RESUMO
ß-arrestin plays a key role in G protein-coupled receptor (GPCR) signaling and desensitization. Despite recent structural advances, the mechanisms that govern receptor-ß-arrestin interactions at the plasma membrane of living cells remain elusive. Here, we combine single-molecule microscopy with molecular dynamics simulations to dissect the complex sequence of events involved in ß-arrestin interactions with both receptors and the lipid bilayer. Unexpectedly, our results reveal that ß-arrestin spontaneously inserts into the lipid bilayer and transiently interacts with receptors via lateral diffusion on the plasma membrane. Moreover, they indicate that, following receptor interaction, the plasma membrane stabilizes ß-arrestin in a longer-lived, membrane-bound state, allowing it to diffuse to clathrin-coated pits separately from the activating receptor. These results expand our current understanding of ß-arrestin function at the plasma membrane, revealing a critical role for ß-arrestin preassociation with the lipid bilayer in facilitating its interactions with receptors and subsequent activation.
Assuntos
Receptores Acoplados a Proteínas G , Transdução de Sinais , beta-Arrestinas , beta-Arrestinas/metabolismo , Membrana Celular/metabolismo , Clatrina/metabolismo , Endocitose , Bicamadas Lipídicas , Receptores Acoplados a Proteínas G/metabolismo , Simulação de Dinâmica MolecularRESUMO
Thermogenic adipocytes possess a therapeutically appealing, energy-expending capacity, which is canonically cold-induced by ligand-dependent activation of ß-adrenergic G protein-coupled receptors (GPCRs). Here, we uncover an alternate paradigm of GPCR-mediated adipose thermogenesis through the constitutively active receptor, GPR3. We show that the N terminus of GPR3 confers intrinsic signaling activity, resulting in continuous Gs-coupling and cAMP production without an exogenous ligand. Thus, transcriptional induction of Gpr3 represents the regulatory parallel to ligand-binding of conventional GPCRs. Consequently, increasing Gpr3 expression in thermogenic adipocytes is alone sufficient to drive energy expenditure and counteract metabolic disease in mice. Gpr3 transcription is cold-stimulated by a lipolytic signal, and dietary fat potentiates GPR3-dependent thermogenesis to amplify the response to caloric excess. Moreover, we find GPR3 to be an essential, adrenergic-independent regulator of human brown adipocytes. Taken together, our findings reveal a noncanonical mechanism of GPCR control and thermogenic activation through the lipolysis-induced expression of constitutively active GPR3.
Assuntos
Tecido Adiposo Marrom/metabolismo , Receptor Constitutivo de Androstano/metabolismo , Lipólise , Receptores Acoplados a Proteínas G/metabolismo , Termogênese , Adipócitos/metabolismo , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Temperatura Baixa , Gorduras na Dieta/farmacologia , Humanos , Camundongos Endogâmicos C57BL , Fenótipo , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , Sistema Nervoso Simpático/metabolismo , Transcrição GênicaRESUMO
G protein-coupled receptors (GPCRs) regulate many cellular and physiological processes, responding to a diverse range of extracellular stimuli including hormones, neurotransmitters, odorants, and light. Decades of biochemical and pharmacological studies have provided fundamental insights into the mechanisms of GPCR signaling. Thanks to recent advances in structural biology, we now possess an atomistic understanding of receptor activation and G protein coupling. However, how GPCRs and G proteins interact in living cells to confer signaling efficiency and specificity remains insufficiently understood. The development of advanced optical methods, including single-molecule microscopy, has provided the means to study receptors and G proteins in living cells with unprecedented spatio-temporal resolution. The results of these studies reveal an unexpected level of complexity, whereby GPCRs undergo transient interactions among themselves as well as with G proteins and structural elements of the plasma membrane to form short-lived signaling nanodomains that likely confer both rapidity and specificity to GPCR signaling. These findings may provide new strategies to pharmaceutically modulate GPCR function, which might eventually pave the way to innovative drugs for common diseases such as diabetes or heart failure.
Assuntos
Membrana Celular/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , AnimaisRESUMO
Mechanisms that control mobilization of cytosolic calcium [Ca2+]i are key for regulation of numerous eukaryotic cell functions. One such paradigmatic mechanism involves activation of phospholipase Cß (PLCß) enzymes by G protein ßγ subunits from activated Gαi-Gßγ heterotrimers. Here, we report identification of a master switch to enable this control for PLCß enzymes in living cells. We find that the Gαi-Gßγ-PLCß-Ca2+ signaling module is entirely dependent on the presence of active Gαq. If Gαq is pharmacologically inhibited or genetically ablated, Gßγ can bind to PLCß but does not elicit Ca2+ signals. Removal of an auto-inhibitory linker that occludes the active site of the enzyme is required and sufficient to empower "stand-alone control" of PLCß by Gßγ. This dependence of Gi-Gßγ-Ca2+ on Gαq places an entire signaling branch of G-protein-coupled receptors (GPCRs) under hierarchical control of Gq and changes our understanding of how Gi-GPCRs trigger [Ca2+]i via PLCß enzymes.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/genética , Fosfolipase C beta/genética , Cálcio/metabolismo , Sinalização do Cálcio/genética , Citosol/metabolismo , Células HEK293 , Humanos , Ligação Proteica/genética , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/genéticaRESUMO
G protein-coupled receptors (GPCRs) mediate the effects of numerous hormones and neurotransmitters and are major pharmacological targets. Classical studies with crude cell lysates or membrane preparations have identified the main biochemical steps involved in GPCR signaling. Moreover, recent studies on purified proteins have provided astounding details at the atomic level of the 3-D structures of receptors in multiple conformations, including in complex with G proteins and ß-arrestins. However, several fundamental questions remain regarding the highly specific effects and rapid nature of GPCR signaling. Recent developments in single-molecule microscopy are providing important contributions to answering these questions. Overall, single-molecule studies have revealed unexpected levels of complexity, with receptors existing in different conformations and dynamically interacting among themselves, their signaling partners, and structural elements of the plasma membrane to produce highly localized signals in space and time. These findings may provide a new basis to develop innovative strategies to modulate GPCR function for pharmacological purposes.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , beta-Arrestinas/metabolismo , Membrana Celular/metabolismo , Humanos , Receptores Acoplados a Proteínas G/química , Transdução de Sinais/fisiologia , Imagem Individual de Molécula/métodosRESUMO
G-protein-coupled receptors mediate the biological effects of many hormones and neurotransmitters and are important pharmacological targets. They transmit their signals to the cell interior by interacting with G proteins. However, it is unclear how receptors and G proteins meet, interact and couple. Here we analyse the concerted motion of G-protein-coupled receptors and G proteins on the plasma membrane and provide a quantitative model that reveals the key factors that underlie the high spatiotemporal complexity of their interactions. Using two-colour, single-molecule imaging we visualize interactions between individual receptors and G proteins at the surface of living cells. Under basal conditions, receptors and G proteins form activity-dependent complexes that last for around one second. Agonists specifically regulate the kinetics of receptor-G protein interactions, mainly by increasing their association rate. We find hot spots on the plasma membrane, at least partially defined by the cytoskeleton and clathrin-coated pits, in which receptors and G proteins are confined and preferentially couple. Imaging with the nanobody Nb37 suggests that signalling by G-protein-coupled receptors occurs preferentially at these hot spots. These findings shed new light on the dynamic interactions that control G-protein-coupled receptor signalling.
Assuntos
Membrana Celular/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Receptores Adrenérgicos/metabolismo , Imagem Individual de Molécula , Animais , Membrana Celular/química , Sobrevivência Celular , Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/química , Invaginações Revestidas da Membrana Celular/metabolismo , Cor , Citoesqueleto/metabolismo , Difusão , Células Endoteliais da Veia Umbilical Humana , Humanos , Cinética , Camundongos , Movimento , Transdução de SinaisRESUMO
The glucagon-like peptide-1 receptor (GLP-1R), a key pharmacological target in type 2 diabetes (T2D) and obesity, undergoes rapid endocytosis after stimulation by endogenous and therapeutic agonists. We have previously highlighted the relevance of this process in fine-tuning GLP-1R responses in pancreatic beta cells to control insulin secretion. In the present study, we demonstrate an important role for the translocation of active GLP-1Rs into liquid-ordered plasma membrane nanodomains, which act as hotspots for optimal coordination of intracellular signaling and clathrin-mediated endocytosis. This process is dynamically regulated by agonist binding through palmitoylation of the GLP-1R at its carboxyl-terminal tail. Biased GLP-1R agonists and small molecule allosteric modulation both influence GLP-1R palmitoylation, clustering, nanodomain signaling, and internalization. Downstream effects on insulin secretion from pancreatic beta cells indicate that these processes are relevant to GLP-1R physiological actions and might be therapeutically targetable.
Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Células Secretoras de Insulina/metabolismo , Animais , Células CHO , Membrana Celular/metabolismo , Análise por Conglomerados , Cricetulus , AMP Cíclico/metabolismo , Diabetes Mellitus Tipo 2 , Endocitose/efeitos dos fármacos , Peptídeo 1 Semelhante ao Glucagon/agonistas , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/fisiologia , Células HEK293 , Humanos , Insulina/metabolismo , Secreção de Insulina/fisiologia , Células Secretoras de Insulina/fisiologia , Lipoilação , Transdução de Sinais/efeitos dos fármacosRESUMO
PURPOSE: Protein kinase A (PKA) subunit defects (in PRKAR1A and PRKACA) are known to contribute to adrenal tumor pathogenesis. We studied the PRKAR1B gene for any genetic changes in bilateral adrenocortical hyperplasia (BAH) and cortisol-producing adrenal adenomas (CPA). METHODS: Exome sequencing and PRKAR1B copy-number variant (CNV) analysis were performed in 74 patients with BAH and 21 with CPA. PKA activity was studied in tumors with defects; sequence variants were investigated in vitro. RESULTS: Three PRKAR1B germline variants (p.I40V, p.A67V, p.A300T) were identified among 74 patients with BAH. PRKAR1B copy-number gains (CNG) were found in 3 of 21 CPAs, one in a tumor carrying a somatic PRKACA "hotspot" pathogenic variant p.L206R. CPAs bearing PRKAR1B CNGs showed higher PRKAR1B messenger RNA (mRNA) levels and reduced PKA activity. Baseline PKA activity was also decreased for p.A67V and p.A300T in vitro, and mutant PRKAR1ß bound PRKACα in fluorescence resonance energy transfer (FRET) recordings of cotransfected HEK293 cells stronger than normal. CONCLUSION: PRKAR1B is yet another PKA subunit that may potentially contribute to adrenal tumor formation. Its involvement in adrenocortical disease may be different from that of other subunits, because PRKAR1B variants and PRKAR1B CNGs were associated with decreased (rather than increased) overall PKA activity in vitro.
Assuntos
Neoplasias das Glândulas Suprarrenais , Síndrome de Cushing , Síndrome de Cushing/genética , Subunidade RIbeta da Proteína Quinase Dependente de AMP Cíclico , Genômica , Células HEK293 , Humanos , MutaçãoRESUMO
In this article, we introduce a new method to detect transient trapping events within a single particle trajectory, thus allowing the explicit accounting of changes in the particle's dynamics over time. Our method is based on new measures of a smoothed recurrence matrix. The newly introduced set of measures takes into account both the spatial and temporal structure of the trajectory. Therefore, it is adapted to study short-lived trapping domains that are not visited by multiple trajectories. Contrary to most existing methods, it does not rely on using a window, sliding along the trajectory, but rather investigates the trajectory as a whole. This method provides useful information to study intracellular and plasma membrane compartmentalisation. Additionally, this method is applied to single particle trajectory data of ß2-adrenergic receptors, revealing that receptor stimulation results in increased trapping of receptors in defined domains, without changing the diffusion of free receptors.
RESUMO
The high expression of somatostatin receptor 2 (SST2) in growth hormone (GH)-secreting tumors represents the rationale for the clinical use of somatostatin analogs (SSAs) in acromegaly. Recently, the cytoskeletal protein Filamin A (FLNA) has emerged as key modulator of the responsiveness of GH-secreting pituitary tumors to SSAs by regulating SST2 signaling and expression. The aim of this study was to explore FLNA involvement in SST2 intracellular trafficking in tumor somatotroph cells. By biotinylation assay, we found that FLNA silencing abolished octreotide-mediated SST2 internalization in rat GH3 cell line (28.0 ± 2.7 vs. 4 ± 4.3% SST2 internalization, control versus FLNA small interfering RNAs (siRNA) cells, respectively, p < 0.001) and human GH-secreting primary cultured cells (70.3 ± 21.1 vs. 24 ± 19.2% SST2 internalization, control versus FLNA siRNA cells, respectively, p < 0.05). In addition, confocal imaging revealed impaired SST2 recycling to the plasma membrane in FLNA silenced GH3 cells. Coimmunoprecipitation and immunofluorescence experiments showed that FLNA, as well as ß-arrestin2, is timely dependent recruited to octreotide-stimulated SST2 receptors both in rat and human tumor somatotroph cells. Although FLNA expression knock down did not prevent the formation of ß-arrestin2-SST2 complex in GH3 cells, it significantly impaired efficient SST2 loading into cytosolic vesicles positive for the early endocytic and recycling markers Rab5 and 4, respectively (33.7 ± 8.9% down to 25.9 ± 6.9%, p < 0.05, and 28.4 ± 7.4% down to 17.6 ± 5.7%, p < 0.01, for SST2-Rab5 and SST2-Rab4 colocalization, respectively, in control versus FLNA siRNA cells). Altogether these data support an important role for FLNA in the mediation of octreotide-induced SST2 trafficking in GH-secreting pituitary tumor cells through Rab5 and 4 sorting endosomes.
Assuntos
Adenoma/metabolismo , Endossomos/fisiologia , Filaminas/fisiologia , Adenoma Hipofisário Secretor de Hormônio do Crescimento/metabolismo , Receptores de Somatostatina/metabolismo , Adenoma/patologia , Animais , Células Cultivadas , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Adenoma Hipofisário Secretor de Hormônio do Crescimento/patologia , Humanos , Octreotida/farmacologia , Transporte Proteico/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacos , Somatotrofos/efeitos dos fármacos , Somatotrofos/metabolismo , Somatotrofos/patologia , Proteínas rab4 de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
µ-Opioid receptors (µ-ORs) play a critical role in the modulation of pain and mediate the effects of the most powerful analgesic drugs. Despite extensive efforts, it remains insufficiently understood how µ-ORs produce specific effects in living cells. We developed new fluorescent ligands based on the µ-OR antagonist E-p-nitrocinnamoylamino-dihydrocodeinone (CACO), that display high affinity, long residence time and pronounced selectivity. Using these ligands, we achieved single-molecule imaging of µ-ORs on the surface of living cells at physiological expression levels. Our results reveal a high heterogeneity in the diffusion of µ-ORs, with a relevant immobile fraction. Using a pair of fluorescent ligands of different color, we provide evidence that µ-ORs interact with each other to form short-lived homodimers on the plasma membrane. This approach provides a new strategy to investigate µ-OR pharmacology at single-molecule level.
Assuntos
Corantes Fluorescentes/química , Hidrocodona/química , Multimerização Proteica , Receptores Opioides mu/química , Imagem Individual de Molécula/métodos , Difusão , Corantes Fluorescentes/farmacologia , Hidrocodona/farmacologia , Ligantes , Estrutura Quaternária de Proteína , Receptores Opioides mu/antagonistas & inibidores , Receptores Opioides mu/metabolismoRESUMO
Glycoprotein hormones (GPHs) are the main regulators of the pituitary-thyroid and pituitary-gonadal axes. Selective interaction between GPHs and their cognate G protein-coupled receptors ensure specificity in GPH signaling. The mechanisms of how these hormones activate glycoprotein hormone receptors (GPHRs) or how mutations and autoantibodies can alter receptor function were unclear. Based on the hypothesis that GPHRs contain an internal agonist, we systematically screened peptide libraries derived from the ectodomain for agonistic activity on the receptors. We show that a peptide (p10) derived from a conserved sequence in the C-terminal part of the extracellular N terminus can activate all GPHRs in vitro and in GPHR-expressing tissues. Inactivating mutations in this conserved region or in p10 can inhibit activation of the thyroid-stimulating hormone receptor by autoantibodies. Our data suggest an activation mechanism where, upon extracellular ligand binding, this intramolecular agonist isomerizes and induces structural changes in the 7-transmembrane helix domain, triggering G protein activation. This mechanism can explain the pathophysiology of activating autoantibodies and several mutations causing endocrine dysfunctions such as Graves disease and hypo- and hyperthyroidism. Our findings highlight an evolutionarily conserved activation mechanism of GPHRs and will further promote the development of specific ligands useful to treat Graves disease and other dysfunctions of GPHRs.
Assuntos
Doenças do Sistema Endócrino/genética , Doenças do Sistema Endócrino/terapia , Glicoproteínas/metabolismo , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/farmacologia , Células COS , Chlorocebus aethiops , AMP Cíclico/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese/genética , Proteínas Mutantes/metabolismo , Mutação/genética , Peptídeos/química , Peptídeos/farmacologia , Estrutura Terciária de Proteína , Receptores de Superfície Celular/agonistas , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Glândula Tireoide/metabolismoRESUMO
BACKGROUND: Corticotropin-independent Cushing's syndrome is caused by tumors or hyperplasia of the adrenal cortex. The molecular pathogenesis of cortisol-producing adrenal adenomas is not well understood. METHODS: We performed exome sequencing of tumor-tissue specimens from 10 patients with cortisol-producing adrenal adenomas and evaluated recurrent mutations in candidate genes in an additional 171 patients with adrenocortical tumors. We also performed genomewide copy-number analysis in 35 patients with cortisol-secreting bilateral adrenal hyperplasias. We studied the effects of these genetic defects both clinically and in vitro. RESULTS: Exome sequencing revealed somatic mutations in PRKACA, which encodes the catalytic subunit of cyclic AMP-dependent protein kinase (protein kinase A [PKA]), in 8 of 10 adenomas (c.617AâC in 7 and c.595_596insCAC in 1). Overall, PRKACA somatic mutations were identified in 22 of 59 unilateral adenomas (37%) from patients with overt Cushing's syndrome; these mutations were not detectable in 40 patients with subclinical hypercortisolism or in 82 patients with other adrenal tumors. Among 35 patients with cortisol-producing hyperplasias, 5 (including 2 first-degree relatives) carried a germline copy-number gain (duplication) of the genomic region on chromosome 19 that includes PRKACA. In vitro studies showed impaired inhibition of both PKA catalytic subunit mutants by the PKA regulatory subunit, whereas cells from patients with germline chromosomal gains showed increased protein levels of the PKA catalytic subunit; in both instances, basal PKA activity was increased. CONCLUSIONS: Genetic alterations of the catalytic subunit of PKA were found to be associated with human disease. Germline duplications of this gene resulted in bilateral adrenal hyperplasias, whereas somatic PRKACA mutations resulted in unilateral cortisol-producing adrenal adenomas. (Funded by the European Commission Seventh Framework Program and others.).
Assuntos
Adenoma/genética , Neoplasias das Glândulas Suprarrenais/genética , Hiperplasia Suprarrenal Congênita/genética , Síndrome de Cushing/etiologia , Proteínas Quinases Dependentes de AMP Cíclico/genética , Mutação em Linhagem Germinativa , Adenoma/complicações , Adenoma/enzimologia , Neoplasias das Glândulas Suprarrenais/complicações , Neoplasias das Glândulas Suprarrenais/enzimologia , Adulto , Domínio Catalítico , Síndrome de Cushing/enzimologia , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Exoma , Humanos , Hidrocortisona/biossíntese , Pessoa de Meia-Idade , Mutação , Conformação Proteica , Análise de Sequência de DNARESUMO
G-protein-coupled receptors (GPCRs) constitute the largest family of receptors and major pharmacological targets. Whereas many GPCRs have been shown to form di-/oligomers, the size and stability of such complexes under physiological conditions are largely unknown. Here, we used direct receptor labeling with SNAP-tags and total internal reflection fluorescence microscopy to dynamically monitor single receptors on intact cells and thus compare the spatial arrangement, mobility, and supramolecular organization of three prototypical GPCRs: the ß(1)-adrenergic receptor (ß(1)AR), the ß(2)-adrenergic receptor (ß(2)AR), and the γ-aminobutyric acid (GABA(B)) receptor. These GPCRs showed very different degrees of di-/oligomerization, lowest for ß(1)ARs (monomers/dimers) and highest for GABA(B) receptors (prevalently dimers/tetramers of heterodimers). The size of receptor complexes increased with receptor density as a result of transient receptor-receptor interactions. Whereas ß(1)-/ß(2)ARs were apparently freely diffusing on the cell surface, GABA(B) receptors were prevalently organized into ordered arrays, via interaction with the actin cytoskeleton. Agonist stimulation did not alter receptor di-/oligomerization, but increased the mobility of GABA(B) receptor complexes. These data provide a spatiotemporal characterization of ß(1)-/ß(2)ARs and GABA(B) receptors at single-molecule resolution. The results suggest that GPCRs are present on the cell surface in a dynamic equilibrium, with constant formation and dissociation of new receptor complexes that can be targeted, in a ligand-regulated manner, to different cell-surface microdomains.
Assuntos
Algoritmos , Modelos Químicos , Complexos Multiproteicos/química , Receptores Adrenérgicos beta 1/química , Receptores Adrenérgicos beta 2/química , Receptores de GABA/química , Animais , Compostos Bicíclicos Heterocíclicos com Pontes , Células CHO , Carbocianinas , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Lipídeos , Microscopia de Fluorescência , Simulação de Dinâmica Molecular , Plasmídeos/genética , Ensaio Radioligante , TiazolidinasRESUMO
The delta opioid receptor (δOR or DOR) is a G protein-coupled receptor (GPCR) showing a promising profile as a drug target for nociception and analgesia. Herein, we design and synthesize new fluorescent antagonist probes with high δOR selectivity that are ideally suited for single-molecule microscopy (SMM) applications in unmodified, untagged receptors. Using our new probes, we investigated wild-type δOR localization and mobility at low physiological receptor densities for the first time. Furthermore, we investigate the potential formation of δOR homodimers, as such a receptor organization might exhibit distinct pharmacological activity, potentially paving the way for innovative pharmacological therapies. Our findings indicate that the majority of δORs labeled with these probes exist as freely diffusing monomers on the cell surface in a simple cell model. This discovery advances our understanding of OR behavior and offers potential implications for future therapeutic research.
Assuntos
Desenho de Fármacos , Corantes Fluorescentes , Receptores Opioides delta , Receptores Opioides delta/metabolismo , Receptores Opioides delta/antagonistas & inibidores , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Humanos , Imagem Individual de Molécula/métodos , Células HEK293 , Animais , Microscopia de FluorescênciaRESUMO
G-protein-coupled receptors (GPCRs) have long been believed to activate G proteins only on the cell surface. However, we have recently shown that, in thyroid cells, the GPCR for the thyroid-stimulating hormone (TSH) can continue stimulating cAMP production after cointernalization with TSH. cAMP signaling by internalized TSH receptors (TSHRs) was persistent, whereas that by cell-surface TSHRs was apparently transient, but the reasons for the transient signaling by cell-surface TSHRs were not investigated. Here, we developed and used fluorescence resonance energy transfer (FRET)-based methods to precisely compare the kinetics of TSH binding and dissociation from cell-surface TSHRs with those of the subsequent termination of cAMP signaling directly in living cells. Our results indicate that both TSH binding to human TSHRs expressed in a human embryonic kidney cell line (HEK 293) and the ensuing cAMP signals are rapidly and fully reversible (t(1/2,off)=2.96±1.04 and 2.70±0.73 min, respectively). The FRET measurement of TSH binding was specific, as shown by the lack of a detectable interaction between TSH and the ß(2)-adrenergic receptor expressed in control cells. Enhancing TSHR internalization by ß-arrestin 2 overexpression did not modify the reversibility of TSHR-cAMP signaling. These findings strengthen the view that the cointernalization of TSH-TSHR complexes to a signaling compartment present in thyroid, but not in HEK 293 cells, is responsible for persistent cAMP signaling.
Assuntos
AMP Cíclico/metabolismo , Receptores da Tireotropina/metabolismo , Transdução de Sinais , Glândula Tireoide/metabolismo , Linhagem Celular , Endocitose , Transferência Ressonante de Energia de Fluorescência , Humanos , Tireotropina/metabolismoRESUMO
The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in glucose homeostasis and food intake. GLP1R agonists (GLP1RA) are widely used in the treatment of diabetes and obesity, yet visualizing the endogenous localization, organization and dynamics of a GPCR has so far remained out of reach. In the present study, we generate mice harboring an enzyme self-label genome-edited into the endogenous Glp1r locus. We also rationally design and test various fluorescent dyes, spanning cyan to far-red wavelengths, for labeling performance in tissue. By combining these technologies, we show that endogenous GLP1R can be specifically and sensitively detected in primary tissue using multiple colors. Longitudinal analysis of GLP1R dynamics reveals heterogeneous recruitment of neighboring cell subpopulations into signaling and trafficking, with differences observed between GLP1RA classes and dual agonists. At the nanoscopic level, GLP1Rs are found to possess higher organization, undergoing GLP1RA-dependent membrane diffusion. Together, these results show the utility of enzyme self-labels for visualization and interrogation of endogenous proteins, and provide insight into the biology of a class B GPCR in primary cells and tissue.
Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1 , Obesidade , Camundongos , Animais , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismoRESUMO
The γ-aminobutyric acid type B (GABAB) receptor is a prototypical family C G protein-coupled receptor (GPCR) that plays a key role in the regulation of synaptic transmission. Although growing evidence suggests that GPCR signaling in neurons might be highly organized in time and space, limited information is available about the mechanisms controlling the nanoscale organization of GABAB receptors and other GPCRs on the neuronal plasma membrane. Using a combination of biochemical assays in vitro, single-particle tracking, and super-resolution microscopy, we provide evidence that the spatial organization and diffusion of GABAB receptors on the plasma membrane are governed by dynamic interactions with filamin A, which tethers the receptors to sub-cortical actin filaments. We further show that GABAB receptors are located together with filamin A in small nanodomains in hippocampal neurons. These interactions are mediated by the first intracellular loop of the GABAB1 subunit and modulate the kinetics of Gαi protein activation in response to GABA stimulation.