RESUMO
The Duffy antigen receptor is a seven-transmembrane (7TM) protein expressed primarily at the surface of red blood cells and displays strikingly promiscuous binding to multiple inflammatory and homeostatic chemokines. It serves as the basis of the Duffy blood group system in humans and also acts as the primary attachment site for malarial parasite Plasmodium vivax and pore-forming toxins secreted by Staphylococcus aureus. Here, we comprehensively profile transducer coupling of this receptor, discover potential non-canonical signaling pathways, and determine the cryoelectron microscopy (cryo-EM) structure in complex with the chemokine CCL7. The structure reveals a distinct binding mode of chemokines, as reflected by relatively superficial binding and a partially formed orthosteric binding pocket. We also observe a dramatic shortening of TM5 and 6 on the intracellular side, which precludes the formation of the docking site for canonical signal transducers, thereby providing a possible explanation for the distinct pharmacological and functional phenotype of this receptor.
RESUMO
Binding of arrestin to phosphorylated G protein-coupled receptors (GPCRs) is crucial for modulating signaling. Once internalized, some GPCRs remain complexed with ß-arrestins, while others interact only transiently; this difference affects GPCR signaling and recycling. Cell-based and in vitro biophysical assays reveal the role of membrane phosphoinositides (PIPs) in ß-arrestin recruitment and GPCR-ß-arrestin complex dynamics. We find that GPCRs broadly stratify into two groups, one that requires PIP binding for ß-arrestin recruitment and one that does not. Plasma membrane PIPs potentiate an active conformation of ß-arrestin and stabilize GPCR-ß-arrestin complexes by promoting a fully engaged state of the complex. As allosteric modulators of GPCR-ß-arrestin complex dynamics, membrane PIPs allow for additional conformational diversity beyond that imposed by GPCR phosphorylation alone. For GPCRs that require membrane PIP binding for ß-arrestin recruitment, this provides a mechanism for ß-arrestin release upon translocation of the GPCR to endosomes, allowing for its rapid recycling.
Assuntos
Arrestinas , Fosfatidilinositóis , beta-Arrestinas/metabolismo , Fosfatidilinositóis/metabolismo , Arrestinas/metabolismo , beta-Arrestina 1/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMO
ß-arrestins bind G protein-coupled receptors to terminate G protein signaling and to facilitate other downstream signaling pathways. Using single-molecule fluorescence resonance energy transfer imaging, we show that ß-arrestin is strongly autoinhibited in its basal state. Its engagement with a phosphopeptide mimicking phosphorylated receptor tail efficiently releases the ß-arrestin tail from its N domain to assume distinct conformations. Unexpectedly, we find that ß-arrestin binding to phosphorylated receptor, with a phosphorylation barcode identical to the isolated phosphopeptide, is highly inefficient and that agonist-promoted receptor activation is required for ß-arrestin activation, consistent with the release of a sequestered receptor C tail. These findings, together with focused cellular investigations, reveal that agonism and receptor C-tail release are specific determinants of the rate and efficiency of ß-arrestin activation by phosphorylated receptor. We infer that receptor phosphorylation patterns, in combination with receptor agonism, synergistically establish the strength and specificity with which diverse, downstream ß-arrestin-mediated events are directed.
Assuntos
Fosfopeptídeos , Receptores Acoplados a Proteínas G , Fosfopeptídeos/metabolismo , Fosforilação , Receptores Acoplados a Proteínas G/metabolismo , beta-Arrestina 1/metabolismo , beta-Arrestinas/metabolismoRESUMO
Heterotrimetic G proteins consist of four subfamilies (Gs, Gi/o, Gq/11, and G12/13) that mediate signaling via G-protein-coupled receptors (GPCRs), principally by receptors binding Gα C termini. G-protein-coupling profiles govern GPCR-induced cellular responses, yet receptor sequence selectivity determinants remain elusive. Here, we systematically quantified ligand-induced interactions between 148 GPCRs and all 11 unique Gα subunit C termini. For each receptor, we probed chimeric Gα subunit activation via a transforming growth factor-α (TGF-α) shedding response in HEK293 cells lacking endogenous Gq/11 and G12/13 proteins, and complemented G-protein-coupling profiles through a NanoBiT-G-protein dissociation assay. Interrogation of the dataset identified sequence-based coupling specificity features, inside and outside the transmembrane domain, which we used to develop a coupling predictor that outperforms previous methods. We used the predictor to engineer designer GPCRs selectively coupled to G12. This dataset of fine-tuned signaling mechanisms for diverse GPCRs is a valuable resource for research in GPCR signaling.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Modelos Biológicos , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Feminino , Células HEK293 , Proteínas Heterotriméricas de Ligação ao GTP/genética , Humanos , Masculino , Células PC-3 , Receptores Acoplados a Proteínas G/genéticaRESUMO
G protein-coupled receptor (GPCR) signaling, mediated by hetero-trimeric G proteins, can be differentially controlled by agonists. At a molecular level, this is thought to occur principally via stabilization of distinct receptor conformations by individual ligands. These distinct conformations control subsequent recruitment of transducer and effector proteins. Here, we report that ligand efficacy at the calcitonin GPCR (CTR) is also correlated with ligand-dependent alterations to G protein conformation. We observe ligand-dependent differences in the sensitivity of the G protein ternary complex to disruption by GTP, due to conformational differences in the receptor-bound G protein hetero-trimer. This results in divergent agonist-dependent receptor-residency times for the hetero-trimeric G protein and different accumulation rates for downstream second messengers. This study demonstrates that factors influencing efficacy extend beyond receptor conformation(s) and expands understanding of the molecular basis for how G proteins control/influence efficacy. This has important implications for the mechanisms that underlie ligand-mediated biased agonism. VIDEO ABSTRACT.
Assuntos
Proteínas de Ligação ao GTP/química , Guanosina Trifosfato/farmacologia , Receptores da Calcitonina/agonistas , Receptores da Calcitonina/química , Difosfato de Adenosina/biossíntese , Animais , Células COS , Chlorocebus aethiops , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Ligantes , Conformação Proteica , Multimerização Proteica , Receptores da Calcitonina/metabolismoRESUMO
Endogenous parathyroid hormone (PTH) and PTH-related peptide (PTHrP) bind to the parathyroid hormone receptor 1 (PTH1R) and activate the stimulatory G-protein (Gs) signaling pathway. Intriguingly, the two ligands have distinct signaling and physiological properties: PTH evokes prolonged Gs activation, whereas PTHrP evokes transient Gs activation with reduced bone-resorption effects. The distinct molecular actions are ascribed to the differences in ligand recognition and dissociation kinetics. Here, we report cryoelectron microscopic structures of six forms of the human PTH1R-Gs complex in the presence of PTH or PTHrP at resolutions of 2.8 -4.1 Å. A comparison of the PTH-bound and PTHrP-bound structures reveals distinct ligand-receptor interactions underlying the ligand affinity and selectivity. Furthermore, five distinct PTH-bound structures, combined with computational analyses, provide insights into the unique and complex process of ligand dissociation from the receptor and shed light on the distinct durations of signaling induced by PTH and PTHrP.
Assuntos
Proteína Relacionada ao Hormônio Paratireóideo , Receptor Tipo 1 de Hormônio Paratireóideo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Humanos , Ligantes , Hormônio Paratireóideo/química , Hormônio Paratireóideo/metabolismo , Hormônio Paratireóideo/farmacologia , Proteína Relacionada ao Hormônio Paratireóideo/química , Proteína Relacionada ao Hormônio Paratireóideo/genética , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismoRESUMO
G protein-coupled receptors (GPCRs) generally accommodate specific ligands in the orthosteric-binding pockets. Ligand binding triggers a receptor allosteric conformational change that leads to the activation of intracellular transducers, G proteins and ß-arrestins. Because these signals often induce adverse effects, the selective activation mechanism for each transducer must be elucidated. Thus, many orthosteric-biased agonists have been developed, and intracellular-biased agonists have recently attracted broad interest. These agonists bind within the receptor intracellular cavity and preferentially tune the specific signalling pathway over other signalling pathways, without allosteric rearrangement of the receptor from the extracellular side1-3. However, only antagonist-bound structures are currently available1,4-6, and there is no evidence to support that biased agonist binding occurs within the intracellular cavity. This limits the comprehension of intracellular-biased agonism and potential drug development. Here we report the cryogenic electron microscopy structure of a complex of Gs and the human parathyroid hormone type 1 receptor (PTH1R) bound to a PTH1R agonist, PCO371. PCO371 binds within an intracellular pocket of PTH1R and directly interacts with Gs. The PCO371-binding mode rearranges the intracellular region towards the active conformation without extracellularly induced allosteric signal propagation. PCO371 stabilizes the significantly outward-bent conformation of transmembrane helix 6, which facilitates binding to G proteins rather than ß-arrestins. Furthermore, PCO371 binds within the highly conserved intracellular pocket, activating 7 out of the 15 class B1 GPCRs. Our study identifies a new and conserved intracellular agonist-binding pocket and provides evidence of a biased signalling mechanism that targets the receptor-transducer interface.
Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP , Imidazolidinas , Receptores Acoplados a Proteínas G , Humanos , Regulação Alostérica , beta-Arrestinas/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Desenvolvimento de Medicamentos , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/ultraestrutura , Imidazolidinas/química , Imidazolidinas/farmacologia , Ligantes , Modelos Moleculares , Conformação Proteica/efeitos dos fármacos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/classificação , Receptores Acoplados a Proteínas G/ultraestrutura , Transdução de SinaisRESUMO
The ß3-adrenergic receptor (ß3AR) is predominantly expressed in adipose tissue and urinary bladder and has emerged as an attractive drug target for the treatment of type 2 diabetes, obesity, and overactive bladder (OAB). Here, we report the cryogenic electron microscopy structure of the ß3AR-Gs signaling complex with the selective agonist mirabegron, a first-in-class drug for OAB. Comparison of this structure with the previously reported ß1AR and ß2AR structures reveals a receptor activation mechanism upon mirabegron binding to the orthosteric site. Notably, the narrower exosite in ß3AR creates a perpendicular pocket for mirabegron. Mutational analyses suggest that a combination of both the exosite shape and the amino-acid-residue substitutions defines the drug selectivity of the ßAR agonists. Our findings provide a molecular basis for ßAR subtype selectivity, allowing the design of more-selective agents with fewer adverse effects.
Assuntos
Acetanilidas/química , Agonistas de Receptores Adrenérgicos beta 3/química , Receptores Adrenérgicos beta 3/química , Receptores Adrenérgicos beta 3/metabolismo , Tiazóis/química , Acetanilidas/metabolismo , Agonistas de Receptores Adrenérgicos beta 3/metabolismo , Animais , Sítios de Ligação , Microscopia Crioeletrônica , Cães , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Receptores Adrenérgicos beta 3/genética , Tiazóis/metabolismoRESUMO
About 150 post-transcriptional RNA modifications have been identified in all kingdoms of life. During RNA catabolism, most modified nucleosides are resistant to degradation and are released into the extracellular space. In this study, we explored the physiological role of these extracellular modified nucleosides and found that N6-methyladenosine (m6A), widely recognized as an epigenetic mark in RNA, acts as a ligand for the human adenosine A3 receptor, for which it has greater affinity than unmodified adenosine. We used structural modeling to define the amino acids required for specific binding of m6A to the human A3 receptor. We also demonstrated that m6A was dynamically released in response to cytotoxic stimuli and facilitated type I allergy in vivo. Our findings implicate m6A as a signaling molecule capable of activating G protein-coupled receptors (GPCRs) and triggering pathophysiological responses, a previously unreported property of RNA modifications.
Assuntos
Adenosina/análogos & derivados , Epigênese Genética , Processamento Pós-Transcricional do RNA , Receptor A3 de Adenosina/metabolismo , Transdução de Sinais , Adenosina/genética , Adenosina/metabolismo , Animais , Feminino , Células HEK293 , Humanos , Masculino , Coelhos , Receptor A3 de Adenosina/genéticaRESUMO
G-protein-coupled receptors (GPCRs), also known as seven transmembrane receptors (7TMRs), typically interact with two distinct signal-transducers, i.e., G proteins and ß-arrestins (ßarrs). Interestingly, there are some non-canonical 7TMRs that lack G protein coupling but interact with ßarrs, although an understanding of their transducer coupling preference, downstream signaling, and structural mechanism remains elusive. Here, we characterize two such non-canonical 7TMRs, namely, the decoy D6 receptor (D6R) and the complement C5a receptor subtype 2 (C5aR2), in parallel with their canonical GPCR counterparts. We discover that D6R and C5aR2 efficiently couple to ßarrs, exhibit distinct engagement of GPCR kinases (GRKs), and activate non-canonical downstream signaling pathways. We also observe that ßarrs adopt distinct conformations for D6R and C5aR2, compared to their canonical GPCR counterparts, in response to common natural agonists. Our study establishes D6R and C5aR2 as ßarr-coupled 7TMRs and provides key insights into their regulation and signaling with direct implication for biased agonism.
Assuntos
Membrana Celular/metabolismo , Conformação Proteica , Transdução de Sinais , beta-Arrestinas/química , Animais , Proteínas de Ligação ao GTP/química , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Transporte Proteico , Receptor da Anafilatoxina C5a/metabolismoRESUMO
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) compose the largest family of transmembrane receptors and are targets of approximately one-third of Food and Drug Administration-approved drugs owing to their involvement in almost all physiologic processes. GPCR signaling occurs through the activation of heterotrimeric G-protein complexes and ß-arrestins, both of which serve as transducers, resulting in distinct cellular responses. Despite seeming simple at first glance, accumulating evidence indicates that activation of either transducer is not a straightforward process as a stimulation of a single molecule has the potential to activate multiple signaling branches. The complexity of GPCR signaling arises from the aspects of G-protein-coupling selectivity, biased signaling, interpathway crosstalk, and variable molecular modifications generating these diverse signaling patterns. Numerous questions relative to these aspects of signaling remained unanswered until the recent development of CRISPR genome-editing technology. Such genome editing technology presents opportunities to chronically eliminate the expression of G-protein subunits, ß-arrestins, G-protein-coupled receptor kinases (GRKs), and many other signaling nodes in the GPCR pathways at one's convenience. Here, we review the practicality of using CRISPR-derived knockout (KO) cells in the experimental contexts of unraveling the molecular details of GPCR signaling mechanisms. To mention a few, KO cells have revealed the contribution of ß-arrestins in ERK activation, Gα protein selectivity, GRK-based regulation of GPCRs, and many more, hence validating its broad applicability in GPCR studies. SIGNIFICANCE STATEMENT: This review emphasizes the practical application of G-protein-coupled receptor (GPCR) transducer knockout (KO) cells in dissecting the intricate regulatory mechanisms of the GPCR signaling network. Currently available cell lines, along with accumulating KO cell lines in diverse cell types, offer valuable resources for systematically elucidating GPCR signaling regulation. Given the association of GPCR signaling with numerous diseases, uncovering the system-based signaling map is crucial for advancing the development of novel drugs targeting specific diseases.
Assuntos
Receptores Acoplados a Proteínas G , Transdução de Sinais , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Humanos , Animais , Linhagem Celular , beta-Arrestinas/metabolismoRESUMO
Arrestin proteins bind to active, phosphorylated G-protein-coupled receptors (GPCRs), thereby preventing G-protein coupling, triggering receptor internalization and affecting various downstream signalling pathways1,2. Although there is a wealth of structural information detailing the interactions between GPCRs and G proteins, less is known about how arrestins engage GPCRs. Here we report a cryo-electron microscopy structure of full-length human neurotensin receptor 1 (NTSR1) in complex with truncated human ß-arrestin 1 (ßarr1(ΔCT)). We find that phosphorylation of NTSR1 is critical for the formation of a stable complex with ßarr1(ΔCT), and identify phosphorylated sites in both the third intracellular loop and the C terminus that may promote this interaction. In addition, we observe a phosphatidylinositol-4,5-bisphosphate molecule forming a bridge between the membrane side of NTSR1 transmembrane segments 1 and 4 and the C-lobe of arrestin. Compared with a structure of a rhodopsin-arrestin-1 complex, in our structure arrestin is rotated by approximately 85° relative to the receptor. These findings highlight both conserved aspects and plasticity among arrestin-receptor interactions.
Assuntos
Modelos Moleculares , Receptores de Neurotensina/química , beta-Arrestina 1/química , Microscopia Crioeletrônica , Humanos , Fosforilação , Estabilidade Proteica , Estrutura Quaternária de Proteína , Receptores de Neurotensina/metabolismo , beta-Arrestina 1/metabolismoRESUMO
Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, including diabetes and obesity1. Structures of active receptors reveal peptide agonists engage deep within the receptor core, leading to an outward movement of extracellular loop 3 and the tops of transmembrane helices 6 and 7, an inward movement of transmembrane helix 1, reorganization of extracellular loop 2 and outward movement of the intracellular side of transmembrane helix 6, resulting in G-protein interaction and activation2-6. Here we solved the structure of a non-peptide agonist, TT-OAD2, bound to the glucagon-like peptide-1 (GLP-1) receptor. Our structure identified an unpredicted non-peptide agonist-binding pocket in which reorganization of extracellular loop 3 and transmembrane helices 6 and 7 manifests independently of direct ligand interaction within the deep transmembrane domain pocket. TT-OAD2 exhibits biased agonism, and kinetics of G-protein activation and signalling that are distinct from peptide agonists. Within the structure, TT-OAD2 protrudes beyond the receptor core to interact with the lipid or detergent, providing an explanation for the distinct activation kinetics that may contribute to the clinical efficacy of this compound series. This work alters our understanding of the events that drive the activation of class B receptors.
Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Isoquinolinas/farmacologia , Fenilalanina/análogos & derivados , Piridinas/farmacologia , Animais , Células CHO , Cricetinae , Cricetulus , Receptor do Peptídeo Semelhante ao Glucagon 1/química , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Humanos , Isoquinolinas/química , Cinética , Modelos Moleculares , Fenilalanina/química , Fenilalanina/farmacologia , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Piridinas/química , Homologia Estrutural de ProteínaRESUMO
FFAR1 is a G-protein-coupled receptor (GPCR) that responds to circulating free fatty acids to enhance glucose-stimulated insulin secretion and release of incretin hormones. Due to the glucose-lowering effect of FFAR1 activation, potent agonists for this receptor have been developed for the treatment of diabetes. Previous structural and biochemical studies of FFAR1 showed multiple sites of ligand binding to the inactive state but left the mechanism of fatty acid interaction and receptor activation unknown. We used cryo-electron microscopy to elucidate structures of activated FFAR1 bound to a Gq mimetic, which were induced either by the endogenous FFA ligand docosahexaenoic acid or γ-linolenic acid and the agonist drug TAK-875. Our data identify the orthosteric pocket for fatty acids and show how both endogenous hormones and synthetic agonists induce changes in helical packing along the outside of the receptor that propagate to exposure of the G-protein-coupling site. These structures show how FFAR1 functions without the highly conserved "DRY" and "NPXXY" motifs of class A GPCRs and also illustrate how the orthosteric site of a receptor can be bypassed by membrane-embedded drugs to confer full activation of G protein signaling.
Assuntos
Ácidos Graxos , Insulina , Insulina/metabolismo , Ligantes , Microscopia Crioeletrônica , Receptores Acoplados a Proteínas G/metabolismo , Ácidos Graxos não Esterificados , GlucoseRESUMO
Drugs targeting the µ-opioid receptor (µOR) are the most effective analgesics available but are also associated with fatal respiratory depression through a pathway that remains unclear. Here we investigated the mechanistic basis of action of lofentanil (LFT) and mitragynine pseudoindoxyl (MP), two µOR agonists with different safety profiles. LFT, one of the most lethal opioids, and MP, a kratom plant derivative with reduced respiratory depression in animal studies, exhibited markedly different efficacy profiles for G protein subtype activation and ß-arrestin recruitment. Cryo-EM structures of µOR-Gi1 complex with MP (2.5 Å) and LFT (3.2 Å) revealed that the two ligands engage distinct subpockets, and molecular dynamics simulations showed additional differences in the binding site that promote distinct active-state conformations on the intracellular side of the receptor where G proteins and ß-arrestins bind. These observations highlight how drugs engaging different parts of the µOR orthosteric pocket can lead to distinct signaling outcomes.
Assuntos
Analgésicos Opioides , Transdução de Sinais , Animais , beta-Arrestinas/metabolismo , Analgésicos Opioides/química , Analgésicos Opioides/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Sítios de LigaçãoRESUMO
Galanin is a neuropeptide expressed in the central and peripheral nervous systems, where it regulates various processes including neuroendocrine release, cognition, and nerve regeneration. Three G-protein coupled receptors (GPCRs) for galanin have been discovered, which is the focus of efforts to treat diseases including Alzheimer's disease, anxiety, and addiction. To understand the basis of the ligand preferences of the receptors and to assist structure-based drug design, we used cryo-electron microscopy (cryo-EM) to solve the molecular structure of GALR2 bound to galanin and a cognate heterotrimeric G-protein, providing a molecular view of the neuropeptide binding site. Mutant proteins were assayed to help reveal the basis of ligand specificity, and structural comparison between the activated GALR2 and inactive hß2AR was used to relate galanin binding to the movements of transmembrane (TM) helices and the G-protein interface.
Assuntos
Galanina/química , Proteínas Heterotriméricas de Ligação ao GTP , Receptor Tipo 2 de Galanina/química , Microscopia Crioeletrônica , Galanina/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Ligantes , Receptor Tipo 2 de Galanina/metabolismoRESUMO
Small intestinal mononuclear cells that express CX3CR1 (CX3CR1+ cells) regulate immune responses1-5. CX3CR1+ cells take up luminal antigens by protruding their dendrites into the lumen1-4,6. However, it remains unclear how dendrite protrusion by CX3CR1+ cells is induced in the intestine. Here we show in mice that the bacterial metabolites pyruvic acid and lactic acid induce dendrite protrusion via GPR31 in CX3CR1+ cells. Mice that lack GPR31, which was highly and selectively expressed in intestinal CX3CR1+ cells, showed defective dendrite protrusions of CX3CR1+ cells in the small intestine. A methanol-soluble fraction of the small intestinal contents of specific-pathogen-free mice, but not germ-free mice, induced dendrite extension of intestinal CX3CR1+ cells in vitro. We purified a GPR31-activating fraction, and identified lactic acid. Both lactic acid and pyruvic acid induced dendrite extension of CX3CR1+ cells of wild-type mice, but not of Gpr31b-/- mice. Oral administration of lactate and pyruvate enhanced dendrite protrusion of CX3CR1+ cells in the small intestine of wild-type mice, but not in that of Gpr31b-/- mice. Furthermore, wild-type mice treated with lactate or pyruvate showed an enhanced immune response and high resistance to intestinal Salmonella infection. These findings demonstrate that lactate and pyruvate, which are produced in the intestinal lumen in a bacteria-dependent manner, contribute to enhanced immune responses by inducing GPR31-mediated dendrite protrusion of intestinal CX3CR1+ cells.
Assuntos
Bactérias/metabolismo , Receptor 1 de Quimiocina CX3C/metabolismo , Extensões da Superfície Celular/metabolismo , Intestino Delgado/citologia , Intestino Delgado/microbiologia , Ácido Láctico/metabolismo , Ácido Pirúvico/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Bactérias/imunologia , Receptor 1 de Quimiocina CX3C/deficiência , Receptor 1 de Quimiocina CX3C/genética , Extensões da Superfície Celular/efeitos dos fármacos , Extensões da Superfície Celular/imunologia , Feminino , Células HEK293 , Humanos , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/imunologia , Ácido Láctico/farmacologia , Lactobacillus helveticus/metabolismo , Masculino , Metanol , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Ácido Pirúvico/farmacologia , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/genética , Salmonella/imunologia , Salmonella/metabolismoRESUMO
Neurotensin receptor 1 (NTSR1) is a G-protein-coupled receptor (GPCR) that engages multiple subtypes of G protein, and is involved in the regulation of blood pressure, body temperature, weight and the response to pain. Here we present structures of human NTSR1 in complex with the agonist JMV449 and the heterotrimeric Gi1 protein, at a resolution of 3 Å. We identify two conformations: a canonical-state complex that is similar to recently reported GPCR-Gi/o complexes (in which the nucleotide-binding pocket adopts more flexible conformations that may facilitate nucleotide exchange), and a non-canonical state in which the G protein is rotated by about 45 degrees relative to the receptor and exhibits a more rigid nucleotide-binding pocket. In the non-canonical state, NTSR1 exhibits features of both active and inactive conformations, which suggests that the structure may represent an intermediate form along the activation pathway of G proteins. This structural information, complemented by molecular dynamics simulations and functional studies, provides insights into the complex process of G-protein activation.
Assuntos
Microscopia Crioeletrônica , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/ultraestrutura , Receptores de Neurotensina/química , Receptores de Neurotensina/ultraestrutura , Sítios de Ligação , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Ligação Proteica , Conformação Proteica , Receptores de Neurotensina/agonistas , Receptores de Neurotensina/metabolismoRESUMO
G proteincoupled receptors (GPCRs) are involved in regulation of manifold physiological processes through coupling to heterotrimeric G proteins upon ligand stimulation. Classical therapeutically active drugs simultaneously initiate several downstream signaling pathways, whereas biased ligands, which stabilize subsets of receptor conformations, elicit more selective signaling. This concept of functional selectivity of a ligand has emerged as an interesting property for the development of new therapeutic molecules. Biased ligands are expected to have superior efficacy and/or reduced side effects by regulating biological functions of GPCRs in a more precise way. In the last decade, 5-HT7 receptor (5-HT7R) has become a promising target for the treatment of neuropsychiatric disorders, sleep and circadian rhythm disorders, and pathological pain. In this study, we showed that Serodolin is unique among a number of agonists and antagonists tested: it behaves as an antagonist/inverse agonist on Gs signaling while inducing ERK activation through a ß-arrestindependent signaling mechanism that requires c-SRC activation. Moreover, we showed that Serodolin clearly decreases hyperalgesia and pain sensation in response to inflammatory, thermal, and mechanical stimulation. This antinociceptive effect could not be observed in 5-HT7R knockout (KO) mice and was fully blocked by administration of SB269-970, a specific 5-HT7R antagonist, demonstrating the specificity of action of Serodolin. Physiological effects of 5-HT7R stimulation have been classically shown to result from Gs-dependent adenylyl cyclase activation. In this study, using a ß-arrestinbiased agonist, we provided insight into the molecular mechanism triggered by 5-HT7R and revealed its therapeutic potential in the modulation of pain response.