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1.
Cell ; 184(4): 931-942.e18, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33571431

RESUMO

The D1- and D2-dopamine receptors (D1R and D2R), which signal through Gs and Gi, respectively, represent the principal stimulatory and inhibitory dopamine receptors in the central nervous system. D1R and D2R also represent the main therapeutic targets for Parkinson's disease, schizophrenia, and many other neuropsychiatric disorders, and insight into their signaling is essential for understanding both therapeutic and side effects of dopaminergic drugs. Here, we report four cryoelectron microscopy (cryo-EM) structures of D1R-Gs and D2R-Gi signaling complexes with selective and non-selective dopamine agonists, including two currently used anti-Parkinson's disease drugs, apomorphine and bromocriptine. These structures, together with mutagenesis studies, reveal the conserved binding mode of dopamine agonists, the unique pocket topology underlying ligand selectivity, the conformational changes in receptor activation, and potential structural determinants for G protein-coupling selectivity. These results provide both a molecular understanding of dopamine signaling and multiple structural templates for drug design targeting the dopaminergic system.


Assuntos
Receptores de Dopamina D1/química , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/química , Receptores de Dopamina D2/metabolismo , Transdução de Sinais , 2,3,4,5-Tetra-Hidro-7,8-Di-Hidroxi-1-Fenil-1H-3-Benzazepina/análogos & derivados , 2,3,4,5-Tetra-Hidro-7,8-Di-Hidroxi-1-Fenil-1H-3-Benzazepina/farmacologia , Sequência de Aminoácidos , Sequência Conservada , Microscopia Crioeletrônica , AMP Cíclico/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Ligantes , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Receptores de Dopamina D1/ultraestrutura , Receptores de Dopamina D2/ultraestrutura , Homologia Estrutural de Proteína
2.
Cell ; 180(1): 64-78.e16, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31923400

RESUMO

Enteric-associated neurons (EANs) are closely associated with immune cells and continuously monitor and modulate homeostatic intestinal functions, including motility and nutrient sensing. Bidirectional interactions between neuronal and immune cells are altered during disease processes such as neurodegeneration or irritable bowel syndrome. We investigated the effects of infection-induced inflammation on intrinsic EANs (iEANs) and the role of intestinal muscularis macrophages (MMs) in this context. Using murine models of enteric infections, we observed long-term gastrointestinal symptoms, including reduced motility and loss of excitatory iEANs, which was mediated by a Nlrp6- and Casp11-dependent mechanism, depended on infection history, and could be reversed by manipulation of the microbiota. MMs responded to luminal infection by upregulating a neuroprotective program via ß2-adrenergic receptor (ß2-AR) signaling and mediated neuronal protection through an arginase 1-polyamine axis. Our results identify a mechanism of neuronal death post-infection and point to a role for tissue-resident MMs in limiting neuronal damage.


Assuntos
Mucosa Intestinal/imunologia , Macrófagos/imunologia , Receptores Adrenérgicos beta 2/metabolismo , Adrenérgicos , Animais , Arginase/metabolismo , Caspases Iniciadoras/imunologia , Caspases Iniciadoras/metabolismo , Sistema Nervoso Entérico/imunologia , Sistema Nervoso Entérico/metabolismo , Feminino , Gastroenteropatias , Microbioma Gastrointestinal , Infecções , Inflamação/imunologia , Mucosa Intestinal/metabolismo , Intestino Delgado/imunologia , Intestinos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microbiota , Neurônios/fisiologia , Receptores Adrenérgicos beta 2/imunologia , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Transdução de Sinais
3.
Cell ; 182(3): 578-593.e19, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32679029

RESUMO

Piloerection (goosebumps) requires concerted actions of the hair follicle, the arrector pili muscle (APM), and the sympathetic nerve, providing a model to study interactions across epithelium, mesenchyme, and nerves. Here, we show that APMs and sympathetic nerves form a dual-component niche to modulate hair follicle stem cell (HFSC) activity. Sympathetic nerves form synapse-like structures with HFSCs and regulate HFSCs through norepinephrine, whereas APMs maintain sympathetic innervation to HFSCs. Without norepinephrine signaling, HFSCs enter deep quiescence by down-regulating the cell cycle and metabolism while up-regulating quiescence regulators Foxp1 and Fgf18. During development, HFSC progeny secretes Sonic Hedgehog (SHH) to direct the formation of this APM-sympathetic nerve niche, which in turn controls hair follicle regeneration in adults. Our results reveal a reciprocal interdependence between a regenerative tissue and its niche at different stages and demonstrate sympathetic nerves can modulate stem cells through synapse-like connections and neurotransmitters to couple tissue production with demands.


Assuntos
Nervo Acessório/fisiologia , Folículo Piloso/citologia , Cabelo/crescimento & desenvolvimento , Proteínas Hedgehog/metabolismo , Norepinefrina/metabolismo , Transdução de Sinais/genética , Células-Tronco/metabolismo , Células-Tronco/fisiologia , Nervo Acessório/citologia , Animais , Ciclo Celular/genética , Temperatura Baixa , Feminino , Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica , Cabelo/citologia , Cabelo/fisiologia , Folículo Piloso/crescimento & desenvolvimento , Folículo Piloso/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Piloereção , RNA-Seq , Receptores Adrenérgicos beta 2/deficiência , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Nicho de Células-Tronco , Células-Tronco/citologia , Sistema Nervoso Simpático/citologia , Sistema Nervoso Simpático/fisiologia , Sinapses/fisiologia
4.
Cell ; 169(3): 407-421.e16, 2017 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-28431242

RESUMO

The phosphorylation of agonist-occupied G-protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) functions to turn off G-protein signaling and turn on arrestin-mediated signaling. While a structural understanding of GPCR/G-protein and GPCR/arrestin complexes has emerged in recent years, the molecular architecture of a GPCR/GRK complex remains poorly defined. We used a comprehensive integrated approach of cross-linking, hydrogen-deuterium exchange mass spectrometry (MS), electron microscopy, mutagenesis, molecular dynamics simulations, and computational docking to analyze GRK5 interaction with the ß2-adrenergic receptor (ß2AR). These studies revealed a dynamic mechanism of complex formation that involves large conformational changes in the GRK5 RH/catalytic domain interface upon receptor binding. These changes facilitate contacts between intracellular loops 2 and 3 and the C terminus of the ß2AR with the GRK5 RH bundle subdomain, membrane-binding surface, and kinase catalytic cleft, respectively. These studies significantly contribute to our understanding of the mechanism by which GRKs regulate the function of activated GPCRs. PAPERCLIP.


Assuntos
Quinase 5 de Receptor Acoplado a Proteína G/química , Mamíferos/metabolismo , Receptores Adrenérgicos beta 2/química , Animais , Camelídeos Americanos , Bovinos , Quinase 5 de Receptor Acoplado a Proteína G/genética , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Humanos , Espectrometria de Massas , Microscopia Eletrônica , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Ratos , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo
5.
Cell ; 164(3): 378-91, 2016 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-26777404

RESUMO

Proper adaptation to environmental perturbations is essential for tissue homeostasis. In the intestine, diverse environmental cues can be sensed by immune cells, which must balance resistance to microorganisms with tolerance, avoiding excess tissue damage. By applying imaging and transcriptional profiling tools, we interrogated how distinct microenvironments in the gut regulate resident macrophages. We discovered that macrophages exhibit a high degree of gene-expression specialization dependent on their proximity to the gut lumen. Lamina propria macrophages (LpMs) preferentially expressed a pro-inflammatory phenotype when compared to muscularis macrophages (MMs), which displayed a tissue-protective phenotype. Upon luminal bacterial infection, MMs further enhanced tissue-protective programs, and this was attributed to swift activation of extrinsic sympathetic neurons innervating the gut muscularis and norepinephrine signaling to ß2 adrenergic receptors on MMs. Our results reveal unique intra-tissue macrophage specialization and identify neuro-immune communication between enteric neurons and macrophages that induces rapid tissue-protective responses to distal perturbations.


Assuntos
Intestino Delgado/fisiologia , Macrófagos/imunologia , Neurônios/citologia , Animais , Linhagem Celular , Mucosa Intestinal/citologia , Mucosa Intestinal/fisiologia , Intestino Delgado/citologia , Intestino Delgado/imunologia , Macrófagos/citologia , Camundongos , Mucosa/citologia , Mucosa/fisiologia , Neuroimunomodulação , Neurônios/fisiologia , Receptores Adrenérgicos beta 2/metabolismo , Infecções por Salmonella/imunologia , Salmonella typhimurium/fisiologia , Organismos Livres de Patógenos Específicos
6.
Cell ; 161(5): 1101-1111, 2015 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-25981665

RESUMO

G-protein-coupled receptors (GPCRs) transduce signals from the extracellular environment to intracellular proteins. To gain structural insight into the regulation of receptor cytoplasmic conformations by extracellular ligands during signaling, we examine the structural dynamics of the cytoplasmic domain of the ß2-adrenergic receptor (ß2AR) using (19)F-fluorine NMR and double electron-electron resonance spectroscopy. These studies show that unliganded and inverse-agonist-bound ß2AR exists predominantly in two inactive conformations that exchange within hundreds of microseconds. Although agonists shift the equilibrium toward a conformation capable of engaging cytoplasmic G proteins, they do so incompletely, resulting in increased conformational heterogeneity and the coexistence of inactive, intermediate, and active states. Complete transition to the active conformation requires subsequent interaction with a G protein or an intracellular G protein mimetic. These studies demonstrate a loose allosteric coupling of the agonist-binding site and G-protein-coupling interface that may generally be responsible for the complex signaling behavior observed for many GPCRs.


Assuntos
Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais , Agonistas Adrenérgicos beta/farmacologia , Sequência de Aminoácidos , Benzoxazinas/farmacologia , Humanos , Isoproterenol/metabolismo , Isoproterenol/farmacologia , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Receptores Adrenérgicos beta 2/química
7.
Nature ; 629(8014): 1182-1191, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38480881

RESUMO

G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating guanine nucleotide exchange in the Gα subunit1. To visualize this mechanism, we developed a time-resolved cryo-EM approach that examines the progression of ensembles of pre-steady-state intermediates of a GPCR-G-protein complex. By monitoring the transitions of the stimulatory Gs protein in complex with the ß2-adrenergic receptor at short sequential time points after GTP addition, we identified the conformational trajectory underlying G-protein activation and functional dissociation from the receptor. Twenty structures generated from sequential overlapping particle subsets along this trajectory, compared to control structures, provide a high-resolution description of the order of main events driving G-protein activation in response to GTP binding. Structural changes propagate from the nucleotide-binding pocket and extend through the GTPase domain, enacting alterations to Gα switch regions and the α5 helix that weaken the G-protein-receptor interface. Molecular dynamics simulations with late structures in the cryo-EM trajectory support that enhanced ordering of GTP on closure of the α-helical domain against the nucleotide-bound Ras-homology domain correlates with α5 helix destabilization and eventual dissociation of the G protein from the GPCR. These findings also highlight the potential of time-resolved cryo-EM as a tool for mechanistic dissection of GPCR signalling events.


Assuntos
Microscopia Crioeletrônica , Subunidades alfa Gs de Proteínas de Ligação ao GTP , Receptores Adrenérgicos beta 2 , Humanos , Sítios de Ligação , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/efeitos dos fármacos , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/ultraestrutura , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacologia , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Receptores Adrenérgicos beta 2/metabolismo , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/ultraestrutura , Fatores de Tempo , Ativação Enzimática/efeitos dos fármacos , Domínios Proteicos , Estrutura Secundária de Proteína , Transdução de Sinais/efeitos dos fármacos
8.
Nature ; 615(7953): 734-741, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36890236

RESUMO

The third intracellular loop (ICL3) of the G protein-coupled receptor (GPCR) fold is important for the signal transduction process downstream of receptor activation1-3. Despite this, the lack of a defined structure of ICL3, combined with its high sequence divergence among GPCRs, complicates characterization of its involvement in receptor signalling4. Previous studies focusing on the ß2 adrenergic receptor (ß2AR) suggest that ICL3 is involved in the structural process of receptor activation and signalling5-7. Here we derive mechanistic insights into the role of ICL3 in ß2AR signalling, observing that ICL3 autoregulates receptor activity through a dynamic conformational equilibrium between states that block or expose the receptor's G protein-binding site. We demonstrate the importance of this equilibrium for receptor pharmacology, showing that G protein-mimetic effectors bias the exposed states of ICL3 to allosterically activate the receptor. Our findings additionally reveal that ICL3 tunes signalling specificity by inhibiting receptor coupling to G protein subtypes that weakly couple to the receptor. Despite the sequence diversity of ICL3, we demonstrate that this negative G protein-selection mechanism through ICL3 extends to GPCRs across the superfamily, expanding the range of known mechanisms by which receptors mediate G protein subtype selective signalling. Furthermore, our collective findings suggest ICL3 as an allosteric site for receptor- and signalling pathway-specific ligands.


Assuntos
Receptores Adrenérgicos beta 2 , Transdução de Sinais , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Ligantes , Sítio Alostérico , Conformação Proteica
9.
Cell ; 152(3): 532-42, 2013 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-23374348

RESUMO

G-protein-coupled receptors (GPCRs) can modulate diverse signaling pathways, often in a ligand-specific manner. The full range of functionally relevant GPCR conformations is poorly understood. Here, we use NMR spectroscopy to characterize the conformational dynamics of the transmembrane core of the ß(2)-adrenergic receptor (ß(2)AR), a prototypical GPCR. We labeled ß(2)AR with (13)CH(3)ε-methionine and obtained HSQC spectra of unliganded receptor as well as receptor bound to an inverse agonist, an agonist, and a G-protein-mimetic nanobody. These studies provide evidence for conformational states not observed in crystal structures, as well as substantial conformational heterogeneity in agonist- and inverse-agonist-bound preparations. They also show that for ß(2)AR, unlike rhodopsin, an agonist alone does not stabilize a fully active conformation, suggesting that the conformational link between the agonist-binding pocket and the G-protein-coupling surface is not rigid. The observed heterogeneity may be important for ß(2)AR's ability to engage multiple signaling and regulatory proteins.


Assuntos
Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/metabolismo , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Conformação Proteica , Transdução de Sinais , Termodinâmica
10.
Cell ; 154(6): 1314-25, 2013 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-24034253

RESUMO

G-protein-coupled receptors (GPCRs) are known to possess two different conformations, active and inactive, and they spontaneously alternate between the two in the absence of ligands. Here, we analyzed the agonist-independent GPCR activity for its possible role in receptor-instructed axonal projection. We generated transgenic mice expressing activity mutants of the ß2-adrenergic receptor, a well-characterized GPCR with the highest homology to odorant receptors (ORs). We found that mutants with altered agonist-independent activity changed the transcription levels of axon-targeting molecules--e.g., Neuropilin-1 and Plexin-A1--but not of glomerular segregation molecules--e.g., Kirrel2 and Kirrel3--thus causing shifts in glomerular locations along the anterior-posterior (A-P) axis. Knockout and in vitro experiments demonstrated that Gs, but not Golf, is responsible for mediating the agonist-independent GPCR activity. We conclude that the equilibrium of conformational transitions set by each OR is the major determinant of expression levels of A-P-targeting molecules.


Assuntos
Axônios/metabolismo , Condutos Olfatórios/embriologia , Receptores Odorantes/metabolismo , Células Receptoras Sensoriais/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/metabolismo , Animais , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Condutos Olfatórios/citologia , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Receptores Odorantes/genética
11.
Nature ; 611(7934): 173-179, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36289326

RESUMO

G-protein-coupled receptors (GPCRs), the largest family of signalling receptors, as well as important drug targets, are known to activate extracellular-signal-regulated kinase (ERK)-a master regulator of cell proliferation and survival1. However, the precise mechanisms that underlie GPCR-mediated ERK activation are not clearly understood2-4. Here we investigated how spatially organized ß2-adrenergic receptor (ß2AR) signalling controls ERK. Using subcellularly targeted ERK activity biosensors5, we show that ß2AR signalling induces ERK activity at endosomes, but not at the plasma membrane. This pool of ERK activity depends on active, endosome-localized Gαs and requires ligand-stimulated ß2AR endocytosis. We further identify an endosomally localized non-canonical signalling axis comprising Gαs, RAF and mitogen-activated protein kinase kinase, resulting in endosomal ERK activity that propagates into the nucleus. Selective inhibition of endosomal ß2AR and Gαs signalling blunted nuclear ERK activity, MYC gene expression and cell proliferation. These results reveal a non-canonical mechanism for the spatial regulation of ERK through GPCR signalling and identify a functionally important endosomal signalling axis.


Assuntos
Adrenérgicos , Endossomos , MAP Quinases Reguladas por Sinal Extracelular , Receptores Adrenérgicos beta 2 , Adrenérgicos/metabolismo , Adrenérgicos/farmacologia , Proliferação de Células , Endossomos/efeitos dos fármacos , Endossomos/enzimologia , Endossomos/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Genes myc , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
12.
Cell ; 151(1): 14-23, 2012 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-23021212

RESUMO

G-protein-coupled receptors serve as key signal transduction conduits, linking extracellular inputs with diverse cellular responses. These receptors eluded structural characterization for decades following their identification. A landmark structure of rhodopsin provided a basis for structure-function studies and homology modeling, but advances in receptor biology suffered from a lack of receptor-specific structural insights. The recent explosion in GPCR structures confirms some features predicted by rhodopsin-based models, and more importantly, it reveals unexpected ligand-binding modes and critical aspects of the receptor activation process. The new structures also promise to foster studies testing emerging models for GPCR function such as receptor dimerization and ligand-biased signaling.


Assuntos
Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Animais , Cristalografia por Raios X , Humanos , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Rodopsina/química , Rodopsina/metabolismo , Relação Estrutura-Atividade
13.
Nature ; 597(7876): 410-414, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34408322

RESUMO

Signals from sympathetic neurons and immune cells regulate adipocytes and thereby contribute to fat tissue biology. Interactions between the nervous and immune systems have recently emerged as important regulators of host defence and inflammation1-4. Nevertheless, it is unclear whether neuronal and immune cells co-operate in brain-body axes to orchestrate metabolism and obesity. Here we describe a neuro-mesenchymal unit that controls group 2 innate lymphoid cells (ILC2s), adipose tissue physiology, metabolism and obesity via a brain-adipose circuit. We found that sympathetic nerve terminals act on neighbouring adipose mesenchymal cells via the ß2-adrenergic receptor to control the expression of glial-derived neurotrophic factor (GDNF) and the activity of ILC2s in gonadal fat. Accordingly, ILC2-autonomous manipulation of the GDNF receptor machinery led to alterations in ILC2 function, energy expenditure, insulin resistance and propensity to obesity. Retrograde tracing and chemical, surgical and chemogenetic manipulations identified a sympathetic aorticorenal circuit that modulates ILC2s in gonadal fat and connects to higher-order brain areas, including the paraventricular nucleus of the hypothalamus. Our results identify a neuro-mesenchymal unit that translates cues from long-range neuronal circuitry into adipose-resident ILC2 function, thereby shaping host metabolism and obesity.


Assuntos
Tecido Adiposo/inervação , Tecido Adiposo/metabolismo , Encéfalo/metabolismo , Imunidade Inata/imunologia , Mesoderma/citologia , Vias Neurais , Neurônios/citologia , Obesidade/metabolismo , Tecido Adiposo/citologia , Animais , Encéfalo/citologia , Sinais (Psicologia) , Citocinas/metabolismo , Metabolismo Energético , Feminino , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Gônadas/metabolismo , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Núcleo Hipotalâmico Paraventricular/metabolismo , Proteínas Proto-Oncogênicas c-ret/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Sistema Nervoso Simpático/citologia , Sistema Nervoso Simpático/metabolismo
14.
Proc Natl Acad Sci U S A ; 121(14): e2304897121, 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38547061

RESUMO

While the existence and functional role of class C G-protein-coupled receptors (GPCR) dimers is well established, there is still a lack of consensus regarding class A and B GPCR multimerization. This lack of consensus is largely due to the inherent challenges of demonstrating the presence of multimeric receptor complexes in a physiologically relevant cellular context. The C-X-C motif chemokine receptor 4 (CXCR4) is a class A GPCR that is a promising target of anticancer therapy. Here, we investigated the potential of CXCR4 to form multimeric complexes with other GPCRs and characterized the relative size of the complexes in a live-cell environment. Using a bimolecular fluorescence complementation (BiFC) assay, we identified the ß2 adrenergic receptor (ß2AR) as an interaction partner. To investigate the molecular scale details of CXCR4-ß2AR interactions, we used a time-resolved fluorescence spectroscopy method called pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS can resolve membrane protein density, diffusion, and multimerization state in live cells at physiological expression levels. We probed CXCR4 and ß2AR homo- and heteromultimerization in model cell lines and found that CXCR4 assembles into multimeric complexes larger than dimers in MDA-MB-231 human breast cancer cells and in HCC4006 human lung cancer cells. We also found that ß2AR associates with CXCR4 multimers in MDA-MB-231 and HCC4006 cells to a higher degree than in COS-7 and CHO cells and in a ligand-dependent manner. These results suggest that CXCR4-ß2AR heteromers are present in human cancer cells and that GPCR multimerization is significantly affected by the plasma membrane environment.


Assuntos
Neoplasias , Receptores Adrenérgicos beta 2 , Receptores CXCR4 , Transdução de Sinais , Animais , Cricetinae , Humanos , Células CHO , Cricetulus , Proteínas de Membrana/metabolismo , Neoplasias/metabolismo , Receptores CXCR4/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Multimerização Proteica
15.
Proc Natl Acad Sci U S A ; 121(18): e2307090121, 2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38648487

RESUMO

G protein-coupled receptors (GPCRs) transduce the effects of many neuromodulators including dopamine, serotonin, epinephrine, acetylcholine, and opioids. The localization of synthetic or endogenous GPCR agonists impacts their action on specific neuronal pathways. In this paper, we show a series of single-protein chain integrator sensors that are highly modular and could potentially be used to determine GPCR agonist localization across the brain. We previously engineered integrator sensors for the mu- and kappa-opioid receptor agonists called M- and K-Single-chain Protein-based Opioid Transmission Indicator Tool (SPOTIT), respectively. Here, we engineered red versions of the SPOTIT sensors for multiplexed imaging of GPCR agonists. We also modified SPOTIT to create an integrator sensor design platform called SPOTIT for all GPCRs (SPOTall). We used the SPOTall platform to engineer sensors for the beta 2-adrenergic receptor (B2AR), the dopamine receptor D1, and the cholinergic receptor muscarinic 2 agonists. Finally, we demonstrated the application of M-SPOTIT and B2AR-SPOTall in detecting exogenously administered morphine, isoproterenol, and epinephrine in the mouse brain via locally injected viruses. The SPOTIT and SPOTall sensor design platform has the potential for unbiased agonist detection of many synthetic and endogenous neuromodulators across the brain.


Assuntos
Receptores Acoplados a Proteínas G , Animais , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Humanos , Camundongos , Células HEK293 , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/metabolismo , Isoproterenol/farmacologia , Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , Morfina/farmacologia , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/diagnóstico por imagem , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/metabolismo , Técnicas Biossensoriais/métodos
16.
Nat Chem Biol ; 20(1): 74-82, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37580554

RESUMO

G-protein-coupled receptors (GPCRs) are a class of integral membrane proteins that detect environmental cues and trigger cellular responses. Deciphering the functional states of GPCRs induced by various ligands has been one of the primary goals in the field. Here we developed an effective universal method for GPCR cryo-electron microscopy structure determination without the need to prepare GPCR-signaling protein complexes. Using this method, we successfully solved the structures of the ß2-adrenergic receptor (ß2AR) bound to antagonistic and agonistic ligands and the adhesion GPCR ADGRL3 in the apo state. For ß2AR, an intermediate state stabilized by the partial agonist was captured. For ADGRL3, the structure revealed that inactive ADGRL3 adopts a compact fold and that large unusual conformational changes on both the extracellular and intracellular sides are required for activation of adhesion GPCRs. We anticipate that this method will open a new avenue for understanding GPCR structure‒function relationships and drug development.


Assuntos
Receptores Adrenérgicos beta 2 , Receptores Acoplados a Proteínas G , Modelos Moleculares , Microscopia Crioeletrônica , Receptores Acoplados a Proteínas G/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Ligantes
17.
Circ Res ; 135(2): e24-e38, 2024 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-38813686

RESUMO

BACKGROUND: Chronically elevated neurohumoral drive, and particularly elevated adrenergic tone leading to ß-adrenergic receptor (ß-AR) overstimulation in cardiac myocytes, is a key mechanism involved in the progression of heart failure. ß1-AR (ß1-adrenergic receptor) and ß2-ARs (ß2-adrenergic receptor) are the 2 major subtypes of ß-ARs present in the human heart; however, they elicit different or even opposite effects on cardiac function and hypertrophy. For example, chronic activation of ß1-ARs drives detrimental cardiac remodeling while ß2-AR signaling is protective. The underlying molecular mechanisms for cardiac protection through ß2-ARs remain unclear. METHODS: ß2-AR signaling mechanisms were studied in isolated neonatal rat ventricular myocytes and adult mouse ventricular myocytes using live cell imaging and Western blotting methods. Isolated myocytes and mice were used to examine the roles of ß2-AR signaling mechanisms in the regulation of cardiac hypertrophy. RESULTS: Here, we show that ß2-AR activation protects against hypertrophy through inhibition of phospholipaseCε signaling at the Golgi apparatus. The mechanism for ß2-AR-mediated phospholipase C inhibition requires internalization of ß2-AR, activation of Gi and Gßγ subunit signaling at endosome and ERK (extracellular regulated kinase) activation. This pathway inhibits both angiotensin II and Golgi-ß1-AR-mediated stimulation of phosphoinositide hydrolysis at the Golgi apparatus ultimately resulting in decreased PKD (protein kinase D) and histone deacetylase 5 phosphorylation and protection against cardiac hypertrophy. CONCLUSIONS: This reveals a mechanism for ß2-AR antagonism of the phospholipase Cε pathway that may contribute to the known protective effects of ß2-AR signaling on the development of heart failure.


Assuntos
Miócitos Cardíacos , Receptores Adrenérgicos beta 2 , Transdução de Sinais , Animais , Masculino , Camundongos , Ratos , Animais Recém-Nascidos , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Células Cultivadas , Endocitose , Complexo de Golgi/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Fosfoinositídeo Fosfolipase C/metabolismo , Proteína Quinase C/metabolismo , Ratos Sprague-Dawley , Receptores Adrenérgicos beta 2/metabolismo
18.
Cell ; 147(5): 1011-23, 2011 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-22118459

RESUMO

Atypical antipsychotic drugs, such as clozapine and risperidone, have a high affinity for the serotonin 5-HT(2A) G protein-coupled receptor (GPCR), the 2AR, which signals via a G(q) heterotrimeric G protein. The closely related non-antipsychotic drugs, such as ritanserin and methysergide, also block 2AR function, but they lack comparable neuropsychological effects. Why some but not all 2AR inhibitors exhibit antipsychotic properties remains unresolved. We now show that a heteromeric complex between the 2AR and the G(i)-linked GPCR, metabotropic glutamate 2 receptor (mGluR2), integrates ligand input, modulating signaling output and behavioral changes. Serotonergic and glutamatergic drugs bind the mGluR2/2AR heterocomplex, which then balances Gi- and Gq-dependent signaling. We find that the mGluR2/2AR-mediated changes in Gi and Gq activity predict the psychoactive behavioral effects of a variety of pharmocological compounds. These observations provide mechanistic insight into antipsychotic action that may advance therapeutic strategies for disorders including schizophrenia and dementia.


Assuntos
Antipsicóticos/farmacologia , Receptores Adrenérgicos beta 2/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Transdução de Sinais , Anfetaminas/farmacologia , Animais , Clozapina/farmacologia , Dimerização , Relação Dose-Resposta a Droga , Lobo Frontal/efeitos dos fármacos , Lobo Frontal/metabolismo , Metisergida/farmacologia , Camundongos , Oócitos , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Xenopus
19.
Nature ; 577(7792): 676-681, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31969699

RESUMO

Empirical and anecdotal evidence has associated stress with accelerated hair greying (formation of unpigmented hairs)1,2, but so far there has been little scientific validation of this link. Here we report that, in mice, acute stress leads to hair greying through the fast depletion of melanocyte stem cells. Using a combination of adrenalectomy, denervation, chemogenetics3,4, cell ablation and knockout of the adrenergic receptor specifically in melanocyte stem cells, we find that the stress-induced loss of melanocyte stem cells is independent of immune attack or adrenal stress hormones. Instead, hair greying results from activation of the sympathetic nerves that innervate the melanocyte stem-cell niche. Under conditions of stress, the activation of these sympathetic nerves leads to burst release of the neurotransmitter noradrenaline (also known as norepinephrine). This causes quiescent melanocyte stem cells to proliferate rapidly, and is followed by their differentiation, migration and permanent depletion from the niche. Transient suppression of the proliferation of melanocyte stem cells prevents stress-induced hair greying. Our study demonstrates that neuronal activity that is induced by acute stress can drive a rapid and permanent loss of somatic stem cells, and illustrates an example in which the maintenance of somatic stem cells is directly influenced by the overall physiological state of the organism.


Assuntos
Vias Autônomas/fisiopatologia , Cor de Cabelo/fisiologia , Melanócitos/patologia , Nicho de Células-Tronco/fisiologia , Células-Tronco/patologia , Estresse Psicológico/fisiopatologia , Sistema Nervoso Simpático/fisiopatologia , Glândulas Suprarrenais/metabolismo , Adrenalectomia , Animais , Vias Autônomas/patologia , Proliferação de Células , Células Cultivadas , Denervação , Feminino , Humanos , Masculino , Melanócitos/citologia , Melanócitos/metabolismo , Camundongos , Norepinefrina/metabolismo , Trauma Psicológico/patologia , Trauma Psicológico/fisiopatologia , Receptores Adrenérgicos beta 2/deficiência , Receptores Adrenérgicos beta 2/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Estresse Psicológico/patologia , Sistema Nervoso Simpático/patologia
20.
Proc Natl Acad Sci U S A ; 120(31): e2302668120, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37490535

RESUMO

Catecholamine-stimulated ß2-adrenergic receptor (ß2AR) signaling via the canonical Gs-adenylyl cyclase-cAMP-PKA pathway regulates numerous physiological functions, including the therapeutic effects of exogenous ß-agonists in the treatment of airway disease. ß2AR signaling is tightly regulated by GRKs and ß-arrestins, which together promote ß2AR desensitization and internalization as well as downstream signaling, often antithetical to the canonical pathway. Thus, the ability to bias ß2AR signaling toward the Gs pathway while avoiding ß-arrestin-mediated effects may provide a strategy to improve the functional consequences of ß2AR activation. Since attempts to develop Gs-biased agonists and allosteric modulators for the ß2AR have been largely unsuccessful, here we screened small molecule libraries for allosteric modulators that selectively inhibit ß-arrestin recruitment to the receptor. This screen identified several compounds that met this profile, and, of these, a difluorophenyl quinazoline (DFPQ) derivative was found to be a selective negative allosteric modulator of ß-arrestin recruitment to the ß2AR while having no effect on ß2AR coupling to Gs. DFPQ effectively inhibits agonist-promoted phosphorylation and internalization of the ß2AR and protects against the functional desensitization of ß-agonist mediated regulation in cell and tissue models. The effects of DFPQ were also specific to the ß2AR with minimal effects on the ß1AR. Modeling, mutagenesis, and medicinal chemistry studies support DFPQ derivatives binding to an intracellular membrane-facing region of the ß2AR, including residues within transmembrane domains 3 and 4 and intracellular loop 2. DFPQ thus represents a class of biased allosteric modulators that targets an allosteric site of the ß2AR.


Assuntos
Arrestina , Transdução de Sinais , beta-Arrestinas/metabolismo , Arrestina/metabolismo , beta-Arrestina 1/genética , beta-Arrestina 1/metabolismo , Receptores Adrenérgicos/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo
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