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1.
Elife ; 92020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32513388

RESUMO

Early Huntington's disease (HD) include over-activation of dopamine D1 receptors (D1R), producing an imbalance in dopaminergic neurotransmission and cell death. To reduce D1R over-activation, we present a strategy based on targeting complexes of D1R and histamine H3 receptors (H3R). Using an HD mouse striatal cell model and HD mouse organotypic brain slices we found that D1R-induced cell death signaling and neuronal degeneration, are mitigated by an H3R antagonist. We demonstrate that the D1R-H3R heteromer is expressed in HD mice at early but not late stages of HD, correlating with HD progression. In accordance, we found this target expressed in human control subjects and low-grade HD patients. Finally, treatment of HD mice with an H3R antagonist prevented cognitive and motor learning deficits and the loss of heteromer expression. Taken together, our results indicate that D1R - H3R heteromers play a pivotal role in dopamine signaling and represent novel targets for treating HD.

3.
Prog Mol Biol Transl Sci ; 169: 213-245, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31952687

RESUMO

This chapter will focus on the therapeutic potential of oligomeric GPCR structures. We will discuss the history of these complexes and their connection with treatment. We will highlight several examples from the literature of complexes of therapeutic or theranostic opportunity. As part of this we will discuss different mechanisms that oligomers use to modulate their partner that could be taken advantage of from a drug targeting perspective. We will also discuss the challenges and hurdles to targeting such complexes.

4.
Angew Chem Int Ed Engl ; 58(50): 17986-17993, 2019 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-31609054

RESUMO

Antibodies have found applications in several fields, including, medicine, diagnostics, and nanotechnology, yet methods to modulate antibody-antigen binding using an external agent remain limited. Here, we have developed photoactive antibody fragments by genetic site-specific replacement of single tyrosine residues with photocaged tyrosine, in an antibody fragment, 7D12. A simple and robust assay is adopted to evaluate the light-mediated binding of 7D12 mutants to its target, epidermal growth factor receptor (EGFR), on the surface of cancer cells. Presence of photocaged tyrosine reduces 7D12-EGFR binding affinity by over 20-fold in two out of three 7D12 mutants studied, and binding is restored upon exposure to 365 nm light. Molecular dynamics simulations explain the difference in effect of photocaging on 7D12-EGFR interaction among the mutants. Finally, we demonstrate the application of photoactive antibodies in delivering fluorophores to EGFR-positive live cancer cells in a light-dependent manner.

5.
J Biol Chem ; 294(45): 16587-16603, 2019 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-31467080

RESUMO

Membrane proteins can associate into larger complexes. Examples include receptor tyrosine complexes, ion channels, transporters, and G protein-coupled receptors (GPCRs). For the latter, there is abundant evidence indicating that GPCRs assemble into complexes, through both homo- and heterodimerization. However, the tools for studying and disrupting these complexes, GPCR or otherwise, are limited. Here, we have developed stabilized interference peptides for this purpose. We have previously reported that tetrahydrocannabinol-mediated cognitive impairment arises from homo- or heterooligomerization between the GPCRs cannabinoid receptor type 1 (CB1R) and 5-hydroxytryptamine 2A (5-HT2AR) receptors. Here, to disrupt this interaction through targeting CB1-5-HT2A receptor heteromers in HEK293 cells and using an array of biochemical techniques, including calcium and cAMP measurements, bimolecular fluorescence complementation assays, and CD-based helicity assessments, we developed a NanoLuc binary technology (NanoBiT)-based reporter assay to screen a small library of aryl-carbon-stapled transmembrane-mimicking peptides produced by solid-phase peptide synthesis. We found that these stapling peptides have increased α-helicity and improved proteolytic resistance without any loss of disrupting activity in vitro, suggesting that this approach may also have utility in vivo In summary, our results provide proof of concept for using NanoBiT to study membrane protein complexes and for stabilizing disrupting peptides to target such membrane complexes through hydrocarbon-mediated stapling. We propose that these peptides could be developed to target previously undruggable GPCR heteromers.

6.
Proc Natl Acad Sci U S A ; 116(9): 3863-3872, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30733293

RESUMO

Although human epidermal growth factor receptor 2 (HER2)-targeted therapies have dramatically improved the clinical outcome of HER2-positive breast cancer patients, innate and acquired resistance remains an important clinical challenge. New therapeutic approaches and diagnostic tools for identification, stratification, and treatment of patients at higher risk of resistance and recurrence are therefore warranted. Here, we unveil a mechanism controlling the oncogenic activity of HER2: heteromerization with the cannabinoid receptor CB2R. We show that HER2 physically interacts with CB2R in breast cancer cells, and that the expression of these heteromers correlates with poor patient prognosis. The cannabinoid Δ9-tetrahydrocannabinol (THC) disrupts HER2-CB2R complexes by selectively binding to CB2R, which leads to (i) the inactivation of HER2 through disruption of HER2-HER2 homodimers, and (ii) the subsequent degradation of HER2 by the proteasome via the E3 ligase c-CBL. This in turn triggers antitumor responses in vitro and in vivo. Selective targeting of CB2R transmembrane region 5 mimicked THC effects. Together, these findings define HER2-CB2R heteromers as new potential targets for antitumor therapies and biomarkers with prognostic value in HER2-positive breast cancer.


Assuntos
Neoplasias da Mama/líquido cefalorraquidiano , Terapia de Alvo Molecular , Receptor CB2 de Canabinoide/genética , Receptor ErbB-2/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Dronabinol/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-cbl/genética , Receptor CB2 de Canabinoide/química , Receptor ErbB-2/química , Transdução de Sinais
7.
Pharmacol Ther ; 192: 20-41, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29953902

RESUMO

The introduction of atypical antipsychotics (AAPs) since the discovery of its prototypical drug clozapine has been a revolutionary pharmacological step for treating psychotic patients as these allow a significant recovery not only in terms of hospitalization and reduction in symptoms severity, but also in terms of safety, socialization and better rehabilitation in the society. Regarding the mechanism of action, AAPs are weak D2 receptor antagonists and they act beyond D2 antagonism, involving other receptor targets which regulate dopamine and other neurotransmitters. Consequently, AAPs present a significant reduction of deleterious side effects like parkinsonism, hyperprolactinemia, apathy and anhedonia, which are all linked to the strong blockade of D2 receptors. This review revisits previous and current findings within the class of AAPs and highlights the differences in terms of receptor properties and clinical activities among them. Furthermore, we propose a continuum spectrum of "atypia" that begins with risperidone (the least atypical) to clozapine (the most atypical), while all the other AAPs fall within the extremes of this spectrum. Clozapine is still considered the gold standard in refractory schizophrenia and in psychoses present in Parkinson's disease, though it has been associated with adverse effects like agranulocytosis (0.7%) and weight gain, pushing the scientific community to find new drugs as effective as clozapine, but devoid of its side effects. To achieve this, it is therefore imperative to characterize and compare in depth the very complex molecular profile of AAPs. We also introduce relatively new concepts like biased agonism, receptor dimerization and neurogenesis to identify better the old and new hallmarks of "atypia". Finally, a detailed confrontation of clinical differences among the AAPs is presented, especially in relation to their molecular targets, and new means like therapeutic drug monitoring are also proposed to improve the effectiveness of AAPs in clinical practice.


Assuntos
Antipsicóticos/farmacologia , Clozapina/farmacologia , Desenho de Fármacos , Receptores Dopaminérgicos/metabolismo , Receptores de Serotonina/metabolismo , Esquizofrenia/tratamento farmacológico , Animais , Antipsicóticos/efeitos adversos , Antipsicóticos/farmacocinética , Antipsicóticos/uso terapêutico , Clozapina/efeitos adversos , Clozapina/uso terapêutico , Humanos , Terapia de Alvo Molecular , Medicina de Precisão , Psicologia Clínica , Receptores Colinérgicos/metabolismo , Receptores de Glutamato/metabolismo , Receptores Histamínicos/metabolismo , Esquizofrenia/metabolismo
8.
BMC Biol ; 16(1): 24, 2018 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-29486745

RESUMO

BACKGROUND: G-protein-coupled receptor (GPCR) heteromeric complexes have distinct properties from homomeric GPCRs, giving rise to new receptor functionalities. Adenosine receptors (A1R or A2AR) can form A1R-A2AR heteromers (A1-A2AHet), and their activation leads to canonical G-protein-dependent (adenylate cyclase mediated) and -independent (ß-arrestin mediated) signaling. Adenosine has different affinities for A1R and A2AR, allowing the heteromeric receptor to detect its concentration by integrating the downstream Gi- and Gs-dependent signals. cAMP accumulation and ß-arrestin recruitment assays have shown that, within the complex, activation of A2AR impedes signaling via A1R. RESULTS: We examined the mechanism by which A1-A2AHet integrates Gi- and Gs-dependent signals. A1R blockade by A2AR in the A1-A2AHet is not observed in the absence of A2AR activation by agonists, in the absence of the C-terminal domain of A2AR, or in the presence of synthetic peptides that disrupt the heteromer interface of A1-A2AHet, indicating that signaling mediated by A1R and A2AR is controlled by both Gi and Gs proteins. CONCLUSIONS: We identified a new mechanism of signal transduction that implies a cross-communication between Gi and Gs proteins guided by the C-terminal tail of the A2AR. This mechanism provides the molecular basis for the operation of the A1-A2AHet as an adenosine concentration-sensing device that modulates the signals originating at both A1R and A2AR.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Purinérgicos P1/metabolismo , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Células HEK293 , Humanos , Estrutura Terciária de Proteína , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/genética , Receptores Purinérgicos P1/química , Receptores Purinérgicos P1/genética
9.
Neuropsychopharmacology ; 43(5): 964-977, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-28102227

RESUMO

The dorsal striatum is a key node for many neurobiological processes such as motor activity, cognitive functions, and affective processes. The proper functioning of striatal neurons relies critically on metabotropic receptors. Specifically, the main adenosine and endocannabinoid receptors present in the striatum, ie, adenosine A2A receptor (A2AR) and cannabinoid CB1 receptor (CB1R), are of pivotal importance in the control of neuronal excitability. Facilitatory and inhibitory functional interactions between striatal A2AR and CB1R have been reported, and evidence supports that this cross-talk may rely, at least in part, on the formation of A2AR-CB1R heteromeric complexes. However, the specific location and properties of these heteromers have remained largely unknown. Here, by using techniques that allowed a precise visualization of the heteromers in situ in combination with sophisticated genetically modified animal models, together with biochemical and pharmacological approaches, we provide a high-resolution expression map and a detailed functional characterization of A2AR-CB1R heteromers in the dorsal striatum. Specifically, our data unveil that the A2AR-CB1R heteromer (i) is essentially absent from corticostriatal projections and striatonigral neurons, and, instead, is largely present in striatopallidal neurons, (ii) displays a striking G protein-coupled signaling profile, where co-stimulation of both receptors leads to strongly reduced downstream signaling, and (iii) undergoes an unprecedented dysfunction in Huntington's disease, an archetypal disease that affects striatal neurons. Altogether, our findings may open a new conceptual framework to understand the role of coordinated adenosine-endocannabinoid signaling in the indirect striatal pathway, which may be relevant in motor function and neurodegenerative diseases.


Assuntos
Corpo Estriado/metabolismo , Estrutura Quaternária de Proteína , Receptor A2A de Adenosina/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Transdução de Sinais , Animais , Humanos , Doença de Huntington/metabolismo , Camundongos , Vias Neurais/metabolismo , Subunidades Proteicas/biossíntese
10.
J Med Chem ; 60(23): 9575-9584, 2017 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-29116785

RESUMO

The 5-HT2CR agonist lorcaserin, clinically approved for the treatment of obesity, causes important side effects mainly related to subtype selectivity. In the search for 5-HT2CR allosteric modulators as safer antiobesity drugs, a chemical library from Vivia Biotech was screened using ExviTech platform. Structural modifications of identified hit VA240 in synthesized analogues 6-41 afforded compound 11 (N-[(1-benzyl-1H-indol-3-yl)methyl]pyridin-3-amine, VA012), which exhibited dose-dependent enhancement of serotonin efficacy, no significant off-target activities, and low binding competition with serotonin or other orthosteric ligands. PAM 11 was very active in feeding inhibition in rodents, an effect that was not related to the activation of 5-HT2AR. A combination of 11 with the SSRI sertraline increased the anorectic effect. Subchronic administration of 11 reduced food intake and body weight gain without causing CNS-related malaise. The behavior of compound 11 identified in this work supports the interest of a serotonin 5-HT2CR PAM as a promising therapeutic approach for obesity.


Assuntos
Fármacos Antiobesidade/química , Fármacos Antiobesidade/farmacologia , Regulação do Apetite/efeitos dos fármacos , Obesidade/tratamento farmacológico , Receptor 5-HT2C de Serotonina/metabolismo , Agonistas do Receptor 5-HT2 de Serotonina/química , Agonistas do Receptor 5-HT2 de Serotonina/farmacologia , Regulação Alostérica/efeitos dos fármacos , Animais , Humanos , Masculino , Camundongos , Piridinas/química , Piridinas/farmacologia , Ratos Wistar , Serotonina/metabolismo , Percepção Gustatória/efeitos dos fármacos
11.
Front Pharmacol ; 8: 616, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29033832

RESUMO

Cyclotides are plant derived, cystine-knot stabilized peptides characterized by their natural abundance, sequence variability and structural plasticity. They are abundantly expressed in Rubiaceae, Psychotrieae in particular. Previously the cyclotide kalata B7 was identified to modulate the human oxytocin and vasopressin G protein-coupled receptors (GPCRs), providing molecular validation of the plants' uterotonic properties and further establishing cyclotides as valuable source for GPCR ligand design. In this study we screened a cyclotide extract derived from the root powder of the South American medicinal plant ipecac (Carapichea ipecacuanha) for its GPCR modulating activity of the corticotropin-releasing factor type 1 receptor (CRF1R). We identified and characterized seven novel cyclotides. One cyclotide, caripe 8, isolated from the most active fraction, was further analyzed and found to antagonize the CRF1R. A nanomolar concentration of this cyclotide (260 nM) reduced CRF potency by ∼4.5-fold. In contrast, caripe 8 did not inhibit forskolin-, or vasopressin-stimulated cAMP responses at the vasopressin V2 receptor, suggesting a CRF1R-specific mode-of-action. These results in conjunction with our previous findings establish cyclotides as modulators of both classes A and B GPCRs. Given the diversity of cyclotides, our data point to other cyclotide-GPCR interactions as potentially important sources of drug-like molecules.

12.
Sci Rep ; 7(1): 7148, 2017 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-28769048

RESUMO

AP-1 is a clathrin adaptor recruited to the trans-Golgi Network where it can interact with specific signals found in the cytosolic tail of cargo proteins to incorporate them into clathrin-coated vesicles for trafficking. The small G protein Arf1 regulates the spatiotemporal recruitment of AP-1 and also drives a conformational change favoring an interaction with cargo proteins. A recent crystal structure and in vitro experiments highlighted potential residues mediating the AP-1/Arf1 interaction and the unlocking of the complex. We have used bioluminescence resonance energy transfer (BRET) to study the Arf1/AP-1 interaction and AP-1 conformational changes in vivo. We identified novel residues required for this interaction in addition to those predicted in the crystal structure. We also studied the conformational changes in AP-1 driven by Arf1 in live cells and found that opening of the complex is prerequisite for oligomerization. Using Arf1 knockout cells generated by CRISPR/Cas9, we demonstrated that residue 172 in Arf1 is necessary for AP-1 activation and is required for the efficient sorting of the lysosomal protein prosaposin. We have used BRET to study the in vivo activation of AP-1. The advantages of BRET include expressing full-length proteins in their native environment that have been fully post-translationally modified.


Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Fator de Transcrição AP-1/metabolismo , Fator 1 de Ribosilação do ADP/química , Fator 1 de Ribosilação do ADP/genética , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Linhagem Celular , Vesículas Revestidas por Clatrina/metabolismo , Expressão Gênica , Genes Reporter , Células HEK293 , Humanos , Mutação , Ligação Proteica , Multimerização Proteica , Transporte Proteico , Fator de Transcrição AP-1/química , Fator de Transcrição AP-1/genética , Rede trans-Golgi/metabolismo
13.
Sci Rep ; 7: 41293, 2017 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-28117458

RESUMO

Serotonin 5-HT6 receptor has been proposed as a promising therapeutic target for cognition enhancement though the development of new antagonists is still needed to validate these molecules as a drug class for the treatment of Alzheimer's disease and other pathologies associated with memory deficiency. As part of our efforts to target the 5-HT6 receptor, new benzimidazole-based compounds have been designed and synthesized. Site-directed mutagenesis and homology models show the importance of a halogen bond interaction between a chlorine atom of the new class of 5-HT6 receptor antagonists identified herein and a backbone carbonyl group in transmembrane domain 4. In vitro pharmacological characterization of 5-HT6 receptor antagonist 7 indicates high affinity and selectivity over a panel of receptors including 5-HT2B subtype and hERG channel, which suggests no major cardiac issues. Compound 7 exhibited in vivo procognitive activity (1 mg/kg, ip) in the novel object recognition task as a model of memory deficit.


Assuntos
Cognição/efeitos dos fármacos , Halogênios/química , Receptores de Serotonina/metabolismo , Antagonistas da Serotonina/farmacologia , Animais , Benzimidazóis/síntese química , Benzimidazóis/química , Benzimidazóis/farmacologia , Humanos , Ligantes , Microssomos Hepáticos/metabolismo , Mutagênese Sítio-Dirigida , Ratos , Receptores de Serotonina/química , Antagonistas da Serotonina/síntese química , Antagonistas da Serotonina/química , Homologia Estrutural de Proteína , Análise e Desempenho de Tarefas
14.
Mol Neurobiol ; 54(6): 4537-4550, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-27370794

RESUMO

Alzheimer's disease (AD) is a neurodegenerative disorder causing progressive memory loss and cognitive dysfunction. Anti-AD strategies targeting cell receptors consider them as isolated units. However, many cell surface receptors cooperate and physically contact each other forming complexes having different biochemical properties than individual receptors. We here report the discovery of dopamine D1, histamine H3, and N-methyl-D-aspartate (NMDA) glutamate receptor heteromers in heterologous systems and in rodent brain cortex. Heteromers were detected by co-immunoprecipitation and in situ proximity ligation assays (PLA) in the rat cortex where H3 receptor agonists, via negative cross-talk, and H3 receptor antagonists, via cross-antagonism, decreased D1 receptor agonist signaling determined by ERK1/2 or Akt phosphorylation, and counteracted D1 receptor-mediated excitotoxic cell death. Both D1 and H3 receptor antagonists also counteracted NMDA toxicity suggesting a complex interaction between NMDA receptors and D1-H3 receptor heteromer function. Likely due to heteromerization, H3 receptors act as allosteric regulator for D1 and NMDA receptors. By bioluminescence resonance energy transfer (BRET), we demonstrated that D1 or H3 receptors form heteromers with NR1A/NR2B NMDA receptor subunits. D1-H3-NMDA receptor complexes were confirmed by BRET combined with fluorescence complementation. The endogenous expression of complexes in mouse cortex was determined by PLA and similar expression was observed in wild-type and APP/PS1 mice. Consistent with allosteric receptor-receptor interactions within the complex, H3 receptor antagonists reduced NMDA or D1 receptor-mediated excitotoxic cell death in cortical organotypic cultures. Moreover, H3 receptor antagonists reverted the toxicity induced by ß1-42-amyloid peptide. Thus, histamine H3 receptors in D1-H3-NMDA heteroreceptor complexes arise as promising targets to prevent neurodegeneration.


Assuntos
Doença de Alzheimer/terapia , Terapia de Alvo Molecular , Neurônios/metabolismo , Neurônios/patologia , Receptores de Dopamina D1/metabolismo , Receptores Histamínicos H3/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Doença de Alzheimer/patologia , Animais , Morte Celular , Córtex Cerebral/patologia , Transferência de Energia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos Transgênicos , Modelos Biológicos , Neuroproteção , Fosforilação , Multimerização Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais
15.
Int J Neuropsychopharmacol ; 20(1): 72-82, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-27744406

RESUMO

Background: Cocaine addiction continues to be a major heath concern, and despite public health intervention there is a lack of efficient pharmacological treatment options. A newly identified potential target are the group I metabotropic glutamate receptors, with allosteric modulators showing particular promise. Methods: We evaluated the capacity of group I metabotropic glutamate receptors to induce functional responses in ex vivo striatal slices from rats with (1) acute cocaine self-administration, (2) chronic cocaine self-administration, and (3) 60 days cocaine self-administration withdrawal by Western blot and extracellular recordings of synaptic transmission. Results: We found that striatal group I metabotropic glutamate receptors are the principal mediator of the mGluR1/5 agonist (RS)-3,5-dihydroxyphenylglycine-induced cAMP responsive-element binding protein phosphorylation. Both acute and chronic cocaine self-administration blunted group I metabotropic glutamate receptor effects on cAMP responsive-element binding protein phosphorylation in the striatum, which correlated with the capacity to induce long-term depression, an effect that was maintained 60 days after chronic cocaine self-administration withdrawal. In the nucleus accumbens, the principal brain region mediating the rewarding effects of drugs, chronic cocaine self-administration blunted group I metabotropic glutamate receptor stimulation of extracellular signal-regulated protein kinases 1/2 and cAMP responsive-element binding protein. Interestingly, the group I metabotropic glutamate receptor antagonist/inverse-agonist, 2-methyl-6-(phenylethynyl)pyridine hydrochloride, led to a specific increase in cAMP responsive-element binding protein phosphorylation after chronic cocaine self-administration, specifically in the nucleus accumbens, but not in the striatum. Conclusions: Prolonged cocaine self-administration, through withdrawal, leads to a blunting of group I metabotropic glutamate receptor responses in the striatum. In addition, specifically in the accumbens, group I metabotropic glutamate receptor signaling to cAMP responsive-element binding protein shifts from an agonist-induced to an antagonist-induced cAMP responsive-element binding protein phosphorylation.


Assuntos
Transtornos Relacionados ao Uso de Cocaína/metabolismo , Cocaína/administração & dosagem , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Inibidores da Captação de Dopamina/administração & dosagem , Receptor de Glutamato Metabotrópico 5/metabolismo , Doença Aguda , Animais , Doença Crônica , Transtornos Relacionados ao Uso de Cocaína/patologia , Corpo Estriado/patologia , Modelos Animais de Doenças , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Ratos Sprague-Dawley , Receptores de Glutamato Metabotrópico/metabolismo , Autoadministração , Síndrome de Abstinência a Substâncias/metabolismo , Síndrome de Abstinência a Substâncias/patologia , Técnicas de Cultura de Tecidos
16.
J Med Chem ; 59(15): 7152-66, 2016 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-27420737

RESUMO

Dimeric/oligomeric states of G-protein coupled receptors have been difficult to target. We report here bivalent ligands consisting of two identical oxytocin-mimetics that induce a three order magnitude boost in G-protein signaling of oxytocin receptors (OTRs) in vitro and a 100- and 40-fold gain in potency in vivo in the social behavior of mice and zebrafish. Through receptor mutagenesis and interference experiments with synthetic peptides mimicking transmembrane helices (TMH), we show that such superpotent behavior follows from the binding of the bivalent ligands to dimeric receptors based on a TMH1-TMH2 interface. Moreover, in this arrangement, only the analogues with a well-defined spacer length (∼25 Å) precisely fit inside a channel-like passage between the two protomers of the dimer. The newly discovered oxytocin bivalent ligands represent a powerful tool for targeting dimeric OTR in neurodevelopmental and psychiatric disorders and, in general, provide a framework to untangle specific arrangements of G-protein coupled receptor dimers.


Assuntos
Desenho de Fármacos , Ocitocina/farmacologia , Receptores de Ocitocina/agonistas , Animais , Dimerização , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Ligantes , Camundongos , Modelos Moleculares , Conformação Molecular , Ocitocina/síntese química , Ocitocina/química , Relação Estrutura-Atividade
17.
Glia ; 64(11): 1962-71, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27462832

RESUMO

Previous findings indicate that reducing brain insulin-like growth factor I receptor (IGF-IR) activity promotes ample neuroprotection. We now examined a possible action of IGF-IR on brain glucose transport to explain its wide protective activity, as energy availability is crucial for healthy tissue function. Using (18) FGlucose PET we found that shRNA interference of IGF-IR in mouse somatosensory cortex significantly increased glucose uptake upon sensory stimulation. In vivo microscopy using astrocyte specific staining showed that after IGF-IR shRNA injection in somatosensory cortex, astrocytes displayed greater increases in glucose uptake as compared to astrocytes in the scramble-injected side. Further, mice with the IGF-IR knock down in astrocytes showed increased glucose uptake in somatosensory cortex upon sensory stimulation. Analysis of underlying mechanisms indicated that IGF-IR interacts with glucose transporter 1 (GLUT1), the main facilitative glucose transporter in astrocytes, through a mechanism involving interactions with the scaffolding protein GIPC and the multicargo transporter LRP1 to retain GLUT1 inside the cell. These findings identify IGF-IR as a key modulator of brain glucose metabolism through its inhibitory action on astrocytic GLUT1 activity. GLIA 2016;64:1962-1971.


Assuntos
Astrócitos/metabolismo , Glucose/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/farmacologia , Animais , Animais Recém-Nascidos , Biotinilação , Encéfalo/citologia , Encéfalo/diagnóstico por imagem , Células Cultivadas , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Glucosamina/análogos & derivados , Glucosamina/farmacologia , Transportador de Glucose Tipo 1/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fator de Crescimento Insulin-Like I/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estimulação Física , Transporte Proteico/genética , RNA Mensageiro/metabolismo , Transfecção , Vibrissas/fisiologia
18.
J Biol Chem ; 291(25): 13048-62, 2016 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-27129257

RESUMO

The truncated non-signaling ghrelin receptor growth hormone secretagogue R1b (GHS-R1b) has been suggested to simply exert a dominant negative role in the trafficking and signaling of the full and functional ghrelin receptor GHS-R1a. Here we reveal a more complex modulatory role of GHS-R1b. Differential co-expression of GHS-R1a and GHS-R1b, both in HEK-293T cells and in striatal and hippocampal neurons in culture, demonstrates that GHS-R1b acts as a dual modulator of GHS-R1a function: low relative GHS-R1b expression potentiates and high relative GHS-R1b expression inhibits GHS-R1a function by facilitating GHS-R1a trafficking to the plasma membrane and by exerting a negative allosteric effect on GHS-R1a signaling, respectively. We found a preferential Gi/o coupling of the GHS-R1a-GHS-R1b complex in HEK-293T cells and, unexpectedly, a preferential Gs/olf coupling in both striatal and hippocampal neurons in culture. A dopamine D1 receptor (D1R) antagonist blocked ghrelin-induced cAMP accumulation in striatal but not hippocampal neurons, indicating the involvement of D1R in the striatal GHS-R1a-Gs/olf coupling. Experiments in HEK-293T cells demonstrated that D1R co-expression promotes a switch in GHS-R1a-G protein coupling from Gi/o to Gs/olf, but only upon co-expression of GHS-R1b. Furthermore, resonance energy transfer experiments showed that D1R interacts with GHS-R1a, but only in the presence of GHS-R1b. Therefore, GHS-R1b not only determines the efficacy of ghrelin-induced GHS-R1a-mediated signaling but also determines the ability of GHS-R1a to form oligomeric complexes with other receptors, promoting profound qualitative changes in ghrelin-induced signaling.


Assuntos
Neurônios/metabolismo , Receptores de Grelina/fisiologia , Transdução de Sinais , Adenilil Ciclases/metabolismo , Animais , Membrana Celular/metabolismo , Grelina/fisiologia , Células HEK293 , Hipocampo/citologia , Humanos , Multimerização Proteica , Subunidades Proteicas/fisiologia , Transporte Proteico , Ratos Sprague-Dawley , Receptores de Dopamina D1/metabolismo
19.
BMC Biol ; 14: 26, 2016 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-27048449

RESUMO

BACKGROUND: G-protein-coupled receptors (GPCRs), in the form of monomers or homodimers that bind heterotrimeric G proteins, are fundamental in the transfer of extracellular stimuli to intracellular signaling pathways. Different GPCRs may also interact to form heteromers that are novel signaling units. Despite the exponential growth in the number of solved GPCR crystal structures, the structural properties of heteromers remain unknown. RESULTS: We used single-particle tracking experiments in cells expressing functional adenosine A1-A2A receptors fused to fluorescent proteins to show the loss of Brownian movement of the A1 receptor in the presence of the A2A receptor, and a preponderance of cell surface 2:2 receptor heteromers (dimer of dimers). Using computer modeling, aided by bioluminescence resonance energy transfer assays to monitor receptor homomerization and heteromerization and G-protein coupling, we predict the interacting interfaces and propose a quaternary structure of the GPCR tetramer in complex with two G proteins. CONCLUSIONS: The combination of results points to a molecular architecture formed by a rhombus-shaped heterotetramer, which is bound to two different interacting heterotrimeric G proteins (Gi and Gs). These novel results constitute an important advance in understanding the molecular intricacies involved in GPCR function.


Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Receptores Purinérgicos P1/química , Receptores Purinérgicos P1/metabolismo , Animais , Células HEK293 , Proteínas Heterotriméricas de Ligação ao GTP/química , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína
20.
J Leukoc Biol ; 99(2): 349-59, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26310829

RESUMO

Regulatory T cells have an important role in immune suppression during HIV-1 infection. As regulatory T cells produce the immunomodulatory molecule adenosine, our aim here was to assess the potential of adenosine removal to revert the suppression of anti-HIV responses exerted by regulatory T cells. The experimental setup consisted of ex vivo cocultures of T and dendritic cells, to which adenosine deaminase, an enzyme that hydrolyzes adenosine, was added. In cells from healthy individuals, adenosine hydrolysis decreased CD4(+)CD25(hi) regulatory T cells. Addition of 5'-N-ethylcarboxamidoadenosine, an adenosine receptor agonist, significantly decreased CD4(+)CD25(lo) cells, confirming a modulatory role of adenosine acting via adenosine receptors. In autologous cocultures of T cells with HIV-1-pulsed dendritic cells, addition of adenosine deaminase led to a significant decrease of HIV-1-induced CD4(+)CD25(hi) forkhead box p3(+) cells and to a significant enhancement of the HIV-1-specific CD4(+) responder T cells. An increase in the effector response was confirmed by the enhanced production of CD4(+) and CD8(+) CD25(-)CD45RO(+) memory cell generation and secretion of Th1 cytokines, including IFN-γ and IL-15 and chemokines MIP-1α/CCL3, MIP-1ß/CCL4, and RANTES/CCL5. These ex vivo results show, in a physiologically relevant model, that adenosine deaminase is able to enhance HIV-1 effector responses markedly. The possibility to revert regulatory T cell-mediated inhibition of immune responses by use of adenosine deaminase, an enzyme that hydrolyzes adenosine, merits attention for restoring T lymphocyte function in HIV-1 infection.


Assuntos
Adenosina Desaminase/farmacologia , Células Dendríticas/efeitos dos fármacos , Infecções por HIV/imunologia , HIV-1 , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Adenosina/metabolismo , Adenosina-5'-(N-etilcarboxamida)/farmacologia , Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/análise , Linfócitos T CD8-Positivos/imunologia , Quimiocinas/metabolismo , Técnicas de Cocultura , Feminino , Fatores de Transcrição Forkhead/análise , Infecções por HIV/patologia , Humanos , Memória Imunológica , Ativação Linfocitária/efeitos dos fármacos , Linfocinas/metabolismo , Masculino , Agonistas do Receptor Purinérgico P1/farmacologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/química , Linfócitos T Reguladores/metabolismo , Células Th1/metabolismo
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