriais , Transferência Ressonante de Energia de Fluorescência/métodos , Agonistas dos Receptores Histamínicos/farmacologia , Antagonistas dos Receptores Histamínicos H3/farmacologia , Histamina/farmacologia , Receptores Histamínicos H3/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , AMP Cíclico/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Imidazóis/farmacologia , Cinética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Plasmídeos/química , Plasmídeos/metabolismo , Receptores Histamínicos H3/genética , Tioureia/análogos & derivados , Tioureia/farmacologia , Transfecção , TrítioRESUMO
Spatial intensity distribution analysis (SpIDA) is a recently developed approach for determining quaternary structure information on fluorophore-labelled proteins of interest in situ. It can be applied to live or fixed cells and native tissue. Using confocal images, SpIDA generates fluorescence intensity histograms that are analysed by super-Poissonian distribution functions to obtain density and quantal brightness values of the fluorophore-labelled protein of interest. This allows both expression level and oligomerisation state of the protein to be determined. We describe the application of SpIDA to investigate the oligomeric state of G protein-coupled receptors (GPCRs) at steady state and following cellular challenge, and consider how SpIDA may be used to explore GPCR quaternary organisation in pathophysiology and to stratify medicines.
Assuntos
Multimerização Proteica , Receptores Acoplados a Proteínas G/química , Imagem Individual de Molécula/métodos , Animais , Humanos , Microscopia de Fluorescência/métodos , Receptores Acoplados a Proteínas G/metabolismoRESUMO
The dopamine D3 receptor (D3R) is a molecular target for both first-generation and several recently-developed antipsychotic agents. Following stable expression of this mEGFP-tagged receptor, Spatial Intensity Distribution Analysis indicated that a substantial proportion of the receptor was present within dimeric/oligomeric complexes and that increased expression levels of the receptor favored a greater dimer to monomer ratio. Addition of the antipsychotics, spiperone or haloperidol, resulted in re-organization of D3R quaternary structure to promote monomerization. This action was dependent on ligand concentration and reversed upon drug washout. By contrast, a number of other antagonists with high affinity at the D3R, did not alter the dimer/monomer ratio. Molecular dynamics simulations following docking of each of the ligands into a model of the D3R derived from the available atomic level structure, and comparisons to the receptor in the absence of ligand, were undertaken. They showed that, in contrast to the other antagonists, spiperone and haloperidol respectively increased the atomic distance between reference α carbon atoms of transmembrane domains IV and V and I and II, both of which provide key interfaces for D3R dimerization. These results offer a molecular explanation for the distinctive ability of spiperone and haloperidol to disrupt D3R dimerization.
Assuntos
Antagonistas de Dopamina/farmacologia , Haloperidol/farmacologia , Multimerização Proteica , Receptores de Dopamina D3/química , Espiperona/farmacologia , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Receptores de Dopamina D3/antagonistas & inibidores , Receptores de Dopamina D3/metabolismoRESUMO
Previous studies have indicated that the G-protein-coupled secretin receptor is present as a homodimer, organized through symmetrical contacts in transmembrane domain IV, and that receptor dimerization is critical for high-potency signalling by secretin. However, whether all of the receptor exists in the dimeric form or if this is regulated is unclear. We used measures of quantal brightness of the secretin receptor tagged with monomeric enhanced green fluorescent protein (mEGFP) and spatial intensity distribution analysis to assess this. Calibration using cells expressing plasma membrane-anchored forms of mEGFP initially allowed us to demonstrate that the epidermal growth factor receptor is predominantly monomeric in the absence of ligand and while wild-type receptor was rapidly converted into a dimeric form by ligand, a mutated form of this receptor remained monomeric. Equivalent studies showed that, at moderate expression levels, the secretin receptor exists as a mixture of monomeric and dimeric forms, with little evidence of higher-order complexity. However, sodium butyrate-induced up-regulation of the receptor resulted in a shift from monomeric towards oligomeric organization. In contrast, a form of the secretin receptor containing a pair of mutations on the lipid-facing side of transmembrane domain IV was almost entirely monomeric. Down-regulation of the secretin receptor-interacting G-protein Gαs did not alter receptor organization, indicating that dimerization is defined specifically by direct protein-protein interactions between copies of the receptor polypeptide, while short-term treatment with secretin had no effect on organization of the wild-type receptor but increased the dimeric proportion of the mutated receptor variant.
Assuntos
Receptores Acoplados a Proteínas G/química , Receptores dos Hormônios Gastrointestinais/química , Animais , Células CHO , Cricetinae , Cricetulus , Proteínas de Fluorescência Verde/genética , Multimerização Proteica , Receptores Acoplados a Proteínas G/genética , Receptores dos Hormônios Gastrointestinais/genéticaRESUMO
Mutations in the gene that encodes the principal l-carnitine transporter, OCTN2, can lead to a reduced intracellular l-carnitine pool and the disease Primary Carnitine Deficiency. l-Carnitine supplementation is used therapeutically to increase intracellular l-carnitine. As AMPK and insulin regulate fat metabolism and substrate uptake, we hypothesized that AMPK-activating compounds and insulin would increase l-carnitine uptake in C2C12 myotubes. The cells express all three OCTN transporters at the mRNA level, and immunohistochemistry confirmed expression at the protein level. Contrary to our hypothesis, despite significant activation of PKB and 2DG uptake, insulin did not increase l-carnitine uptake at 100 nM. However, l-carnitine uptake was modestly increased at a dose of 150 nM insulin. A range of AMPK activators that increase intracellular calcium content [caffeine (10 mM, 5 mM, 1 mM, 0.5 mM), A23187 (10 µM)], inhibit mitochondrial function [sodium azide (75 µM), rotenone (1 µM), berberine (100 µM), DNP (500 µM)], or directly activate AMPK [AICAR (250 µM)] were assessed for their ability to regulate l-carnitine uptake. All compounds tested significantly inhibited l-carnitine uptake. Inhibition by caffeine was not dantrolene (10 µM) sensitive despite dantrolene inhibiting caffeine-mediated calcium release. Saturation curve analysis suggested that caffeine did not competitively inhibit l-carnitine transport. To assess the potential role of AMPK in this process, we assessed the ability of the AMPK inhibitor Compound C (10 µM) to rescue the effect of caffeine. Compound C offered a partial rescue of l-carnitine uptake with 0.5 mM caffeine, suggesting that AMPK may play a role in the inhibitory effects of caffeine. However, caffeine likely inhibits l-carnitine uptake by alternative mechanisms independently of calcium release. PKA activation or direct interference with transporter function may play a role.
Assuntos
Carnitina/antagonistas & inibidores , Ativadores de Enzimas/farmacologia , Mioblastos/efeitos dos fármacos , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Berberina/farmacologia , Transporte Biológico/efeitos dos fármacos , Cafeína/farmacologia , Calcimicina/farmacologia , Cálcio/metabolismo , Carnitina/metabolismo , Linhagem Celular , Dantroleno/farmacologia , Ativação Enzimática/efeitos dos fármacos , Expressão Gênica , Insulina/farmacologia , Camundongos , Mioblastos/citologia , Mioblastos/enzimologia , Proteínas de Transporte de Cátions Orgânicos/genética , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ribonucleotídeos/farmacologia , Rotenona/farmacologia , Azida Sódica/farmacologia , Membro 5 da Família 22 de Carreadores de SolutoRESUMO
Although rhodopsin-like G protein-coupled receptors can exist as both monomers and non-covalently associated dimers/oligomers, the steady-state proportion of each form and whether this is regulated by receptor ligands are unknown. Herein we address these topics for the M1 muscarinic acetylcholine receptor, a key molecular target for novel cognition enhancers, by using spatial intensity distribution analysis. This method can measure fluorescent particle concentration and assess oligomerization states of proteins within defined regions of living cells. Imaging and analysis of the basolateral surface of cells expressing some 50 molecules·µm(-2) human muscarinic M1 receptor identified a â¼75:25 mixture of receptor monomers and dimers/oligomers. Both sustained and shorter term treatment with the selective M1 antagonist pirenzepine resulted in a large shift in the distribution of receptor species to favor the dimeric/oligomeric state. Although sustained treatment with pirenzepine also resulted in marked up-regulation of the receptor, simple mass action effects were not the basis for ligand-induced stabilization of receptor dimers/oligomers. The related antagonist telenzepine also produced stabilization and enrichment of the M1 receptor dimer population, but the receptor subtype non-selective antagonists atropine and N-methylscopolamine did not. In contrast, neither pirenzepine nor telenzepine altered the quaternary organization of the related M3 muscarinic receptor. These data provide unique insights into the selective capacity of receptor ligands to promote and/or stabilize receptor dimers/oligomers and demonstrate that the dynamics of ligand regulation of the quaternary organization of G protein-coupled receptors is markedly more complex than previously appreciated. This may have major implications for receptor function and behavior.
Assuntos
Atropina/farmacologia , Antagonistas Muscarínicos/farmacologia , Pirenzepina/análogos & derivados , Pirenzepina/farmacologia , Multimerização Proteica/efeitos dos fármacos , Receptor Muscarínico M1/antagonistas & inibidores , Linhagem Celular , Humanos , Receptor Muscarínico M1/química , Receptor Muscarínico M1/metabolismoRESUMO
The questions of whether G protein-coupled receptors exist as monomers, dimers, and/or oligomers and if these species interconvert in a ligand-dependent manner are among the most contentious current issues in biology. When employing spatial intensity distribution analysis to laser scanning confocal microscope images of cells stably expressing either a plasma membrane-associated form of monomeric enhanced green fluorescent protein (eGFP) or a tandem version of this fluorophore, the eGFP tandem was identified as a dimer. Similar studies on cells stably expressing an eGFP-tagged form of the epidermal growth factor receptor demonstrated that, although largely a monomer in the basal state, this receptor rapidly became predominantly dimeric upon the addition of its ligand epidermal growth factor. In cells induced to express an eGFP-tagged form of the serotonin 5-hydroxytryptamine 2C (5-HT2C) receptor, global analysis of construct quantal brightness was consistent with the predominant form of the receptor being dimeric. However, detailed spatial intensity distribution analysis demonstrated the presence of multiple forms ranging from monomers to higher-order oligomers. Furthermore, treatment with chemically distinct 5-HT2C receptor antagonists resulted in a time-dependent change in the quaternary organization to one in which there was a preponderance of receptor monomers. This antagonist-mediated effect was reversible, because washout of the ligand resulted in the regeneration of many of the oligomeric forms of the receptor.
Assuntos
Complexos Multiproteicos/metabolismo , Multimerização Proteica/fisiologia , Receptor 5-HT2C de Serotonina/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Complexos Multiproteicos/genética , Multimerização Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína , Receptor 5-HT2C de Serotonina/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Antagonistas do Receptor 5-HT2 de Serotonina/farmacologiaRESUMO
Robo (Roundabout) receptors and their Slit polypeptide ligands are known to play key roles in neuronal development and have been implicated in both angiogenesis and cancer. Like the other family members, Robo1 is a large single transmembrane domain polypeptide containing a series of well-defined extracellular elements. However, the intracellular domain lacks structural definition and little is known about the quaternary structure of Robo receptors or how binding of a Slit might affect this. To address these questions combinations of both autofluorescent protein-based FRET imaging and time-resolved FRET were employed. Both approaches identified oligomeric organization of Robo1 that did not require the presence of the intracellular domain. SpIDA (spatial intensity distribution analysis) of eGFP-tagged forms of Robo1 indicated that for a C-terminally deleted version approximately two-thirds of the receptor was present as a dimer and one-third as a monomer. By contrast, full-length Robo1 was present almost exclusively as a dimer. In each case this was unaffected by the addition of Slit2, although parallel studies demonstrated the biological activity of Slit2 and its interaction with Robo1. Deletion of both the immunoglobulin and fibronectin type III extracellular repeats prevented dimer formation, with the immunoglobulin repeats providing the bulk of the protein-protein interaction affinity.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Multimerização Proteica/fisiologia , Receptores Imunológicos/química , Receptores Imunológicos/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Leucócitos Mononucleares/metabolismo , Ligantes , Ligação Proteica/fisiologia , Proteínas RoundaboutRESUMO
TUG-891 [3-(4-((4-fluoro-4'-methyl-[1,1'-biphenyl]-2-yl)methoxy)phenyl)propanoic acid] was recently described as a potent and selective agonist for the long chain free fatty acid (LCFA) receptor 4 (FFA4; previously G protein-coupled receptor 120, or GPR120). Herein, we have used TUG-891 to further define the function of FFA4 and used this compound in proof of principle studies to indicate the therapeutic potential of this receptor. TUG-891 displayed similar signaling properties to the LCFA α-linolenic acid at human FFA4 across various assay end points, including stimulation of Ca²âº mobilization, ß-arrestin-1 and ß-arrestin-2 recruitment, and extracellular signal-regulated kinase phosphorylation. Activation of human FFA4 by TUG-891 also resulted in rapid phosphorylation and internalization of the receptor. While these latter events were associated with desensitization of the FFA4 signaling response, removal of TUG-891 allowed both rapid recycling of FFA4 back to the cell surface and resensitization of the FFA4 Ca²âº signaling response. TUG-891 was also a potent agonist of mouse FFA4, but it showed only limited selectivity over mouse FFA1, complicating its use in vivo in this species. Pharmacologic dissection of responses to TUG-891 in model murine cell systems indicated that activation of FFA4 was able to mimic many potentially beneficial therapeutic properties previously reported for LCFAs, including stimulating glucagon-like peptide-1 secretion from enteroendocrine cells, enhancing glucose uptake in 3T3-L1 adipocytes, and inhibiting release of proinflammatory mediators from RAW264.7 macrophages, which suggests promise for FFA4 as a therapeutic target for type 2 diabetes and obesity. Together, these results demonstrate both potential but also significant challenges that still need to be overcome to therapeutically target FFA4.
Assuntos
Compostos de Bifenilo/farmacologia , Fenilpropionatos/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Células 3T3-L1 , Animais , Arrestinas/fisiologia , Cálcio/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Glucose/metabolismo , Células HEK293 , Humanos , Camundongos , Fosforilação , beta-Arrestina 1 , beta-Arrestina 2 , beta-ArrestinasRESUMO
Induction of the suppressor of cytokine signalling 3 (SOCS-3) gene is vital to the normal control of inflammatory signalling. In order to understand these processes we investigated the role of the proto-oncogene component of the AP-1 transcription factor complex, c-Jun, in the regulation of SOCS-3 gene induction. We found that cyclic AMP stimulation of HUVECs promoted phosphorylation and activation of JNK MAP kinase and its substrate c-Jun. The JNK responsive element of the human SOCS-3 promoter mapped to a putative AP-1 site within 1000bp of the transcription start site. The PKC inhibitors, GF-109203X, Gö-6983 and Ro-317549, were all found to inhibit AP-1 transcriptional activity, transcriptional activation of this minimal SOCS-3 promoter and SOCS-3 gene induction in HUVECs. Interestingly, Ro-317549 treatment was also found to promote PKC-dependent activation of ERK and JNK MAP kinases and promote JNK-dependent hyper-phosphorylation of c-Jun, whereas GF-109203X and Gö-6983 had little effect. Despite this, all three PKC inhibitors were found to be effective inhibitors of c-Jun DNA-binding activity. The JNK-dependent hyper-phosphorylation of c-Jun in response to Ro-317549 treatment of HUVECs does therefore not interfere with its ability to inhibit c-Jun activity and acts as an effective inhibitor of c-Jun-dependent SOCS-3 gene induction.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Indóis/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Maleimidas/farmacologia , Proteínas Proto-Oncogênicas c-jun/antagonistas & inibidores , Proteínas Supressoras da Sinalização de Citocina/genética , Ativação Transcricional/efeitos dos fármacos , AMP Cíclico/antagonistas & inibidores , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Ativação Enzimática/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/metabolismoRESUMO
Proteinase-activated receptors 4 (PAR(4)) is a class A G protein-coupled receptor (GPCR) recognized through the ability of serine proteases such as thrombin and trypsin to mediate receptor activation. Due to the irreversible nature of activation, a fresh supply of receptor is required to be mobilized to the cell surface for responsiveness to agonist to be sustained. Unlike other PAR subtypes, the mechanisms regulating receptor trafficking of PAR(4) remain unknown. Here, we report novel features of the intracellular trafficking of PAR(4) to the plasma membrane. PAR(4) was poorly expressed at the plasma membrane and largely retained in the endoplasmic reticulum (ER) in a complex with the COPI protein subunit ß-COP1. Analysis of the PAR(4) protein sequence identified an arginine-based (RXR) ER retention sequence located within intracellular loop-2 (R(183)AR â A(183)AA), mutation of which allowed efficient membrane delivery of PAR(4). Interestingly, co-expression with PAR(2) facilitated plasma membrane delivery of PAR(4), an effect produced through disruption of ß-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3ζ. Intermolecular FRET studies confirmed heterodimerization between PAR(2) and PAR(4). PAR(2) also enhanced glycosylation of PAR(4) and activation of PAR(4) signaling. Our results identify a novel regulatory role for PAR(2) in the anterograde traffic of PAR(4). PAR(2) was shown to both facilitate and abrogate protein interactions with PAR(4), impacting upon receptor localization and cell signal transduction. This work is likely to impact markedly upon the understanding of the receptor pharmacology of PAR(4) in normal physiology and disease.
Assuntos
Membrana Celular/metabolismo , Multimerização Proteica/fisiologia , Receptor PAR-2/metabolismo , Receptores de Trombina/metabolismo , Transdução de Sinais/fisiologia , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Membrana Celular/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Ligação Proteica , Sinais Direcionadores de Proteínas/fisiologia , Transporte Proteico/fisiologia , Receptor PAR-2/genética , Receptores de Trombina/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Intramolecular fluorescence resonance energy transfer (FRET) sensors able to detect changes in distance or orientation between the 3rd intracellular loop and C-terminal tail of the human orexin OX(1) and OX(2) G protein-coupled receptors following binding of agonist ligands were produced and expressed stably. These were directed to the plasma membrane and, despite the substantial sequence alterations introduced, in each case were able to elevate [Ca(2+)](i), promote phosphorylation of the ERK1/2 MAP kinases and become internalized effectively upon addition of the native orexin peptides. Detailed characterization of the OX(1) sensor demonstrated that it was activated with rank order of potency orexin A > orexin B > orexin A 16-33, that it bound antagonist ligands with affinity similar to the wild-type receptor, and that mutation of a single residue, D203A, greatly reduced the binding and function of orexin A but not antagonist ligands. Addition of orexin A to individual cells expressing an OX(1) sensor resulted in a time- and concentration-dependent reduction in FRET signal consistent with mass-action and potency/affinity estimates for the peptide. Compared with the response kinetics of a muscarinic M(3) acetylcholine receptor sensor upon addition of agonist, response of the OX(1) and OX(2) sensors to orexin A was slow, consistent with a multistep binding and activation process. Such sensors provide means to assess the kinetics of receptor activation and how this may be altered by mutation and sequence variation of the receptors.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neuropeptídeos/farmacologia , Peptídeos/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/agonistas , Receptores de Neuropeptídeos/metabolismo , Substituição de Aminoácidos , Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Cinética , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mutação de Sentido Incorreto , Receptores de Orexina , Orexinas , Fosforilação/efeitos dos fármacos , Estrutura Secundária de Proteína , Receptor Muscarínico M3/agonistas , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores de Neuropeptídeos/genéticaRESUMO
Agonist-induced internalization was observed for both inducible and constitutively expressed forms of the cannabinoid CB(1) receptor. These were also internalized by the peptide orexin A, which has no direct affinity for the cannabinoid CB(1) receptor, but only when the orexin OX(1) receptor was co-expressed along with the cannabinoid CB(1) receptor. This effect of orexin A was concentration-dependent and blocked by OX(1) receptor antagonists. Moreover, the ability of orexin A to internalize the CB(1) receptor was also blocked by CB(1) receptor antagonists. Remarkably, orexin A was substantially more potent in producing internalization of the CB(1) receptor than in causing internalization of the bulk OX(1) receptor population, and this was true in cells in which the CB(1) receptor was maintained at a constant level, whereas levels of OX(1) could be varied and vice versa. Both co-immunoprecipitation and cell surface, homogenous time-resolved fluorescence resonance energy transfer based on covalent labeling of N-terminal "SNAP" and "CLIP" tags present in the extracellular N-terminal domain of the receptors confirmed the capacity of these two receptors to heteromultimerize. These studies confirm the capacity of the CB(1) and OX(1) receptors to interact directly and demonstrate that this complex has unique regulatory characteristics. The higher potency of the agonist orexin A to regulate the CB(1)-OX(1) heteromer compared with the OX(1)-OX(1) homomer present in the same cells and the effects of CB(1) receptor antagonists on the function of orexin A suggest an interplay between these two systems that may modulate appetite, feeding, and wakefulness.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neuropeptídeos/metabolismo , Multimerização Proteica/fisiologia , Subunidades Proteicas/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/metabolismo , Linhagem Celular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Neuropeptídeos/genética , Neuropeptídeos/farmacologia , Neurotransmissores/genética , Neurotransmissores/metabolismo , Neurotransmissores/farmacologia , Receptores de Orexina , Orexinas , Multimerização Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Receptor CB1 de Canabinoide/genética , Receptores Acoplados a Proteínas G/genética , Receptores de Neuropeptídeos/genéticaRESUMO
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 TestesRESUMO
It is unclear what proportion of a G-protein-coupled receptor is present in cells as dimers or oligomers. Saturation bioluminescence resonance energy transfer studies demonstrated the orexin OX(1) receptor to be present in such complexes. Forms of this receptor containing a minimal epitope tag, with the C-terminus linked to yellow fluorescent protein or modified at the N-terminus to incorporate a SNAP tag, migrated in SDS/PAGE gels as monomers, indicating a lack of covalent interactions. Solubilization with dodecylmaltoside, followed by Blue native-PAGE, indicated that the receptor constructs migrated predominantly as anticipated for dimeric species with evidence for further, higher-order, complexes, and this was true over a wide range of expression levels. Addition of SDS prior to separation by Blue native-PAGE resulted in much of the previously dimeric, and all of the higher-order, complexes being dissociated and now migrating at the size predicted for monomeric species. Expression of forms of the OX(1) receptor capable of generating enzyme complementation confirmed that solubilization itself did not result in interaction artefacts. Addition of the endogenous agonist orexin A enhanced the proportion of higher-order OX(1) receptor complexes, whereas selective OX(1) antagonists increased the proportion the OX(1) receptor migrating in Blue native-PAGE as a monomer. The antagonist effects were produced in a concentration-dependent manner, consistent with the affinity of the ligands for the receptor. Homogeneous time-resolved fluorescence resonance energy transfer studies using Tag-Lite™ reagents on cells expressing the SNAP-tagged OX(1) receptor identified cell-surface OX(1) homomers. Predominantly at low receptor expression levels, orexin A increased such fluorescence resonance energy transfer signals, also consistent with ligand-induced reorganization of the homomeric complex.
Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/metabolismo , Linhagem Celular , Doxiciclina/farmacologia , Eletroforese em Gel de Poliacrilamida , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Neuropeptídeos/farmacologia , Receptores de Orexina , Orexinas , Multimerização Proteica , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Receptores de Neuropeptídeos/agonistas , Receptores de Neuropeptídeos/antagonistas & inibidores , Receptores de Neuropeptídeos/genética , Dodecilsulfato de Sódio/farmacologiaRESUMO
BACKGROUND AND PURPOSE: Many G protein-coupled receptors internalize following agonist binding. The studies were designed to identify novel means to effectively quantify this process using the orexin OX(1) receptor and the cannabinoid CB(1) receptor as exemplars. EXPERIMENTAL APPROACH: The human OX(1) and CB(1) receptors were modified to incorporate both epitope tags and variants (SNAP and CLIP) of the enzyme O(6)-alkylguanine-DNA-alkyltransferase within their extracellular, N-terminal domain. Cells able to regulate expression of differing amounts of these constructs upon addition of an antibiotic were developed and analysed. KEY RESULTS: Cell surface forms of each receptor construct were detected by both antibody recognition of the epitope tags and covalent binding of fluorophores to the O(6)-alkylguanine-DNA-alkyltransferase variants. Receptor internalization in response to agonists but not antagonists could be monitored by each approach but sensitivity was up to six- to 10-fold greater than other approaches when employing a novel, time-resolved fluorescence probe for the SNAP tag. Sensitivity was not enhanced, however, for the CLIP tag, possibly due to higher levels of nonspecific binding. CONCLUSIONS AND IMPLICATIONS: These studies demonstrate that highly sensitive and quantitative assays that monitor cell surface CB(1) and OX(1) receptors and their internalization by agonists can be developed based on introduction of variants of O(6)-alkylguanine-DNA-alkyltransferase into the N-terminal domain of the receptor. This should be equally suitable for other G protein-coupled receptors.
Assuntos
Alquil e Aril Transferases/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/metabolismo , Alquil e Aril Transferases/química , Benzoxazóis/metabolismo , Benzoxazóis/farmacologia , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Clonagem Molecular , Cicloexanóis/metabolismo , Cicloexanóis/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Ligantes , Naftiridinas , Neuropeptídeos/metabolismo , Neuropeptídeos/farmacologia , Receptores de Orexina , Orexinas , Compostos de Fenilureia/metabolismo , Compostos de Fenilureia/farmacologia , Fosforilação/efeitos dos fármacos , Piperidinas/metabolismo , Piperidinas/farmacologia , Plasmídeos , Pirazóis/metabolismo , Pirazóis/farmacologia , Pirrolidinas/metabolismo , Pirrolidinas/farmacologia , Receptor CB1 de Canabinoide/agonistas , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptor CB1 de Canabinoide/química , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/química , Receptores de Neuropeptídeos/agonistas , Receptores de Neuropeptídeos/antagonistas & inibidores , Receptores de Neuropeptídeos/química , Rimonabanto , Tiazóis/metabolismo , Tiazóis/farmacologia , Ureia/análogos & derivados , Ureia/metabolismo , Ureia/farmacologiaRESUMO
The role of G protein coupled receptors (GPCRs) in numerous physiological processes that may be disrupted or modified in disease makes them key targets for the development of new therapeutic medicines. A wide variety of resonance energy transfer (RET) techniques such as fluorescence RET and bioluminescence RET have been developed in recent years to detect protein-protein interactions in living cells. Furthermore, these techniques are now being exploited to screen for novel compounds that activate or block GPCRs and to search for new, previously undiscovered signaling pathways activated by well-known pharmacologically classified drugs. The high resolution that can be achieved with these RET methods means that they are well suited to study both intramolecular conformational changes in response to ligand binding at the receptor level and intermolecular interactions involving protein translocation in subcellular compartments resulting from external stimuli. In this review we highlight the latest advances in these technologies to illustrate general principles.