RESUMO
G protein-coupled receptor associated sorting protein 1 (GPRASP1) belongs to a family of 10 proteins that display sequence homologies in their C-terminal region. Several members including GPRASP1 also display a short repeated sequence called the GASP motif that is critically involved in protein-protein interactions with G protein-coupled receptors (GPCRs). Here, we characterized anti-GASP motif antibodies and investigated their potential inhibitory functions. We first showed that our in-house anti-GPRASP1 rabbit polyclonal serum contains anti-GASP motif antibodies and purified them by affinity chromatography. We further showed that these antibodies can detect GPRASP1 and GPRASP2 in Western blot, immunoprecipitation and immunofluorescence experiments while a mutant of GPRASP2, in which the most conserved hydrophobic core of the GASP motifs is mutated, was no more detected. Further characterization of anti-GASP motif antibodies by ELISA and Surface Plasmon Resonance assays suggests that GASP motifs function as multivalent epitopes. Finally, we set-up an Amplified Luminescent Proximity Homogeneous AlphaScreen® assay to detect the interaction between purified ADRB2 receptor and the central domain of GPRASP1 and showed that anti-GASP motif antibodies efficiently inhibit this interaction. Altogether, our results suggest that anti-GASP motif antibodies could represent a valuable tool to neutralize the interaction of GPRASP1 and GPRASP2 with different GPCRs.
Assuntos
Proteínas de Transporte , Receptores Acoplados a Proteínas G , Animais , Coelhos , Transporte Proteico/fisiologiaRESUMO
The physiological and pathophysiological relevance of the angiotensin II type 1 (AT1) G protein-coupled receptor no longer needs to be proven in the cardiovascular system. The renin-angiotensin system and the AT1 receptor are the targets of several classes of therapeutics (such as angiotensin converting enzyme inhibitors or angiotensin receptor blockers, ARBs) used as first-line treatments in cardiovascular diseases. The importance of AT1 in the regulation of the cerebrovascular system is also acknowledged. However, despite numerous beneficial effects in preclinical experiments, ARBs do not induce satisfactory curative results in clinical stroke studies. A better understanding of AT1 signaling and the development of biased AT1 agonists, able to selectively activate the ß-arrestin transduction pathway rather than the Gq pathway, have led to new therapeutic strategies to target detrimental effects of AT1 activation. In this paper, we review the involvement of AT1 in cerebrovascular diseases as well as recent advances in the understanding of its molecular dynamics and biased or non-biased signaling. We also describe why these alternative signaling pathways induced by ß-arrestin biased AT1 agonists could be considered as new therapeutic avenues for cerebrovascular diseases.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Doenças Cardiovasculares/tratamento farmacológico , Receptor Tipo 1 de Angiotensina/química , Receptor Tipo 1 de Angiotensina/metabolismo , Animais , Lesões Encefálicas Traumáticas/metabolismo , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/fisiopatologia , Humanos , Aneurisma Intracraniano/metabolismo , Terapia de Alvo Molecular/métodos , Transdução de Sinais/efeitos dos fármacos , Acidente Vascular Cerebral/metabolismo , beta-Arrestinas/agonistas , beta-Arrestinas/metabolismoRESUMO
Several cytoplasmic proteins that are involved in G protein-coupled receptor signaling cascades are known to translocate to the plasma membrane upon receptor activation, such as beta-arrestin2. Based on this example and in order to identify new cytoplasmic proteins implicated in the ON-and-OFF cycle of G protein-coupled receptor, a live-imaging screen of fluorescently labeled cytoplasmic proteins was performed using translocation criteria. The screening of 193 fluorescently tagged human proteins identified eight proteins that responded to activation of the tachykinin NK2 receptor by a change in their intracellular localization. Previously we have presented the functional characterization of one of these proteins, REDD1, that translocates to the plasma membrane. Here we report the results of the entire screening. The process of cell activation was recorded on videos at different time points and all the videos can be visualized on a dedicated website. The proteins BAIAP3 and BIN1, partially translocated to the plasma membrane upon activation of NK2 receptors. Proteins ARHGAP12 and PKM2 translocated toward membrane blebs. Three proteins that associate with the cytoskeleton were of particular interest : PLEKHH2 rearranged from individual dots located near the cell-substrate adhesion surface into lines of dots. The speriolin-like protein, SPATC1L, redistributed to cell-cell junctions. The Chloride intracellular Channel protein, CLIC2, translocated from actin-enriched plasma membrane bundles to cell-cell junctions upon activation of NK2 receptors. CLIC2, and one of its close paralogs, CLIC4, were further shown to respond with the same translocation pattern to muscarinic M3 and lysophosphatidic LPA receptors. This screen allowed us to identify potential actors in signaling pathways downstream of G protein-coupled receptors and could be scaled-up for high-content screening.
Assuntos
Bioensaio , Imagem Molecular/métodos , Receptores da Neurocinina-2/genética , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Proteínas do Citoesqueleto , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transporte Proteico , Receptores da Neurocinina-2/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Hormônios Tireóideos/genética , Hormônios Tireóideos/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Ligação a Hormônio da TireoideRESUMO
The mTORC1 kinase promotes cell growth in response to growth factors by activation of receptor tyrosine kinase. It is regulated by the cellular energy level and the availability of nutrients. mTORC1 activity is also inhibited by cellular stresses through overexpression of REDD1 (regulated in development and DNA damage responses). We report the identification of REDD1 in a fluorescent live-imaging screen aimed at discovering new proteins implicated in G-protein-coupled receptor signaling, based on translocation criteria. Using a sensitive and quantitative plasma membrane localization assay based on bioluminescent resonance energy transfer, we further show that a panel of endogenously expressed GPCRs, through a Ca(2+)/calmodulin pathway, triggers plasma membrane translocation of REDD1 but not of its homolog REDD2. REDD1 and REDD2 share a conserved mTORC1-inhibitory motif characterized at the functional and structural level and differ most in their N-termini. We show that the N-terminus of REDD1 and its mTORC1-inhibitory motif participate in the GPCR-evoked dynamic interaction of REDD1 with the plasma membrane. We further identify REDD1 as a novel effector in GPCR signaling. We show that fast activation of mTORC1 by GPCRs correlates with fast and maximal translocation of REDD1 to the plasma membrane. Overexpression of functional REDD1 leads to a reduction of mTORC1 activation by GPCRs. By contrast, depletion of endogenous REDD1 protein unleashes mTORC1 activity. Thus, translocation to the plasma membrane appears to be an inactivation mechanism of REDD1 by GPCRs, which probably act by sequestering its functional mTORC1-inhibitory motif that is necessary for plasma membrane targeting.
Assuntos
Membrana Celular/metabolismo , Complexos Multiproteicos/metabolismo , Receptores da Neurocinina-2/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Sinalização do Cálcio , Calmodulina/metabolismo , Ativação Enzimática , Células HEK293 , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Dados de Sequência Molecular , Domínios e Motivos de Interação entre Proteínas , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteínas/metabolismo , Fatores de Transcrição/químicaRESUMO
The design of bifunctional compounds is a promising approach toward the development of strong analgesics with reduced side effects. We here report the optimization of the previously published lead peptide KGFF09, which contains opioid receptor agonist and neuropeptide FF receptor antagonist pharmacophores and is shown to induce potent antinociception and reduced side effects. We evaluated the novel hybrid peptides for their in vitro activity at MOP, NPFFR1, and NPFFR2 and selected four of them (DP08/14/32/50) for assessment of their acute antinociceptive activity in mice. We further selected DP32 and DP50 and observed that their antinociceptive activity is mostly peripherally mediated; they produced no respiratory depression, no hyperalgesia, significantly less tolerance, and strongly attenuated withdrawal syndrome, as compared to morphine and the recently FDA-approved TRV130. Overall, these data suggest that MOP agonist/NPFF receptor antagonist hybrids might represent an interesting strategy to develop novel analgesics with reduced side effects.
Assuntos
Receptores de Neuropeptídeos , Receptores Opioides mu , Animais , Receptores Opioides mu/agonistas , Receptores Opioides mu/antagonistas & inibidores , Receptores Opioides mu/metabolismo , Camundongos , Receptores de Neuropeptídeos/agonistas , Receptores de Neuropeptídeos/antagonistas & inibidores , Receptores de Neuropeptídeos/metabolismo , Masculino , Analgésicos/farmacologia , Analgésicos/química , Analgésicos/uso terapêutico , Analgésicos/síntese química , Humanos , Relação Estrutura-Atividade , Analgésicos Opioides/farmacologia , Analgésicos Opioides/químicaRESUMO
GPRASP (GPCR-associated sorting protein)/ARMCX (ARMadillo repeat-Containing proteins on the X chromosome) family is composed of 10 proteins, whose genes are located on a small locus of the X chromosome except one. They possess at least two armadillo-like repeats on their carboxylterminal homologous sequence, but they can be subdivided on specific sequence features. Subfamily 1 (GPRASP1, GPRASP2, GPRASP3, ARMCX4 and ARMCX5) displays additional repeated motifs while a mitochondrial targeting transmembrane domain is present in subfamily 2 (ARMC10, ARMCX1, ARMCX2, ARMCX3 and ARMCX6). Although their roles are not yet fully understood, the recent identification of several interacting partners has shed new light on the processes in which GPRASP/ARMCX proteins are implicated. Among the interacting partners of proteins from subfamily 1, many are GPCRs. GPRASP1 binds trafficking proteins, such as Beclin2 and the Dysbindin-HRS-Gαs complex, to participate in GPCR post-endocytic sorting. Moreover, in vitro as well as in vivo experiments indicate that GPRASP1 is a critical player in the adaptive responses related to chronic treatments with GPCR agonists. GPRASP2 seems to play a key role in the signaling of the hedgehog pathway in the primary cilium through a Smoothened-GPRASP2-Pifo complex. Identified small compound inhibitors of this complex could treat drug-resistant smoothened derived cancer forms. Deletion of GPRASP2 in mice causes neurodevelopmental alteration and affects mGluR5 regulation, reflected by autism-like behavior. Several members of subfamily 2, in complex with TRAK2 and MIRO, are involved in the trafficking of mitochondria in axons and in the regulation of their size and division, influencing the cell cycle. The essential role of GPRASP/ARMCX proteins in cellular physiology is supported by human cases of deletions, causing male neonatal lethality by pulmonary delayed development, dysmorphic face, and psychiatric and intellectual impacts in females.
Assuntos
Proteínas do Domínio Armadillo/química , Neoplasias/metabolismo , Proteínas de Transporte Vesicular/química , Animais , Proteínas do Domínio Armadillo/metabolismo , Humanos , Pneumopatias/metabolismo , Transtornos Psicóticos/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
BACKGROUND AND PURPOSE: Angiotensin II (AngII) and NO regulate the cerebral circulation. AngII AT1 receptors exert ligand-dependent and ligand-independent (myogenic tone [MT]) vasoconstriction of cerebral vessels. NO induces post-translational modifications of proteins such as S-nitrosation (redox modification of cysteine residues). In cultured cells, S-nitrosation decreases AngII's affinity for the AT1 receptor. The present work evaluated the functional consequences of S-nitrosation on both AngII-dependent and AngII-independent cerebrovascular responses. EXPERIMENTAL APPROACH: S-Nitrosation was induced in rat isolated middle cerebral arteries by pretreatment with the NO donors, S-nitrosoglutathione (GSNO) or sodium nitroprusside (SNP). Agonist-dependent activation of AT1 receptors was evaluated by obtaining concentration-response curves to AngII. Ligand-independent activation of AT1 receptors was evaluated by calculating MT (active vs. passive diameter) at pressures ranging from 20 to 200 mmHg in the presence or not of a selective AT1 receptor inverse agonist. KEY RESULTS: GSNO or SNP completely abolished the AngII-dependent AT1 receptor-mediated vasoconstriction of cerebral arteries. GSNO had no impact on responses to other vasoconstrictors sharing (phenylephrine, U46619) or not (5-HT) the same signalling pathway. MT was reduced by GSNO, and the addition of losartan did not further decrease MT, suggesting that GSNO blocks AT1 receptor-dependent MT. Ascorbate (which reduces S-nitrosated compounds) restored the response to AngII but not the soluble GC inhibitor ODQ, suggesting that these effects are mediated by S-nitrosation rather than by S-nitrosylation. CONCLUSIONS AND IMPLICATIONS: In rat middle cerebral arteries, GSNO pretreatment specifically affects the AT1 receptor and reduces both AngII-dependent and AngII-independent activation, most likely through AT1 receptor S-nitrosation.
Assuntos
Artérias Cerebrais/efeitos dos fármacos , Receptor Tipo 1 de Angiotensina/metabolismo , S-Nitrosoglutationa/farmacologia , Angiotensina II/farmacologia , Animais , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Ligantes , Masculino , Óxido Nítrico/metabolismo , Nitrosação/efeitos dos fármacos , Ratos , Ratos Wistar , S-Nitrosoglutationa/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Numerous antibodies targeting G protein-coupled receptors (GPCRs) have been described as non-specific among the polyclonal antibodies against angiotensin II type 1 receptor (AT1). We have tested the newly developed AT1 receptor mouse monoclonal antibody for its specificity. Human embryonic kidney (HEK293) cells, which do not endogenously express AT1 receptor, were transfected in order to overexpress a fluorescently labeled enhanced green fluorescent protein (EGFP)-tagged human AT1 receptor. Western blot and immunofluorescence assays were performed to test the specificity of the Santa Cruz monoclonal antibody sc-57036. These results were compared to the ones obtained with the polyclonal sc-1173 anti-AT1 receptor antibodies that have already been described as non-specific. While the positive controls using GFP antibodies detected the EGFP-tagged AT1 receptor, both polyclonal and monoclonal anti-AT1 receptor antibodies failed to specifically recognize the corresponding band by Western blot, as similar bands were revealed in either transfected or non-transfected cells. It also failed to detect AT1 receptor in immunofluorescence experiments. The lack of target recognition of the monoclonal AT1 receptor antibody in our experimental conditions suggests that this antibody could give misleading results such as misidentification of the protein. To our knowledge, no specific antibodies targeting AT1 receptors have been developed so far and the field is thus in need of new technical developments.
Assuntos
Anticorpos Monoclonais/farmacologia , Receptor Tipo 1 de Angiotensina/metabolismo , Animais , Células HEK293 , Humanos , Camundongos , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/imunologia , TransfecçãoRESUMO
Fluorescence resonance energy transfer (FRET) between an adequate pair of fluorophores is an indication of closer proximity than colocalization and is used by biologists to study fluorescently modified protein interactions inside cells. We present a method for visualization of FRET images acquired by confocal sensitized emission, involving excitation of the donor fluorophore and detection of the energy transfer as an emission from the acceptor fluorophore into the FRET channel. Authentic FRET signal measurements require the correction from the FRET channel of the undesired bleed-through signals (BT) resulting from both the leak-through of the donor emission and the direct acceptor emission. Our method reduces the interference of the user to a minimum by analyzing the entire image, pixel by pixel. It proposes imaging treatments and the display of control images to validate the BT calculation and the image corrections. It displays FRET images as a function of the colocalization of the two fluorescent partners. Finally, it proposes an alternative to normalization of the FRET intensities to compare FRET signal variations between samples. This method called "FRET and Colocalization Analyzer" has been implemented in a Plug-in of the freely available ImageJ software. It is particularly adapted when transient expression of the fluorescent proteins is used thereby giving very variable expression levels or when the colocalization of the two partners is varying in proportion, in amount, and in size, as a function of time. The method and program are validated using the analysis of the spatio-temporal interactions between a G-protein coupled receptor, the tachykinin NK2 receptor, and the beta-arrestin 2 as an example.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Processamento de Imagem Assistida por Computador , Microscopia Confocal/métodos , Arrestinas/metabolismo , Corantes Fluorescentes , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Software , beta-Arrestina 2 , beta-ArrestinasRESUMO
Neuropeptide Y binds to G-protein coupled receptors whose action results in inhibition of adenylyl cyclase activity. Using HEK293 cells stably expressing the native neuropeptide Y Y1 receptors, we found that the NPY agonist elicits a transient phosphorylation of the extracellular signal-regulated kinases (ERK1/2). We first show that ERK1/2 activation following Y1 receptor stimulation is dependent on heterotrimeric Gi/o since it is completely inhibited by pre-treatment with pertussis toxin. In addition, ERK1/2 activation is internalization-independent since mutant Y1 receptors unable to recruit ß-arrestins, can still activate ERK signaling to the same extent as wild-type receptors. We next show that this activation of the MAPK pathway is inhibited by the MEK inhibitor U0126, is not dependent on calcium signaling at the Y1 receptor (no effect upon inhibition of phospholipase C, protein kinase C or protein kinase D) but instead dependent on Gß/γ and associated signaling pathways that activate PI3-kinase. Although inhibition of the epidermal-growth factor receptor tyrosine kinase did not influence NPY-induced ERK1/2 activation, we show that the inhibition of insulin growth factor receptor IGFR by AG1024 completely blocks activation of ERK1/2 by the Y1 receptor. This Gß/γ-PI3K-AG1024-sensitive pathway does not involve activation of IGFR through the release of a soluble ligand by metalloproteinases since it is not affected by the metalloproteinase inhibitor marimastat. Finally, we found that a similar pathway, sensitive to wortmannin-AG1024 but insensitive to marimastat, is implicated in activation of ERK signaling in HEK293 cells by endogenously expressed GPCRs coupled to Gq-protein (muscarinic M3 receptors) or coupled to Gs-protein (endothelin ETB receptors). Our analysis is the first to show that ß-arrestin recruitment to the NPY Y1 receptor is not necessary for MAPK activation by this receptor but that transactivation of the IGFR receptor is required.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptor IGF Tipo 2/metabolismo , Receptores de Neuropeptídeo Y/metabolismo , Butadienos/farmacologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Neuropeptídeo Y/farmacologia , Nitrilas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Receptor IGF Tipo 1/agonistas , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 2/antagonistas & inibidores , Receptor IGF Tipo 2/genética , Receptores de Neuropeptídeo Y/genética , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional , Tirfostinas/farmacologiaRESUMO
The human NPY Y(1) receptor undergoes fast agonist-induced internalization via clathrin-coated pits then recycles back to the cell membrane. In an attempt to identify the molecular determinants involved in this process, we studied several C-terminal truncation mutants tagged with EFGP. In the absence of agonist, Y(1) receptors lacking the last 32 C-terminal amino acids (Y(1)Δ32) are constitutively internalized, unlike full-length Y(1) receptors. At steady state, internalized Y(1)Δ32 receptors co-localize with transferrin, a marker of early and recycling endosomes. Inhibition of constitutive internalization of Y(1)Δ32 receptors by hypertonic sucrose or by co-expression of Rab5aS34N, a dominant negative form of the small GTPase Rab5a or depletion of all three isoforms of Rab5 indicates the involvement of clathrin-coated pits. In contrast, a truncated receptor lacking the last 42 C-terminal amino acids (Y(1)Δ42) does not constitutively internalize, consistent with the possibility that there is a molecular determinant responsible for constitutive internalization located in the last 10 amino acids of Y(1)Δ32 receptors. We show that the agonist-independent internalization of Y(1)Δ32 receptors involves a tyrosine-based motif YXXΦ. The potential role of this motif in the behaviour of full-length Y(1) receptors has also been explored. Our results indicate that a C-terminal tyrosine-based motif is critical for the constitutive internalization of truncated Y(1)Δ32 receptors. We suggest that this motif is masked in full-length Y(1) receptors which do not constitutively internalize in the absence of agonist.
Assuntos
Receptores de Neuropeptídeo Y/metabolismo , Tirosina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Clatrina/química , Clatrina/metabolismo , Células HEK293 , Humanos , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Neuropeptídeo Y/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores de Neuropeptídeo Y/agonistas , Receptores de Neuropeptídeo Y/genética , Transdução de Sinais , Transferrina/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
Regulation of cellular responses to external stimuli such as hormones, neurotransmitters, or cytokines is achieved through the control of all steps of the complex cascade starting with synthesis, going through maturation steps, release, distribution, degradation and/or uptake of the signalling molecule interacting with the target protein. One possible way of regulation, referred to as scavenging or neutralization of the ligand, has been increasingly studied, especially for small protein ligands. It shows innovative potential in chemical biology approaches as well as in disease treatment. Neutralization of protein ligands, as for example cytokines or chemokines can lead to the validation of signalling pathways under physiological or pathophysiological conditions, and in certain cases, to the development of therapeutic molecules now used in autoimmune diseases, chronic inflammation and cancer treatment. This review explores the field of ligand neutralization and tries to determine to what extent small chemical molecules could substitute for neutralizing antibodies in therapeutic approaches.
Assuntos
Anticorpos Neutralizantes/uso terapêutico , Quimiocinas/uso terapêutico , Citocinas/uso terapêutico , Inflamação/tratamento farmacológico , Quimiocinas/imunologia , Quimiocinas/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Ligação Proteica , Transdução de Sinais/imunologia , Transdução de Sinais/fisiologiaRESUMO
Activated human neuropeptide Y Y(1) receptors rapidly desensitize and internalize through clathrin-coated pits and recycle from early and recycling endosomes, unlike Y(2) receptors that neither internalize nor desensitize. To identify motifs implicated in Y(1) receptor desensitization and trafficking, mutants with varying C-terminal truncations or a substituted Y(2) C-terminus were constructed. Point mutations of key putative residues were made in a C-terminal conserved motif [phi-H-(S/T)-(E/D)-V-(S/T)-X-T] that we have identified and in the second intracellular i2 loop. Receptors were analyzed by functional assays, spectrofluorimetric measurements on living cells, flow cytometry, confocal imaging and bioluminescence resonance energy transfer assays for beta-arrestin activation and adaptor protein (AP-2) complex recruitment. Inhibitory GTP-binding protein-dependent signaling of Y(1) receptors to adenylyl cyclase and desensitization was unaffected by C-terminal truncations or mutations, while C-terminal deletion mutants of 42 and 61 amino acids no longer internalized. Substitutions of Thr357, Asp358, Ser360 and Thr362 by Ala in the C-terminus abolished both internalization and beta-arrestin activation but not desensitization. A Pro145 substitution by His in an i2 consensus motif reported to mediate phosphorylation-independent recruitment of beta-arrestins affected neither desensitization, internalization or recycling kinetics of activated Y(1) receptors nor beta-arrestin activation. Interestingly, combining Pro145 substitution by His and C-terminal substitutions significantly attenuates Y(1) desensitization. In the Y(2) receptor, replacement of His155 with Pro at this position in the i2 loop motif promotes agonist-mediated desensitization, beta-arrestin activation, internalization and recycling. Overall, our results indicate that beta-arrestin-mediated desensitization and internalization of Y(1) and Y(2) receptors are differentially regulated by the C-terminal motif and the i2 loop consensus motif.
Assuntos
Receptores de Neuropeptídeo Y/química , Receptores de Neuropeptídeo Y/metabolismo , Adenilil Ciclases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Arrestinas/metabolismo , Transporte Biológico Ativo , Linhagem Celular , AMP Cíclico/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Cinética , Microscopia Confocal , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Receptores de Neuropeptídeo Y/agonistas , Receptores de Neuropeptídeo Y/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Transfecção , beta-ArrestinasRESUMO
The chemokine CXCL12 and the receptor CXCR4 play pivotal roles in normal vascular and neuronal development, in inflammatory responses, and in infectious diseases and cancer. For instance, CXCL12 has been shown to mediate human immunodeficiency virus-induced neurotoxicity, proliferative retinopathy and chronic inflammation, whereas its receptor CXCR4 is involved in human immunodeficiency virus infection, cancer metastasis and in the rare disease known as the warts, hypogammaglobulinemia, immunodeficiency, and myelokathexis (WHIM) syndrome. As we screened chemical libraries to find inhibitors of the interaction between CXCL12 and the receptor CXCR4, we identified synthetic compounds from the family of chalcones that reduce binding of CXCL12 to CXCR4, inhibit calcium responses mediated by the receptor, and prevent CXCR4 internalization in response to CXCL12. We found that the chemical compounds display an original mechanism of action as they bind to the chemokine but not to CXCR4. The highest affinity molecule blocked chemotaxis of human peripheral blood lymphocytes ex vivo. It was also active in vivo in a mouse model of allergic eosinophilic airway inflammation in which we detected inhibition of the inflammatory infiltrate. The compound showed selectivity for CXCL12 and not for CCL5 and CXCL8 chemokines and blocked CXCL12 binding to its second receptor, CXCR7. By analogy to the effect of neutralizing antibodies, this molecule behaves as a small organic neutralizing compound that may prove to have valuable pharmacological and therapeutic potential.
Assuntos
Quimiocina CXCL12/metabolismo , Regulação da Expressão Gênica , Cálcio/metabolismo , Calorimetria , Linhagem Celular , Proliferação de Células , Chalconas/metabolismo , Quimiocina CCL5/metabolismo , Quimiocina CXCL12/fisiologia , Humanos , Inflamação , Ligantes , Ligação Proteica , Receptores CXCR/metabolismo , Receptores CXCR4/metabolismo , Espectrometria de Fluorescência/métodosRESUMO
By combining real time measurements of agonist binding, by fluorescence resonance energy transfer, and of subsequent responses, we proposed previously that the neurokinin NK2 receptor preexists in equilibrium between three states: inactive, calcium-triggering, and cAMP-producing. Thr(24) and Phe(26) of the NK2 receptor extracellular domain are considered to interact with neuropeptide agonists based on the reduction of affinity when they are substituted by alanine. Using fluorescence resonance energy transfer, we now quantify the binding kinetics of two Texas Red-modified neurokinin A agonists to the fluorescent wild-type (Y-NK2wt) and the mutant (Y-NK2mut) receptor carrying Thr(24) --> Ala and Phe(26) --> Ala mutations. TR1-neurokinin A binds with a fast component and a slow component to the Y-NK2wt receptor and triggers both a calcium and a cAMP response. In contrast, on the mutant receptor, it binds in a single fast step with a lower apparent affinity and activates only the calcium response. Another agonist, TRC4-neurokinin A, binds to both wild-type and mutant receptors in a single fast step, with similar affinities and kinetics and promotes only calcium signaling. Kinetic modeling of ligand binding and receptor interconversions is carried out to analyze phenotypic changes in terms of binding alterations or changes in the transitions between conformational states. We show that the binding and response properties of the Y-NK2mut receptor are best described according to a phenotype where a reduction of the transition between the inactive and the active states occurs.
Assuntos
Cálcio/metabolismo , AMP Cíclico/fisiologia , Receptores da Neurocinina-2/química , Transferência Ressonante de Energia de Fluorescência , Humanos , Cinética , Modelos Teóricos , Mutação , Neurocinina A/metabolismo , Conformação Proteica , Receptores da Neurocinina-1/metabolismo , Receptores da Neurocinina-2/agonistas , Receptores da Neurocinina-2/fisiologia , Relação Estrutura-Atividade , Substância P/metabolismoRESUMO
The G-protein coupled receptor (GPCR) human mu opioid receptor (hMOR) fused to the carboxy-terminus of the enhanced green fluorescent protein (EGFP) has been successfully and stably expressed in Drosophila Schneider 2 cells under the control of an inducible metallothionein promoter. Polyclonal cells expressing EGFPhMOR display high-affinity, saturable, and specific binding sites for the opioid antagonist diprenorphine. Competition studies with opioid agonists and antagonists defined the pharmacological profile of a mu opioid receptor similar to that observed in mammalian cells, suggesting proper folding of EGFPhMOR in a high-affinity state in Drosophila cells. The functionality of the fusion protein was demonstrated by the ability of agonist to reduce forskolin-stimulated cyclic AMP production and to induce [35S]GTPgammaS incorporation. The EGFPhMOR protein had the expected molecular weight (70kDa), as demonstrated by protein immunoblotting with anti-EGFP and anti-C-terminus hMOR antibodies. However, quantitative EGFP fluorescence intensity analysis revealed that the total level of expressed EGFPhMOR is 8-fold higher than the level of diprenorphine binding sites, indicating that part of the receptor is not in a high-affinity state. This may in part be due to a population of receptors localized in intracellular compartments, as shown by the distribution of fluorescence between the plasma membrane and the cell interior. This study shows that EGFP is a valuable and versatile tool for monitoring and quantifying expression levels as well as for optimizing and characterizing an expression system. Optimization of the Drosophila Schneider 2 cell expression system will allow large-scale purification of GPCRs, thus enabling structural studies to be undertaken.
Assuntos
Proteínas Luminescentes/biossíntese , Receptores Opioides mu/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Animais , Ligação Competitiva/efeitos dos fármacos , Western Blotting , Linhagem Celular , Clonagem Molecular , Colforsina/farmacologia , Sulfato de Cobre/farmacologia , AMP Cíclico/metabolismo , DNA Complementar/genética , Diprenorfina/metabolismo , Diprenorfina/farmacologia , Drosophila/citologia , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Proteínas de Fluorescência Verde , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Humanos , Proteínas Luminescentes/genética , Metalotioneína/genética , Microscopia Confocal , Morfina/farmacologia , Naloxona/farmacologia , Naltrexona/farmacologia , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Peptídeos Opioides , Toxina Pertussis/farmacologia , Reação em Cadeia da Polimerase , Ligação Proteica/efeitos dos fármacos , Receptores Acoplados a Proteínas G/biossíntese , Receptores Acoplados a Proteínas G/genética , Receptores Opioides mu/genética , Receptores Opioides mu/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de Fluorescência , TermodinâmicaRESUMO
A functional fluorescent neurokinin NK2 receptor, EGFP-NK2, was previously used to follow, by fluorescence resonance energy transfer measurements in living cells, the binding of its fluorescently labeled agonist, bodipy-neurokinin A (NKA). Local agonist application suggested that the activation and desensitization of the NK2 receptors were compartmentalized at the level of the plasma membrane. In this study, fluorescence recovery after photobleaching experiments are carried out at variable observation radius (vrFRAP) to probe EGFP-NK2 receptor mobility and confinement. Experiments are carried out at 20 degrees C to maintain the number of receptors constant at the cell surface during recordings. In the absence of agonist, 35% EGFP-NK2 receptors diffuse within domains of 420 +/- 80 nm in radius with the remaining 65% of receptors able to diffuse with a long range lateral diffusion coefficient between the domains. When cells are incubated with a saturating concentration of NKA, 30% EGFP-NK2 receptors become immobilized in small domains characterized by a radius equal to 170 +/- 50 nm. Biochemical experiments show that the confinement of EGFP-NK2 receptor is not due to its association with rafts at any given time. Colocalization of the receptor with beta-arrestin and transferrin supports that the small domains, containing 30% of activated EGFP-NK2, correspond to clathrin-coated pre-pits. The similar amount of confined EGFP-NK2 receptors found before and after activation (30-35%) is discussed in term of putative transient interactions of the receptors with preexisting scaffolds of signaling molecules.
Assuntos
Membrana Celular/metabolismo , Receptores da Neurocinina-2/química , Arrestinas/metabolismo , Compostos de Boro/farmacologia , Linhagem Celular , Clatrina/química , Difusão , Relação Dose-Resposta a Droga , Recuperação de Fluorescência Após Fotodegradação , Corantes Fluorescentes/farmacologia , Vetores Genéticos , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Humanos , Immunoblotting , Lipídeos/química , Microscopia Confocal , Microscopia de Fluorescência , Modelos Químicos , Neurocinina A/química , Octoxinol/farmacologia , Estrutura Terciária de Proteína , Receptores da Neurocinina-2/metabolismo , Temperatura , Fatores de Tempo , Transfecção , Transferrina/metabolismo , Xantenos/farmacologia , beta-ArrestinasRESUMO
Desensitization of G protein-coupled receptors (GPCRs) involves receptor phosphorylation and reduction in the number of receptors at the cell surface. The neuropeptide Y (NPY) Y(1) receptor undergoes fast desensitization. We examined agonist-induced signaling and internalization using NPY Y(1) receptors fused to green fluorescent protein (EGFP). When expressed in HEK293 cells, EGFP-hNPY Y(1) receptors were localized at the plasma membrane, desensitized rapidly as assessed using calcium responses, and had similar properties compared to hNPY Y(1) receptors. Upon agonist challenge, the EGFP signal decreased rapidly (t(1/2) = 107 +/- 3 s) followed by a slow recovery. This decrease was blocked by BIBP3226, a Y(1) receptor antagonist, or by pertussis toxin, in agreement with Y(1) receptor activation. Internalization of EGFP-hNPY Y(1) receptors to acidic endosomal compartments likely accounts for the decrease in the EGFP signal, being absent after pretreatment with monensin. Concanavalin A and hypertonic sucrose, which inhibit clathrin-mediated endocytosis, blocked the decrease in fluorescence. After agonist, intracellular EGFP signals were punctate and co-localized with transferrin-Texas Red, a marker of clathrin-associated internalization and recycling, but not with LysoTracker Red, a lysosomal pathway marker, supporting receptor trafficking to recycling endosomes rather than the late endosomal/lysosomal pathway. Pulse-chase experiments revealed no receptor degradation after internalization. The slow recovery of fluorescence was unaffected by cycloheximide or actinomycin D, indicating that de novo synthesis of receptors was not limiting. Use of a multicompartment model to fit our fluorescence data allows simultaneous determination of internalization and recycling rate constants. We propose that rapid internalization of receptors via the clathrin-coated pits recycling pathway may largely account for the rapid desensitization of NPY Y(1) receptors.