RESUMO
G protein-coupled receptors (GPCRs) relay extracellular stimuli into specific cellular functions. Cells express many different GPCRs, but all these GPCRs signal to only a few second messengers such as cAMP. It is largely unknown how cells distinguish between signals triggered by different GPCRs to orchestrate their complex functions. Here, we demonstrate that individual GPCRs signal via receptor-associated independent cAMP nanodomains (RAINs) that constitute self-sufficient, independent cell signaling units. Low concentrations of glucagon-like peptide 1 (GLP-1) and isoproterenol exclusively generate highly localized cAMP pools around GLP-1- and ß2-adrenergic receptors, respectively, which are protected from cAMP originating from other receptors and cell compartments. Mapping local cAMP concentrations with engineered GPCR nanorulers reveals gradients over only tens of nanometers that define the size of individual RAINs. The coexistence of many such RAINs allows a single cell to operate thousands of independent cellular signals simultaneously, rather than function as a simple "on/off" switch.
Assuntos
Receptores Acoplados a Proteínas G , Transdução de Sinais , Fenômenos Fisiológicos Celulares , AMP Cíclico , Peptídeo 1 Semelhante ao Glucagon , Receptores Adrenérgicos beta 2 , Receptores Acoplados a Proteínas G/química , Sistemas do Segundo MensageiroRESUMO
Cells relay a plethora of extracellular signals to specific cellular responses by using only a few second messengers, such as cAMP. To explain signaling specificity, cAMP-degrading phosphodiesterases (PDEs) have been suggested to confine cAMP to distinct cellular compartments. However, measured rates of fast cAMP diffusion and slow PDE activity render cAMP compartmentalization essentially impossible. Using fluorescence spectroscopy, we show that, contrary to earlier data, cAMP at physiological concentrations is predominantly bound to cAMP binding sites and, thus, immobile. Binding and unbinding results in largely reduced cAMP dynamics, which we term "buffered diffusion." With a large fraction of cAMP being buffered, PDEs can create nanometer-size domains of low cAMP concentrations. Using FRET-cAMP nanorulers, we directly map cAMP gradients at the nanoscale around PDE molecules and the areas of resulting downstream activation of cAMP-dependent protein kinase (PKA). Our study reveals that spatiotemporal cAMP signaling is under precise control of nanometer-size domains shaped by PDEs that gate activation of downstream effectors.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Transdução de Sinais , Análise de Célula Única/métodos , Simulação por Computador , AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/química , Citoplasma/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Modelos Moleculares , Diester Fosfórico Hidrolases/química , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes , Análise Espaço-Temporal , Espectrometria de FluorescênciaRESUMO
The phagocytes of the innate immune system, macrophages and neutrophils, contribute to antibacterial defense, but their functional specialization and cooperation is unclear. Here, we report that three distinct phagocyte subsets play highly coordinated roles in bacterial urinary tract infection. Ly6C(-) macrophages acted as tissue-resident sentinels that attracted circulating neutrophils and Ly6C(+) macrophages. Such Ly6C(+) macrophages played a previously undescribed helper role: once recruited to the site of infection, they produced the cytokine TNF, which caused Ly6C(-) macrophages to secrete CXCL2. This chemokine activated matrix metalloproteinase-9 in neutrophils, allowing their entry into the uroepithelium to combat the bacteria. In summary, the sentinel macrophages elicit the powerful antibacterial functions of neutrophils only after confirmation by the helper macrophages, reminiscent of the licensing role of helper T cells in antiviral adaptive immunity. These findings identify helper macrophages and TNF as critical regulators in innate immunity against bacterial infections in epithelia.
Assuntos
Infecções Bacterianas/imunologia , Macrófagos/imunologia , Neutrófilos/imunologia , Infecções Urinárias/imunologia , Animais , Antígenos Ly/metabolismo , Quimiocina CXCL2/imunologia , Feminino , Doenças do Sistema Imunitário , Cinética , Transtornos Leucocíticos , Macrófagos/citologia , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Neutrófilos/citologia , Organismos Livres de Patógenos Específicos , Fator de Necrose Tumoral alfa/imunologiaRESUMO
G protein-coupled receptors are the largest and pharmacologically most important receptor family and are involved in the regulation of most cell functions. Most of them reside exclusively at the cell surface, from where they signal via heterotrimeric G proteins to control the production of second messengers such as cAMP and IP3 as well as the activity of several ion channels. However, they may also internalize upon agonist stimulation or constitutively reside in various intracellular locations. Recent evidence indicates that their function differs depending on their precise cellular localization. This is because the signals they produce, notably cAMP and Ca2+, are mostly bound to cell proteins that significantly reduce their mobility, allowing the generation of steep concentration gradients. As a result, signals generated by the receptors remain confined to nanometer-sized domains. We propose that such nanometer-sized domains represent the basic signaling units in a cell and a new type of target for drug development.
Assuntos
Desenvolvimento de Medicamentos , Transdução de Sinais , Humanos , Membrana CelularRESUMO
Muscarinic acetylcholine receptors are prototypical G protein-coupled receptors (GPCRs), members of a large family of 7 transmembrane receptors mediating a wide variety of extracellular signals. We show here, in cultured cells and in a murine model, that the carboxyl terminal fragment of the muscarinic M2 receptor, comprising the transmembrane regions 6 and 7 (M2tail), is expressed by virtue of an internal ribosome entry site localized in the third intracellular loop. Single-cell imaging and import in isolated yeast mitochondria reveals that M2tail, whose expression is up-regulated in cells undergoing integrated stress response, does not follow the normal route to the plasma membrane, but is almost exclusively sorted to the mitochondria inner membrane: here, it controls oxygen consumption, cell proliferation, and the formation of reactive oxygen species (ROS) by reducing oxidative phosphorylation. Crispr/Cas9 editing of the key methionine where cap-independent translation begins in human-induced pluripotent stem cells (hiPSCs), reveals the physiological role of this process in influencing cell proliferation and oxygen consumption at the endogenous level. The expression of the C-terminal domain of a GPCR, capable of regulating mitochondrial function, constitutes a hitherto unknown mechanism notably unrelated to its canonical signaling function as a GPCR at the plasma membrane. This work thus highlights a potential novel mechanism that cells may use for controlling their metabolism under variable environmental conditions, notably as a negative regulator of cell respiration.
Assuntos
Respiração Celular , Mitocôndrias , Receptor Muscarínico M2 , Animais , Humanos , Camundongos , Proliferação de Células , Células HEK293 , Células-Tronco Pluripotentes Induzidas/metabolismo , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Consumo de Oxigênio , Espécies Reativas de Oxigênio/metabolismo , Receptor Muscarínico M2/metabolismo , Receptor Muscarínico M2/genética , Estresse FisiológicoRESUMO
Receptor-activity-modifying proteins (RAMPs) are ubiquitously expressed membrane proteins that associate with different G protein-coupled receptors (GPCRs), including the parathyroid hormone 1 receptor (PTH1R), a class B GPCR and an important modulator of mineral ion homeostasis and bone metabolism. However, it is unknown whether and how RAMP proteins may affect PTH1R function. Using different optical biosensors to measure the activation of PTH1R and its downstream signaling, we describe here that RAMP2 acts as a specific allosteric modulator of PTH1R, shifting PTH1R to a unique preactivated state that permits faster activation in a ligand-specific manner. Moreover, RAMP2 modulates PTH1R downstream signaling in an agonist-dependent manner, most notably increasing the PTH-mediated Gi3 signaling sensitivity. Additionally, RAMP2 increases both PTH- and PTHrP-triggered ß-arrestin2 recruitment to PTH1R. Employing homology modeling, we describe the putative structural molecular basis underlying our functional findings. These data uncover a critical role of RAMPs in the activation and signaling of a GPCR that may provide a new venue for highly specific modulation of GPCR function and advanced drug design.
Assuntos
Proteína 2 Modificadora da Atividade de Receptores , Receptor Tipo 1 de Hormônio Paratireóideo , Transdução de Sinais , Técnicas Biossensoriais , Ligantes , Hormônio Paratireóideo/metabolismo , Proteína 2 Modificadora da Atividade de Receptores/genética , Proteína 2 Modificadora da Atividade de Receptores/metabolismo , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , beta-Arrestina 2/metabolismoRESUMO
A key question in receptor signaling is how specificity is realized, particularly when different receptors trigger the same biochemical pathway(s). A notable case is the two ß-adrenergic receptor (ß-AR) subtypes, ß1 and ß2, in cardiomyocytes. They are both coupled to stimulatory Gs proteins, mediate an increase in cyclic adenosine monophosphate (cAMP), and stimulate cardiac contractility; however, other effects, such as changes in gene transcription leading to cardiac hypertrophy, are prominent only for ß1-AR but not for ß2-AR. Here, we employ highly sensitive fluorescence spectroscopy approaches, in combination with a fluorescent ß-AR antagonist, to determine the presence and dynamics of the endogenous receptors on the outer plasma membrane as well as on the T-tubular network of intact adult cardiomyocytes. These techniques allow us to visualize that the ß2-AR is confined to and diffuses within the T-tubular network, as opposed to the ß1-AR, which is found to diffuse both on the outer plasma membrane as well as on the T-tubules. Upon overexpression of the ß2-AR, this compartmentalization is lost, and the receptors are also seen on the cell surface. Such receptor segregation depends on the development of the T-tubular network in adult cardiomyocytes since both the cardiomyoblast cell line H9c2 and the cardiomyocyte-differentiated human-induced pluripotent stem cells express the ß2-AR on the outer plasma membrane. These data support the notion that specific cell surface targeting of receptor subtypes can be the basis for distinct signaling and functional effects.
Assuntos
Membrana Celular/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Imagem Molecular , Miócitos Cardíacos/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Animais , Linhagem Celular , Membrana Celular/genética , Humanos , Camundongos , Camundongos Transgênicos , Receptores Adrenérgicos beta 1/genética , Receptores Adrenérgicos beta 2/genéticaRESUMO
Root exudation is a major pathway of organic carbon input into soils. It affects soil physical properties, element solubility as well as speciation, and impacts the microbial community in the rhizosphere. Root exudates contain a large number of primary and secondary plant metabolites, and the amount and composition are highly variable depending on plant species and developmental stage. Detailed information about exudate composition will allow for a better understanding of exudate-driven rhizosphere processes and their feedback loops. Although non-targeted metabolomics by high-resolution mass spectrometry is an established tool to characterize root exudate composition, the extent and depth of the information obtained depends strongly on the analytical approach applied. Here, two genotypes of Zea mays L., differing in root hair development, were used to compare six mass spectrometric approaches for the analysis of root exudates. Reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography combined with time-of-flight mass spectrometry (LC-TOF-MS), as well as direct infusion Fourier-transform ion cyclotron resonance mass spectrometry (DI-FT-ICR-MS), were applied with positive and negative ionization mode. By using the same statistical workflow, the six approaches resulted in different numbers of detected molecular features, ranging from 176 to 889, with a fraction of 48 to 69% of significant features (fold change between the two genotypes of > 2 and p-value < 0.05). All approaches revealed the same trend between genotypes, namely up-regulation of most metabolites in the root hair defective mutant (rth3). These results were in agreement with the higher total carbon and nitrogen exudation rate of the rth3-mutant as compared to the corresponding wild-type maize (WT). However, only a small fraction of features were commonly found across the different analytical approaches (20-79 features, 13-31% of the rth3-mutant up-regulated molecular formulas), highlighting the need for different mass spectrometric approaches to obtain a more comprehensive view into the composition of root exudates. In summary, 111 rth3-mutant up-regulated compounds (92 different molecular formulas) were detected with at least two different analytical approaches, while no WT up-regulated compound was found by both, LC-TOF-MS and DI-FT-ICR-MS. Zea mays L. exudate features obtained with multiple analytical approaches in our study were matched against the metabolome database of Zea mays L. (KEGG) and revealed 49 putative metabolites based on their molecular formula.
Assuntos
Metaboloma , Metabolômica , Metabolômica/métodos , Espectrometria de Massas/métodos , Exsudatos e Transudatos , Carbono/análise , Raízes de Plantas/químicaRESUMO
The SARS-CoV-2 pandemic has shown the importance of rapid and comprehensive diagnostic tools. While there are numerous rapid antigen tests available, rapid serological assays for the detection of neutralizing antibodies are and will be needed to determine not only the amount of antibodies formed after infection or vaccination but also their neutralizing potential, preventing the cell entry of SARS-CoV-2. Current active-virus neutralization assays require biosafety level 3 facilities, while virus-free surrogate assays are more versatile in applications, but still take typically several hours until results are available. To overcome these disadvantages, we developed a competitive chemiluminescence immunoassay that enables the detection of neutralizing SARS-CoV-2 antibodies within 7 min. The neutralizing antibodies bind to the viral receptor binding domain (RBD) and inhibit the binding to the human angiotensin-converting enzyme 2 (ACE2) receptor. This competitive binding inhibition test was characterized with a set of 80 samples, which could all be classified correctly. The assay results favorably compare to those obtained with a more time-intensive ELISA-based neutralization test and a commercial surrogate neutralization assay. Our test could further be used to detect individuals with a high total IgG antibody titer, but only a low neutralizing titer, as well as for monitoring neutralizing antibodies after vaccinations. This effective performance in SARS-CoV-2 seromonitoring delineates the potential for the test to be adapted to other diseases in the future.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/diagnóstico , Ensaio de Imunoadsorção Enzimática , Imunoensaio , Luminescência , Automação LaboratorialRESUMO
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
Although class A G protein-coupled receptors (GPCRs) can function as monomers, many of them form dimers and oligomers, but the mechanisms and functional relevance of such oligomerization is ill understood. Here, we investigate this problem for the CXC chemokine receptor 4 (CXCR4), a GPCR that regulates immune and hematopoietic cell trafficking, and a major drug target in cancer therapy. We combine single-molecule microscopy and fluorescence fluctuation spectroscopy to investigate CXCR4 membrane organization in living cells at densities ranging from a few molecules to hundreds of molecules per square micrometer of the plasma membrane. We observe that CXCR4 forms dynamic, transient homodimers, and that the monomer-dimer equilibrium is governed by receptor density. CXCR4 inverse agonists that bind to the receptor minor pocket inhibit CXCR4 constitutive activity and abolish receptor dimerization. A mutation in the minor binding pocket reduced the dimer-disrupting ability of these ligands. In addition, mutating critical residues in the sixth transmembrane helix of CXCR4 markedly diminished both basal activity and dimerization, supporting the notion that CXCR4 basal activity is required for dimer formation. Together, these results link CXCR4 dimerization to its density and to its activity. They further suggest that inverse agonists binding to the minor pocket suppress both dimerization and constitutive activity and may represent a specific strategy to target CXCR4.
Assuntos
Dimerização , Microscopia de Fluorescência/métodos , Receptores CXCR4/química , Receptores CXCR4/metabolismo , Membrana Celular/metabolismo , Células HEK293 , Humanos , Ligantes , Simulação de Acoplamento Molecular , Mutação , Conformação Proteica , Multimerização Proteica , Receptores CXCR4/genética , Receptores CXCR4/imunologia , Receptores de QuimiocinasRESUMO
G-protein-coupled receptors (GPCRs) are key signaling proteins that mostly function as monomers, but for several receptors constitutive dimer formation has been described and in some cases is essential for function. Using single-molecule microscopy combined with super-resolution techniques on intact cells, we describe here a dynamic monomer-dimer equilibrium of µ-opioid receptors (µORs), where dimer formation is driven by specific agonists. The agonist DAMGO, but not morphine, induces dimer formation in a process that correlates both temporally and in its agonist- and phosphorylation-dependence with ß-arrestin2 binding to the receptors. This dimerization is independent from, but may precede, µOR internalization. These data suggest a new level of GPCR regulation that links dimer formation to specific agonists and their downstream signals.
Assuntos
Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , Imagem Individual de Molécula/métodos , Animais , Células CHO , Cricetulus , Ala(2)-MePhe(4)-Gly(5)-Encefalina/química , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Transferência Ressonante de Energia de Fluorescência , Morfina/química , Morfina/farmacologia , Mutação , Naloxona/química , Naloxona/farmacologia , Naltrexona/análogos & derivados , Naltrexona/química , Naltrexona/farmacologia , Antagonistas de Entorpecentes/química , Antagonistas de Entorpecentes/farmacologia , Fosforilação , Multimerização Proteica , Receptores Opioides mu/antagonistas & inibidores , Receptores Opioides mu/genética , beta-Arrestinas/metabolismoRESUMO
(ß-)Arrestins are important regulators of G-protein-coupled receptors (GPCRs). They bind to active, phosphorylated GPCRs and thereby shut off 'classical' signalling to G proteins, trigger internalization of GPCRs via interaction with the clathrin machinery and mediate signalling via 'non-classical' pathways. In addition to two visual arrestins that bind to rod and cone photoreceptors (termed arrestin1 and arrestin4), there are only two (non-visual) ß-arrestin proteins (ß-arrestin1 and ß-arrestin2, also termed arrestin2 and arrestin3), which regulate hundreds of different (non-visual) GPCRs. Binding of these proteins to GPCRs usually requires the active form of the receptors plus their phosphorylation by G-protein-coupled receptor kinases (GRKs). The binding of receptors or their carboxy terminus as well as certain truncations induce active conformations of (ß-)arrestins that have recently been solved by X-ray crystallography. Here we investigate both the interaction of ß-arrestin with GPCRs, and the ß-arrestin conformational changes in real time and in living human cells, using a series of fluorescence resonance energy transfer (FRET)-based ß-arrestin2 biosensors. We observe receptor-specific patterns of conformational changes in ß-arrestin2 that occur rapidly after the receptor-ß-arrestin2 interaction. After agonist removal, these changes persist for longer than the direct receptor interaction. Our data indicate a rapid, receptor-type-specific, two-step binding and activation process between GPCRs and ß-arrestins. They further indicate that ß-arrestins remain active after dissociation from receptors, allowing them to remain at the cell surface and presumably signal independently. Thus, GPCRs trigger a rapid, receptor-specific activation/deactivation cycle of ß-arrestins, which permits their active signalling.
Assuntos
Arrestinas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Arrestinas/química , Técnicas Biossensoriais , Bovinos , Linhagem Celular , Membrana Celular/metabolismo , Sobrevivência Celular , Cristalografia por Raios X , Transferência Ressonante de Energia de Fluorescência , Humanos , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Receptores Acoplados a Proteínas G/química , Transdução de Sinais , Especificidade por Substrato , Fatores de Tempo , beta-ArrestinasRESUMO
G protein-coupled receptors (GPCRs) are key biological switches that transmit both internal and external stimuli into the cell interior. Among the GPCRs, the "light receptor" rhodopsin has been shown to activate with a rearrangement of the transmembrane (TM) helix bundle within â¼1 ms, while all other receptors are thought to become activated within â¼50 ms to seconds at saturating concentrations. Here, we investigate synchronous stimulation of a dimeric GPCR, the metabotropic glutamate receptor type 1 (mGluR1), by two entirely different methods: (i) UV light-triggered uncaging of glutamate in intact cells or (ii) piezo-driven solution exchange in outside-out patches. Submillisecond FRET recordings between labels at intracellular receptor sites were used to record conformational changes in the mGluR1. At millimolar ligand concentrations, the initial rearrangement between the mGluR1 subunits occurs at a speed of τ1 â¼ 1-2 ms and requires the occupancy of both binding sites in the mGluR1 dimer. These rapid changes were followed by significantly slower conformational changes in the TM domain (τ2 â¼ 20 ms). Receptor deactivation occurred with time constants of â¼40 and â¼900 ms for the inter- and intrasubunit conformational changes, respectively. Together, these data show that, at high glutamate concentrations, the initial intersubunit activation of mGluR1 proceeds with millisecond speed, that there is loose coupling between this initial step and activation of the TM domain, and that activation and deactivation follow a cyclic pathway, including-in addition to the inactive and active states-at least two metastable intermediate states.
Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Dimerização , Células HEK293 , Humanos , Cinética , Receptores Acoplados a Proteínas G/efeitos da radiaçãoRESUMO
Insect neuropeptide receptors, including allatostatin receptor type C (AstR-C), a G protein-coupled receptor, are among the potential targets for designing next-generation pesticides that despite their importance in offering a new mode-of-action have been overlooked. Focusing on AstR-C of Thaumetopoea pityocampa, a common pest in Mediterranean countries, by employing resonance energy transfer-based methods, we showed Gαi/o coupling and ß-arrestin recruitment of the receptor at sub-nanomolar and nanomolar ranges of the endogenous ligand, AST-C, respectively. Molecular docking and molecular dynamics simulation studies revealed the importance of extracellular loop 2 in AstRC/AST-C interaction, and a combination of in silico and in vitro approaches showed the substantial role of Q2716.55 in G protein-dependent activation of AstR-C possibly via contributing to the flexibility of the receptor's structure. The functional and structural insights obtained on T. pit AstR-C positively assist future efforts in developing environmentally friendly pest control agents that are needed urgently.
Assuntos
Proteínas de Insetos/química , Lepidópteros , Neuropeptídeos , Receptores de Neuropeptídeos/química , Animais , Simulação de Acoplamento Molecular , Controle de Pragas , Receptores Acoplados a Proteínas GRESUMO
G protein-coupled receptors (GPCRs) are biologic switches that transduce extracellular stimuli into intracellular responses in the cell. Temporally resolving GPCR transduction pathways is key to understanding how cell signaling occurs. Here, we investigate the kinetics and dynamics of the activation and early signaling steps of the CXC chemokine receptor (CXCR) 4 in response to its natural ligands CXC chemokine ligand (CXCL) 12 and macrophage migration inhibitory factor (MIF), using Förster resonance energy transfer-based approaches. We show that CXCR4 presents a multifaceted response to CXCL12, with receptor activation (≈0.6 seconds) followed by a rearrangement in the receptor/G protein complex (≈1 seconds), a slower dimer rearrangement (≈1.7 seconds), and prolonged G protein activation (≈4 seconds). In comparison, MIF distinctly modulates every step of the transduction pathway, indicating distinct activation mechanisms and reflecting the different pharmacological properties of these two ligands. Our study also indicates that CXCR4 exhibits some degree of ligand-independent activity, a relevant feature for drug development. SIGNIFICANCE STATEMENT: The CXC chemokine ligand (CXCL) 12/CXC chemokine receptor (CXCR) 4 axis represents a well-established therapeutic target for cancer treatment. We demonstrate that CXCR4 exhibits a multifaceted response that involves dynamic receptor dimer rearrangements and that is kinetically embedded between receptor-G protein complex rearrangements and G protein activation. The alternative endogenous ligand macrophage migration inhibitory factor behaves opposite to CXCL12 in each assay studied and does not lead to G protein activation. This detailed understanding of the receptor activation may aid in the development of more specific drugs against this target.
Assuntos
Quimiocina CXCL12/metabolismo , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Receptores CXCR4/química , Receptores CXCR4/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Cinética , Ligação Proteica , Multimerização Proteica , Transdução de SinaisRESUMO
BACKGROUND: Approximately 40% of prescribed drugs exert their activity via GTP-binding protein-coupled receptors (GPCRs). Once activated, these receptors cause transient changes in the concentration of second messengers, e.g., cyclic adenosine 3',5'-monophosphate (cAMP). Specific and efficacious genetically encoded biosensors have been developed to monitor cAMP fluctuations with high spatial and temporal resolution in living cells or tissue. A well characterized biosensor for cAMP is the Förster resonance energy transfer (FRET)-based Epac1-camps protein. Pharmacological characterization of newly developed ligands acting at GPCRs often includes numerical quantification of the second messenger amount that was produced. RESULTS: To quantify cellular cAMP concentrations, we bacterially over-expressed and purified Epac1-camps and applied the purified protein in a cell-free detection assay for cAMP in a multi-well format. We found that the biosensor can detect as little as 0.15 pmol of cAMP, and that the sensitivity is not impaired by non-physiological salt concentrations or pH values. Notably, the assay tolerated desiccation and storage of the protein without affecting Epac1-camps cyclic nucleotide sensitivity. CONCLUSIONS: We found that determination cAMP in lysates obtained from cell assays or tissue samples by purified Epac1-camps is a robust, fast, and sensitive assay suitable for routine and high throughput analyses.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Fluorescência , Nucleotídeos Cíclicos , Técnicas Biossensoriais/métodos , Técnicas de Cultura de Células , Clonagem Molecular , AMP Cíclico , Escherichia coli/genética , Fatores de Troca do Nucleotídeo Guanina , Concentração de Íons de Hidrogênio , Nucleotídeos Cíclicos/genética , Receptores Acoplados a Proteínas G , Proteínas RecombinantesRESUMO
The melanocortin 4 receptor (MC4R) is a key player in hypothalamic weight regulation and energy expenditure as part of the leptin-melanocortin pathway. Mutations in this G protein coupled receptor (GPCR) are the most common cause for monogenetic obesity, which appears to be mediated by changes in the anorectic action of MC4R via GS-dependent cyclic adenosine-monophosphate (cAMP) signaling as well as other signaling pathways. To study potential bias in the effects of MC4R mutations between the different signaling pathways, we investigated three major MC4R mutations: a GS loss-of-function (S127L) and a GS gain-of-function mutant (H158R), as well as the most common European single nucleotide polymorphism (V103I). We tested signaling of all four major G protein families plus extracellular regulated kinase (ERK) phosphorylation and ß-arrestin2 recruitment, using the two endogenous agonists, α- and ß-melanocyte stimulating hormone (MSH), along with a synthetic peptide agonist (NDP-α-MSH). The S127L mutation led to a full loss-of-function in all investigated pathways, whereas V103I and H158R were clearly biased towards the Gq/11 pathway when challenged with the endogenous ligands. These results show that MC4R mutations can cause vastly different changes in the various MC4R signaling pathways and highlight the importance of a comprehensive characterization of receptor mutations.
Assuntos
Mutação , Receptor Tipo 4 de Melanocortina/genética , Receptor Tipo 4 de Melanocortina/metabolismo , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Leptina/metabolismo , Ligantes , Melanocortinas/metabolismo , Modelos Teóricos , Obesidade/genética , Fosforilação , Polimorfismo de Nucleotídeo Único , Receptores Acoplados a Proteínas G/metabolismo , alfa-MSH/metabolismoRESUMO
G protein-coupled receptors (GPCRs) are regulated by complex molecular mechanisms, both in physiologic and pathologic conditions, and their signaling can be intricate. Many factors influence their signaling behavior, including the type of ligand that activates the GPCR, the presence of interacting partners, the kinetics involved, or their location. The two CXC-type chemokine receptors, CXC chemokine receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3), both members of the GPCR superfamily, are important and established therapeutic targets in relation to cancer, human immunodeficiency virus infection, and inflammatory diseases. Therefore, it is crucial to understand how the signaling of these receptors works to be able to specifically target them. In this review, we discuss how the signaling pathways activated by CXCR4 and ACKR3 can vary in different situations. G protein signaling of CXCR4 depends on the cellular context, and discrepancies exist depending on the cell lines used. ACKR3, as an atypical chemokine receptor, is generally reported to not activate G proteins but can broaden its signaling spectrum upon heteromerization with other receptors, such as CXCR4, endothelial growth factor receptor, or the α 1-adrenergic receptor (α 1-AR). Also, CXCR4 forms heteromers with CC chemokine receptor (CCR) 2, CCR5, the Na+/H+ exchanger regulatory factor 1, CXCR3, α 1-AR, and the opioid receptors, which results in differential signaling from that of the monomeric subunits. In addition, CXCR4 is present on membrane rafts but can go into the nucleus during cancer progression, probably acquiring different signaling properties. In this review, we also provide an overview of the currently known critical amino acids involved in CXCR4 and ACKR3 signaling.