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1.
Mol Cell ; 84(20): 3997-4015.e7, 2024 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-39366376

RESUMO

The spatial organization of inositol 1,4,5-trisphosphate (IP3)-evoked Ca2+ signals underlies their versatility. Low stimulus intensities evoke Ca2+ puffs, localized Ca2+ signals arising from a few IP3 receptors (IP3Rs) within a cluster tethered beneath the plasma membrane. More intense stimulation evokes global Ca2+ signals. Ca2+ signals propagate regeneratively as the Ca2+ released stimulates more IP3Rs. How is this potentially explosive mechanism constrained to allow local Ca2+ signaling? We developed methods that allow IP3 produced after G-protein coupled receptor (GPCR) activation to be intercepted and replaced by flash photolysis of a caged analog of IP3. We find that phosphatidylinositol 4,5-bisphosphate (PIP2) primes IP3Rs to respond by partially occupying their IP3-binding sites. As GPCRs stimulate IP3 formation, they also deplete PIP2, relieving the priming stimulus. Loss of PIP2 resets IP3R sensitivity and delays the transition from local to global Ca2+ signals. Dual regulation of IP3Rs by PIP2 and IP3 through GPCRs controls the transition from local to global Ca2+ signals.


Assuntos
Sinalização do Cálcio , Cálcio , Receptores de Inositol 1,4,5-Trifosfato , Inositol 1,4,5-Trifosfato , Fosfatidilinositol 4,5-Difosfato , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Humanos , Cálcio/metabolismo , Animais , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Sítios de Ligação , Células HEK293 , Membrana Celular/metabolismo
2.
Nat Rev Mol Cell Biol ; 19(10): 638-653, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30104700

RESUMO

G protein-coupled receptors (GPCRs) are the largest group of cell surface receptors in humans that signal in response to diverse inputs and regulate a plethora of cellular processes. Hence, they constitute one of the primary drug target classes. Progress in our understanding of GPCR dynamics, activation and signalling has opened new possibilities for selective drug development. A key advancement has been provided by the concept of biased agonism, which describes the ability of ligands acting at the same GPCR to elicit distinct cellular signalling profiles by preferentially stabilizing different active conformational states of the receptor. Application of this concept raises the prospect of 'designer' biased agonists as optimized therapeutics with improved efficacy and/or reduced side-effect profiles. However, this application will require a detailed understanding of the spectrum of drug actions and a structural understanding of the drug-receptor interactions that drive distinct pharmacologies. The recent revolution in GPCR structural biology provides unprecedented insights into ligand binding, conformational dynamics and the control of signalling outcomes. These insights, together with new approaches to multi-dimensional analysis of drug action, are allowing refined classification of drugs according to their pharmacodynamic profiles, which can be linked to receptor structure and predictions of preclinical drug efficacy.


Assuntos
Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Animais , Humanos , Ligantes , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
3.
Nature ; 587(7835): 650-656, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33149304

RESUMO

G-protein-coupled receptors (GPCRs) are membrane proteins that modulate physiology across human tissues in response to extracellular signals. GPCR-mediated signalling can differ because of changes in the sequence1,2 or expression3 of the receptors, leading to signalling bias when comparing diverse physiological systems4. An underexplored source of such bias is the generation of functionally diverse GPCR isoforms with different patterns of expression across different tissues. Here we integrate data from human tissue-level transcriptomes, GPCR sequences and structures, proteomics, single-cell transcriptomics, population-wide genetic association studies and pharmacological experiments. We show how a single GPCR gene can diversify into several isoforms with distinct signalling properties, and how unique isoform combinations expressed in different tissues can generate distinct signalling states. Depending on their structural changes and expression patterns, some of the detected isoforms may influence cellular responses to drugs and represent new targets for developing drugs with improved tissue selectivity. Our findings highlight the need to move from a canonical to a context-specific view of GPCR signalling that considers how combinatorial expression of isoforms in a particular cell type, tissue or organism collectively influences receptor signalling and drug responses.


Assuntos
Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcriptoma , Bases de Dados Factuais , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Terapia de Alvo Molecular , Especificidade de Órgãos/efeitos dos fármacos , Isoformas de Proteínas/genética , Proteômica , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/genética , Análise de Célula Única
5.
Nat Methods ; 17(8): 777-787, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32661425

RESUMO

G-protein-coupled receptors (GPCRs) are involved in numerous physiological processes and are the most frequent targets of approved drugs. The explosion in the number of new three-dimensional (3D) molecular structures of GPCRs (3D-GPCRome) over the last decade has greatly advanced the mechanistic understanding and drug design opportunities for this protein family. Molecular dynamics (MD) simulations have become a widely established technique for exploring the conformational landscape of proteins at an atomic level. However, the analysis and visualization of MD simulations require efficient storage resources and specialized software. Here we present GPCRmd (http://gpcrmd.org/), an online platform that incorporates web-based visualization capabilities as well as a comprehensive and user-friendly analysis toolbox that allows scientists from different disciplines to visualize, analyze and share GPCR MD data. GPCRmd originates from a community-driven effort to create an open, interactive and standardized database of GPCR MD simulations.


Assuntos
Simulação de Dinâmica Molecular , Receptores Acoplados a Proteínas G/química , Software , Metaboloma , Modelos Moleculares , Conformação Proteica
6.
Biochem Soc Trans ; 51(1): 13-20, 2023 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-36688421

RESUMO

G protein-coupled receptor (GPCR) family members can sense an extraordinary variety of biomolecules to activate intracellular signalling cascades that modulate key aspects of cell physiology. Apart from their crucial role in maintaining cell homeostasis, these critical sensory and modulatory properties have made GPCRs the most successful drug target class to date. However, establishing direct links between receptor activation of specific intracellular partners and individual physiological outcomes is still an ongoing challenge. By studying this receptor signalling complexity at increasing resolution through the development of novel biosensors and high-throughput techniques, a growing number of studies are revealing how receptor function can be diversified in a spatial, temporal or cell-specific manner. This mini-review will introduce recent examples of this context-dependent receptor signalling and discuss how it can impact our understanding of receptor function in health and disease, and contribute to the search of more selective, efficacious and safer GPCR drug candidates.


Assuntos
Receptores Acoplados a Proteínas G , Transdução de Sinais , Receptores Acoplados a Proteínas G/fisiologia , Membrana Celular , Sistemas de Liberação de Medicamentos , Ligantes
8.
Molecules ; 26(3)2021 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-33530327

RESUMO

While selective inhibition is one of the key assets for a small molecule drug, many diseases can only be tackled by simultaneous inhibition of several proteins. An example where achieving selectivity is especially challenging are ligands targeting human kinases. This difficulty arises from the high structural conservation of the kinase ATP binding sites, the area targeted by most inhibitors. We investigated the possibility to identify novel small molecule ligands with pre-defined binding profiles for a series of kinase targets and anti-targets by in silico docking. The candidate ligands originating from these calculations were assayed to determine their experimental binding profiles. Compared to previous studies, the acquired hit rates were low in this specific setup, which aimed at not only selecting multi-target kinase ligands, but also designing out binding to anti-targets. Specifically, only a single profiled substance could be verified as a sub-micromolar, dual-specific EGFR/ErbB2 ligand that indeed avoided its selected anti-target BRAF. We subsequently re-analyzed our target choice and in silico strategy based on these findings, with a particular emphasis on the hit rates that can be expected from a given target combination. To that end, we supplemented the structure-based docking calculations with bioinformatic considerations of binding pocket sequence and structure similarity as well as ligand-centric comparisons of kinases. Taken together, our results provide a multi-faceted picture of how pocket space can determine the success of docking in multi-target drug discovery efforts.


Assuntos
Simulação de Acoplamento Molecular/métodos , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Simulação por Computador , Descoberta de Drogas , Receptores ErbB/química , Receptores ErbB/metabolismo , Humanos , Ligantes , Modelos Moleculares , Conformação Molecular , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas B-raf/metabolismo , Relação Estrutura-Atividade
9.
Mol Pharmacol ; 87(4): 740-6, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25661038

RESUMO

Detection of biased agonists for the serotonin 5-HT2A receptor can guide the discovery of safer and more efficient antipsychotic drugs. However, the rational design of such drugs has been hampered by the difficulty detecting the impact of small structural changes on signaling bias. To overcome these difficulties, we characterized the dynamics of ligand-receptor interactions of known biased and balanced agonists using molecular dynamics simulations. Our analysis revealed that interactions with residues S5.46 and N6.55 discriminate compounds with different functional selectivity. Based on our computational predictions, we selected three derivatives of the natural balanced ligand serotonin and experimentally validated their ability to act as biased agonists. Remarkably, our approach yielded compounds promoting an unprecedented level of signaling bias at the 5-HT2A receptor, which could help interrogate the importance of particular pathways in conditions like schizophrenia.


Assuntos
Antipsicóticos/química , Simulação de Dinâmica Molecular , Receptor 5-HT2A de Serotonina/química , Agonistas do Receptor 5-HT2 de Serotonina/química , Animais , Antipsicóticos/farmacologia , Ligação Competitiva , Células CHO , Cricetulus , Desenho de Fármacos , Humanos , Simulação de Acoplamento Molecular , Receptor 5-HT2A de Serotonina/metabolismo , Serotonina/análogos & derivados , Serotonina/química , Serotonina/farmacologia , Agonistas do Receptor 5-HT2 de Serotonina/farmacologia
10.
Science ; 382(6677): eadh1859, 2023 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-38127743

RESUMO

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) bind to extracellular ligands and drugs and modulate intracellular responses through conformational changes. Despite their importance as drug targets, the molecular origins of pharmacological properties such as efficacy (maximum signaling response) and potency (the ligand concentration at half-maximal response) remain poorly understood for any ligand-receptor-signaling system. We used the prototypical adrenaline-ß2 adrenergic receptor-G protein system to reveal how specific receptor residues decode and translate the information encoded in a ligand to mediate a signaling response. We present a data science framework to integrate pharmacological and structural data to uncover structural changes and allosteric networks relevant for ligand pharmacology. These methods can be tailored to study any ligand-receptor-signaling system, and the principles open possibilities for designing orthosteric and allosteric compounds with defined signaling properties.


Assuntos
Agonistas de Receptores Adrenérgicos beta 2 , Receptores Adrenérgicos beta 2 , Humanos , Agonistas de Receptores Adrenérgicos beta 2/química , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Regulação Alostérica , Técnicas Biossensoriais , Ligantes , Conformação Proteica , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/genética , Transdução de Sinais , Técnicas de Transferência de Energia por Ressonância de Bioluminescência
11.
Am J Physiol Lung Cell Mol Physiol ; 302(8): L736-45, 2012 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-22287614

RESUMO

Sphingosine-1-phosphate (S1P) is an immunomodulatory lipid mediator that plays an important role in lymphocyte trafficking. Elevated levels of S1P are found in bronchoalveolar lavage (BAL) fluid of patients with asthma; however, its role in disease is not known. FTY720, a synthetic analog of S1P, has been shown to abrogate allergic inflammation and airway hyperresponsiveness following acute allergen challenge. However, its effects on asthmatic airway remodeling induced by repeated allergen exposure are unknown. Ovalbumin (OVA)-sensitized rats were challenged on days 14, 19, and 24 after sensitization. FTY720 or vehicle (PBS) therapy was administered 1 h prior to each challenge. BAL fluid and quantitative histological analysis were performed 48 h after the last challenge. FTY720 inhibited OVA-induced features of airway remodeling including increased airway smooth muscle mass and bronchial neovascularization, without affecting lymphocyte numbers in secondary lymphoid organs. Furthermore, CD3+ cells adjacent to airway smooth muscle bundles were increased in OVA-challenged rats but the increase was inhibited by FTY720. There was an expansion of bronchus-associated lymphoid tissue following FTY720 treatment of OVA-challenged animals. Real-time quantitative PCR revealed that Th2-associated transcription factors were inhibited following FTY720 therapy. Airway remodeling is a cardinal feature of severe asthma. These results demonstrate that allergen-driven airway remodeling can be inhibited by FTY720, offering potential new therapies for the treatment of severe asthma.


Assuntos
Remodelação das Vias Aéreas/efeitos dos fármacos , Alérgenos/efeitos adversos , Imunossupressores/uso terapêutico , Lisofosfolipídeos/agonistas , Lisofosfolipídeos/uso terapêutico , Propilenoglicóis/uso terapêutico , Esfingosina/análogos & derivados , Animais , Antiasmáticos/uso terapêutico , Asma/tratamento farmacológico , Brônquios/irrigação sanguínea , Brônquios/efeitos dos fármacos , Brônquios/patologia , Líquido da Lavagem Broncoalveolar , Complexo CD3/análise , Cloridrato de Fingolimode , Masculino , Músculo Liso/efeitos dos fármacos , Ratos , Índice de Gravidade de Doença , Esfingosina/agonistas , Esfingosina/uso terapêutico , Resultado do Tratamento
12.
Cell Rep ; 34(12): 108862, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33761344

RESUMO

The Melanocortin-4 Receptor (MC4R) plays a pivotal role in energy homeostasis. We used human MC4R mutations associated with an increased or decreased risk of obesity to dissect mechanisms that regulate MC4R function. Most obesity-associated mutations impair trafficking to the plasma membrane (PM), whereas obesity-protecting mutations either accelerate recycling to the PM or decrease internalization, resulting in enhanced signaling. MC4R mutations that do not affect canonical Gαs protein-mediated signaling, previously considered to be non-pathogenic, nonetheless disrupt agonist-induced internalization, ß-arrestin recruitment, and/or coupling to Gαs, establishing their causal role in severe obesity. Structural mapping reveals ligand-accessible sites by which MC4R couples to effectors and residues involved in the homodimerization of MC4R, which is disrupted by multiple obesity-associated mutations. Human genetic studies reveal that endocytosis, intracellular trafficking, and homodimerization regulate MC4R function to a level that is physiologically relevant, supporting the development of chaperones, agonists, and allosteric modulators of MC4R for weight loss therapy.


Assuntos
Peso Corporal/genética , Endocitose , Variação Genética , Multimerização Proteica , Receptor Tipo 4 de Melanocortina/genética , Animais , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP , Células HEK293 , Humanos , Modelos Biológicos , Proteínas Mutantes/metabolismo , Mutação/genética , Fosforilação , Receptor Tipo 4 de Melanocortina/química , Transdução de Sinais , beta-Arrestinas/metabolismo
13.
FEBS J ; 287(4): 736-748, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31386272

RESUMO

The δ-opioid receptor (δ-OR) couples to Gi/Go proteins to modulate a variety of responses in the nervous system. Τhe regulator of G protein signalling 4 (RGS4) was previously shown to directly interact within the C-terminal region of δ-OR using its N-terminal domain to negatively modulate opioid receptor signalling. Herein, using molecular dynamics simulations and in vitro pull-down experiments we delimit this interaction to 12 helix 8 residues of δ-ΟR and to the first 17 N-terminal residues (NT) of RGS4. Monitoring the complex arrangement and stabilization between RGS4 and δ-OR by molecular dynamics simulations combined with mutagenesis studies, we defined that two critical interactions are formed: one between Phe329 of helix8 of δ-ΟR and Pro9 of the NT of RGS4 and the other a salt bridge between Glu323 of δ-ΟR and Lys17 of RGS4. Our observations allow drafting for the first time a structural model of a ternary complex including the δ-opioid receptor, a G protein and a RGS protein. Furthermore, the high degree of conservation among opioid receptors of the RGS4-binding region, points to a conserved interaction mode between opioid receptors and this important regulatory protein.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Proteínas RGS/química , Receptores Opioides delta/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Clonagem Molecular , Sequência Conservada , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas RGS/genética , Proteínas RGS/metabolismo , Receptores Opioides delta/genética , Receptores Opioides delta/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
14.
Neuron ; 108(6): 1146-1162.e10, 2020 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-33086039

RESUMO

Astrocytes tile the central nervous system and are widely implicated in brain diseases, but the molecular mechanisms by which astrocytes contribute to brain disorders remain incompletely explored. By performing astrocyte gene expression analyses following 14 experimental perturbations of relevance to the striatum, we discovered that striatal astrocytes mount context-specific molecular responses at the level of genes, pathways, and upstream regulators. Through data mining, we also identified astrocyte pathways in Huntington's disease (HD) that were reciprocally altered with respect to the activation of striatal astrocyte G protein-coupled receptor (GPCR) signaling. Furthermore, selective striatal astrocyte stimulation of the Gi-GPCR pathway in vivo corrected several HD-associated astrocytic, synaptic, and behavioral phenotypes, with accompanying improvement of HD-associated astrocyte signaling pathways, including those related to synaptogenesis and neuroimmune functions. Overall, our data show that astrocytes are malleable, using context-specific responses that can be dissected molecularly and used for phenotypic benefit in brain disorders.


Assuntos
Astrócitos/metabolismo , Corpo Estriado/metabolismo , Doença de Huntington/metabolismo , Animais , Mineração de Dados , Humanos , Doença de Huntington/genética , Camundongos , Neurônios/metabolismo , Transdução de Sinais/fisiologia , Sinapses/metabolismo
15.
Nat Commun ; 10(1): 667, 2019 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-30737406

RESUMO

Class F receptors are considered valuable therapeutic targets due to their role in human disease, but structural changes accompanying receptor activation remain unexplored. Employing population and cancer genomics data, structural analyses, molecular dynamics simulations, resonance energy transfer-based approaches and mutagenesis, we identify a conserved basic amino acid in TM6 in Class F receptors that acts as a molecular switch to mediate receptor activation. Across all tested Class F receptors (FZD4,5,6,7, SMO), mutation of the molecular switch confers an increased potency of agonists by stabilizing an active conformation as assessed by engineered mini G proteins as conformational sensors. Disruption of the switch abrogates the functional interaction between FZDs and the phosphoprotein Dishevelled, supporting conformational selection as a prerequisite for functional selectivity. Our studies reveal the molecular basis of a common activation mechanism conserved in all Class F receptors, which facilitates assay development and future discovery of Class F receptor-targeting drugs.


Assuntos
Modelos Teóricos , Simulação de Dinâmica Molecular , Receptores Acoplados a Proteínas G/metabolismo , Citometria de Fluxo , Células HEK293 , Humanos , Immunoblotting , Proteína Quinase 1 Ativada por Mitógeno , Proteína Quinase 3 Ativada por Mitógeno , Modelos Moleculares , Filogenia , Ligação Proteica , Conformação Proteica , Receptores Acoplados a Proteínas G/genética
17.
Nat Biotechnol ; 37(2): 169-178, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30607034

RESUMO

Existing high-throughput methods to identify RNA-binding proteins (RBPs) are based on capture of polyadenylated RNAs and cannot recover proteins that interact with nonadenylated RNAs, including long noncoding RNA, pre-mRNAs and bacterial RNAs. We present orthogonal organic phase separation (OOPS), which does not require molecular tagging or capture of polyadenylated RNA, and apply it to recover cross-linked protein-RNA and free protein, or protein-bound RNA and free RNA, in an unbiased way. We validated OOPS in HEK293, U2OS and MCF10A human cell lines, and show that 96% of proteins recovered were bound to RNA. We show that all long RNAs can be cross-linked to proteins, and recovered 1,838 RBPs, including 926 putative novel RBPs. OOPS is approximately 100-fold more efficient than existing methods and can enable analyses of dynamic RNA-protein interactions. We also characterize dynamic changes in RNA-protein interactions in mammalian cells following nocodazole arrest, and present a bacterial RNA-interactome for Escherichia coli. OOPS is compatible with downstream proteomics and RNA sequencing, and can be applied in any organism.


Assuntos
RNA Mensageiro/química , Proteínas de Ligação a RNA/isolamento & purificação , RNA/isolamento & purificação , Linhagem Celular Tumoral , Análise por Conglomerados , Reagentes de Ligações Cruzadas/química , Escherichia coli , Glicoproteínas/química , Células HEK293 , Humanos , Nocodazol/química , Ligação Proteica , Proteoma , Proteômica , RNA/química , RNA Bacteriano/química , RNA Longo não Codificante/química , Proteínas de Ligação a RNA/química , Análise de Sequência de RNA , Timidina/química , Transcriptoma
18.
Cell Signal ; 42: 114-126, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29056499

RESUMO

Muscarinic receptors (M-Rs) for acetylcholine (ACh) belong to the class A of G protein-coupled receptors. M-Rs are activated by orthosteric agonists that bind to a specific site buried in the M-R transmembrane helix bundle. In the active conformation, receptor function can be modulated either by allosteric modulators, which bind to the extracellular receptor surface or by the membrane potential via an unknown mechanism. Here, we compared the modulation of M1-Rs and M3-Rs induced by changes in voltage to their allosteric modulation by chemical compounds. We quantified changes in receptor signaling in single HEK 293 cells with a FRET biosensor for the Gq protein cycle. In the presence of ACh, M1-R signaling was potentiated by voltage, similarly to positive allosteric modulation by benzyl quinolone carboxylic acid. Conversely, signaling of M3-R was attenuated by voltage or the negative allosteric modulator gallamine. Because the orthosteric site is highly conserved among M-Rs, but allosteric sites vary, we constructed "allosteric site" M3/M1-R chimeras and analyzed their voltage dependencies. Exchanging the entire allosteric sites eliminated the voltage sensitivity of ACh responses for both receptors, but did not affect their modulation by allosteric compounds. Furthermore, a point mutation in M3-Rs caused functional uncoupling of the allosteric and orthosteric sites and abolished voltage dependence. Molecular dynamics simulations of the receptor variants indicated a subtype-specific crosstalk between both sites, involving the conserved tyrosine lid structure of the orthosteric site. This molecular crosstalk leads to receptor subtype-specific voltage effects.


Assuntos
Acetilcolina/química , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Trietiodeto de Galamina/química , Hidroxiquinolinas/química , Receptores Muscarínicos/química , Proteínas Recombinantes de Fusão/química , Acetilcolina/farmacologia , Sítio Alostérico , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Técnicas Biossensoriais , Quimera/genética , Quimera/metabolismo , Transferência Ressonante de Energia de Fluorescência , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Trietiodeto de Galamina/farmacologia , Expressão Gênica , Células HEK293 , Humanos , Hidroxiquinolinas/farmacologia , Cinética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Simulação de Dinâmica Molecular , Técnicas de Patch-Clamp , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Receptores Muscarínicos/genética , Receptores Muscarínicos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
19.
Nat Commun ; 8: 14505, 2017 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-28220900

RESUMO

Cholesterol is a key component of cell membranes with a proven modulatory role on the function and ligand-binding properties of G-protein-coupled receptors (GPCRs). Crystal structures of prototypical GPCRs such as the adenosine A2A receptor (A2AR) have confirmed that cholesterol finds stable binding sites at the receptor surface suggesting an allosteric role of this lipid. Here we combine experimental and computational approaches to show that cholesterol can spontaneously enter the A2AR-binding pocket from the membrane milieu using the same portal gate previously suggested for opsin ligands. We confirm the presence of cholesterol inside the receptor by chemical modification of the A2AR interior in a biotinylation assay. Overall, we show that cholesterol's impact on A2AR-binding affinity goes beyond pure allosteric modulation and unveils a new interaction mode between cholesterol and the A2AR that could potentially apply to other GPCRs.


Assuntos
Membrana Celular/química , Colesterol/química , Domínios Proteicos , Receptores Acoplados a Proteínas G/química , Animais , Sítios de Ligação , Ligação Competitiva , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Colesterol/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Ratos , Receptor A2A de Adenosina/química , Receptor A2A de Adenosina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo
20.
Eur J Pharmacol ; 815: 138-146, 2017 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-28899696

RESUMO

The serotonin 2A (5-HT2A) receptor is a G-protein coupled receptor (GPCR) with a conserved disulfide bridge formed by Cys148 (transmembrane helix 3, TM3) and Cys227 (extracellular loop 2, ECL-2). We hypothesized that disulfide bridges may determine serotonin 5-HT2A receptor functions such as receptor activation, functional selectivity and ligand recognition. We used the reducing agent dithiothreitol (DTT) to determine how the reduction of disulfide bridges affects radioligand binding, second messenger mobilization and receptor dimerization. A DTT-induced decrease in the number of binding sites (1190 ± 63.55 fmol/mg protein for control cells compared with 921.2 ± 60.84 fmol/mg protein for DTT-treated cells) as well as in the efficacy of both signalling pathways characterized was observed, although the affinity and potency were unchanged. Bioluminiscence resonance energy transfer (BRET) assays revealed the DTT treatment did not modify the homodimeric nature of serotonin 5-HT2A receptors. In molecular dynamic simulations, the ECL-2 of the receptor with a broken cysteine bond adopts a wider variety of conformations, some of which protrude deeper into the receptor orthosteric binding pocket leading to collapse of the pocket. A shrunken binding pocket would be incapable of accommodating lysergic acid diethylamide (LSD). Our findings suggest that the decrease of efficacy may be due to disruption of disulfide bridge between TM3 and ECL-2. This reveals the integrity of the ECL-2 epitope, which should be explored in the development of novel ligands acting as allosteric modulators of serotonin 5-HT2A receptors.


Assuntos
Dissulfetos/química , Multimerização Proteica , Receptor 5-HT2A de Serotonina/química , Receptor 5-HT2A de Serotonina/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Cricetinae , Cricetulus , Ditiotreitol/farmacologia , Humanos , Ligantes , Modelos Moleculares , Proteínas Nucleares/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína
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