RESUMO
The endothelin receptor type B (ETB) exhibits promiscuous coupling with various heterotrimeric G protein subtypes including Gs, Gi/o, Gq/11, and G12/13. Recent fluorescence and structural studies have raised questions regarding the coupling efficiencies and determinants of these G protein subtypes. Herein, by utilizing an integrative approach, combining hydrogen/deuterium exchange mass spectrometry and NanoLuc Binary Technology-based cellular systems, we investigated conformational changes of Gs, Gi, and Gq triggered by ETB activation. ETB coupled to Gi and Gq but not with Gs. We underscored the critical roles of specific regions, including the C terminus of Gα and intracellular loop 2 (ICL2) of ETB in ETB-Gi1 or ETB-Gq coupling. Although The C terminus of Gα is essential for ETB-Gi1 and ETB-Gq coupling, ETB ICL2 influences Gq-coupling but not Gi1-coupling. Our results suggest a differential coupling efficiency of ETB with Gs, Gi1, and Gq, accompanied by distinct conformational changes in G proteins upon ETB-induced activation.
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Pyroglutamylated RF-amide peptide (QRFP) is a peptide hormone with a C-terminal RF-amide motif. QRFP selectively activates a class A G-protein-coupled receptor (GPCR) GPR103 to exert various physiological functions such as energy metabolism and appetite regulation. Here, we report the cryo-electron microscopy structure of the QRFP26-GPR103-Gq complex at 3.19 Å resolution. QRFP26 adopts an extended structure bearing no secondary structure, with its N-terminal and C-terminal sides recognized by extracellular and transmembrane domains of GPR103 respectively. This movement, reminiscent of class B1 GPCRs except for orientation and structure of the ligand, is critical for the high-affinity binding and receptor specificity of QRFP26. Mutagenesis experiments validate the functional importance of the binding mode of QRFP26 by GPR103. Structural comparisons with closely related receptors, including RY-amide peptide-recognizing GPCRs, revealed conserved and diversified peptide recognition mechanisms, providing profound insights into the biological significance of RF-amide peptides. Collectively, this study not only advances our understanding of GPCR-ligand interactions, but also paves the way for the development of novel therapeutics targeting metabolic and appetite disorders and emergency medical care.
Assuntos
Microscopia Crioeletrônica , Receptores Acoplados a Proteínas G , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/química , Humanos , Células HEK293 , Ligação Proteica , Ligantes , Peptídeos e Proteínas de Sinalização IntercelularRESUMO
The integrin family is a transmembrane receptor that plays critical roles in the cell-cell and cell-extracellular matrix adhesion, signal transduction such as cell cycle regulation, organization of the intracellular cytoskeleton, and immune responses. Consequently, dysfunction of integrins is associated with a wide range of human diseases, including cancer and immune diseases, which makes integrins therapeutic targets for drug discovery. Here we report the cryo-EM structure of the human α-I domain-containing full-length integrin αEß7, which is expressed in the leukocytes of the immune system and a drug target for inflammatory bowel disease (IBD). The structure reveals the half-bent conformation, an intermediate between the close and the open conformation, while the α-I domain responsible for the ligand binding covers the headpiece domain by a unique spatial arrangement. Our results provide the structural information for the drug design targeting IBD.
Assuntos
Microscopia Crioeletrônica , Modelos Moleculares , Domínios Proteicos , Humanos , Integrinas/metabolismo , Integrinas/química , Integrinas/ultraestrutura , Conformação ProteicaRESUMO
G-protein-coupled receptors (GPCRs) constitute a prominent superfamily in humans and are categorized into six classes (A-F) that play indispensable roles in cellular communication and therapeutics. Nonetheless, their structural comprehension has been limited by challenges in high-resolution data acquisition. This review highlights the transformative impact of cryogenic electron microscopy (cryo-EM) on the structural determinations of GPCR-G-protein complexes. Specific technologies, such as nanobodies and mini-G-proteins, stabilize complexes and facilitate structural determination. We discuss the structural alterations upon receptor activation in different GPCR classes, revealing their diverse mechanisms. This review highlights the robust foundation for comprehending GPCR function and pave the way for future breakthroughs in drug discovery and therapeutic targeting.
Assuntos
Microscopia Crioeletrônica , Receptores Acoplados a Proteínas G , Microscopia Crioeletrônica/métodos , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/química , Conformação Proteica , Animais , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/metabolismo , Anticorpos de Domínio Único/imunologia , Modelos MolecularesRESUMO
GPR34 is a recently identified G-protein coupled receptor, which has an immunomodulatory role and recognizes lysophosphatidylserine (LysoPS) as a putative ligand. Here, we report cryo-electron microscopy structures of human GPR34-Gi complex bound with one of two ligands bound: either the LysoPS analogue S3E-LysoPS, or M1, a derivative of S3E-LysoPS in which oleic acid is substituted with a metabolically stable aromatic fatty acid surrogate. The ligand-binding pocket is laterally open toward the membrane, allowing lateral entry of lipidic agonists into the cavity. The amine and carboxylate groups of the serine moiety are recognized by the charged residue cluster. The acyl chain of S3E-LysoPS is bent and fits into the L-shaped hydrophobic pocket in TM4-5 gap, and the aromatic fatty acid surrogate of M1 fits more appropriately. Molecular dynamics simulations further account for the LysoPS-regioselectivity of GPR34. Thus, using a series of structural and physiological experiments, we provide evidence that chemically unstable 2-acyl LysoPS is the physiological ligand for GPR34. Overall, we anticipate the present structures will pave the way for development of novel anticancer drugs that specifically target GPR34.
Assuntos
Ácidos Graxos , Lisofosfolipídeos , Humanos , Microscopia Crioeletrônica , Ácidos Graxos/metabolismo , Ligantes , Lisofosfolipídeos/metabolismo , Receptores de Lisofosfolipídeos/agonistas , Receptores de Lisofosfolipídeos/metabolismoRESUMO
ß-arrestins (ßarrs) are multifunctional proteins involved in signaling and regulation of seven transmembrane receptors (7TMRs), and their interaction is driven primarily by agonist-induced receptor activation and phosphorylation. Here, we present seven cryo-electron microscopy structures of ßarrs either in the basal state, activated by the muscarinic receptor subtype 2 (M2R) through its third intracellular loop, or activated by the ßarr-biased decoy D6 receptor (D6R). Combined with biochemical, cellular, and biophysical experiments, these structural snapshots allow the visualization of atypical engagement of ßarrs with 7TMRs and also reveal a structural transition in the carboxyl terminus of ßarr2 from a ß strand to an α helix upon activation by D6R. Our study provides previously unanticipated molecular insights into the structural and functional diversity encoded in 7TMR-ßarr complexes with direct implications for exploring novel therapeutic avenues.
Assuntos
Domínios e Motivos de Interação entre Proteínas , Receptores Acoplados a Proteínas G , beta-Arrestinas , beta-Arrestinas/química , Microscopia Crioeletrônica , Receptores Acoplados a Proteínas G/química , Transdução de Sinais , Conformação Proteica em Folha beta , Conformação Proteica em alfa-Hélice , HumanosRESUMO
Cryo-EM single particle analysis has recently facilitated the high-resolution structural determination of numerous GPCR-G complexes. Diverse methodologies have been devised with this trend, and in the case of GPCR-Gi complexes, scFv16, an antibody that recognizes the intricate interface of the complex, has been mainly implemented to stabilize the complex. However, owing to their flexibility and heterogeneity, structural determinations of GPCR-Gi complexes remain both challenging and resource-intensive. By employing eGαt, which exhibits binding affinity to modified nanobody Nb35, the cryo-EM structure of Rhodopsin-eGαt complex was previously reported. Using this modified G protein, we determined the structure of the ETB-eGt complex bound to the modified Nb35. The determined structure of ETB receptor was the same as the previously reported ETB-Gi complex, and the resulting dataset demonstrated significantly improved anisotropy. This modified G protein will be utilized for the structural determination of other GPCR-Gi complexes.
Assuntos
Proteínas de Ligação ao GTP , Rodopsina , Microscopia Crioeletrônica , Proteínas de Ligação ao GTP/metabolismo , Rodopsina/metabolismo , Anticorpos/metabolismoRESUMO
Endothelins and their receptors, type A (ETA) and type B (ETB), modulate vital cellular processes, including growth, survival, invasion and angiogenesis, through multiple G proteins. This review highlights the structural determinations of these receptors by X-ray crystallography and cryo-electron microscopy, and their activation mechanisms by endothelins. Explorations of the conformational changes upon receptor activation have provided insights into the unique G-protein coupling feature of the endothelin receptors. The review further delves into the binding modes of the clinical antagonist and the inverse agonists. These findings significantly contribute to understanding the mechanism of G-protein activation and have potential implications for drug development, particularly in the context of vasodilatory antagonists and agonists targeting the endothelin receptors.
Assuntos
Agonismo Inverso de Drogas , Endotelinas , Microscopia Crioeletrônica , Endotelinas/metabolismo , Receptores de Endotelina/química , Receptores de Endotelina/metabolismo , Transdução de SinaisRESUMO
The endothelin ETB receptor is a promiscuous G-protein coupled receptor that is activated by vasoactive peptide endothelins. ETB signaling induces reactive astrocytes in the brain and vasorelaxation in vascular smooth muscle. Consequently, ETB agonists are expected to be drugs for neuroprotection and improved anti-tumor drug delivery. Here, we report the cryo-electron microscopy structure of the endothelin-1-ETB-Gi complex at 2.8 Å resolution, with complex assembly stabilized by a newly established method. Comparisons with the inactive ETB receptor structures revealed how endothelin-1 activates the ETB receptor. The NPxxY motif, essential for G-protein activation, is not conserved in ETB, resulting in a unique structural change upon G-protein activation. Compared with other GPCR-G-protein complexes, ETB binds Gi in the shallowest position, further expanding the diversity of G-protein binding modes. This structural information will facilitate the elucidation of G-protein activation and the rational design of ETB agonists.
Assuntos
Endotelina-1 , Endotelinas , Endotelina-1/metabolismo , Microscopia Crioeletrônica , Receptor de Endotelina B/metabolismo , Endotelinas/metabolismo , Proteínas de Ligação ao GTP/metabolismoRESUMO
Energy transfer from light-harvesting ketocarotenoids to the light-driven proton pump xanthorhodopsins has been previously demonstrated in two unique cases: an extreme halophilic bacterium1 and a terrestrial cyanobacterium2. Attempts to find carotenoids that bind and transfer energy to abundant rhodopsin proton pumps3 from marine photoheterotrophs have thus far failed4-6. Here we detected light energy transfer from the widespread hydroxylated carotenoids zeaxanthin and lutein to the retinal moiety of xanthorhodopsins and proteorhodopsins using functional metagenomics combined with chromophore extraction from the environment. The light-harvesting carotenoids transfer up to 42% of the harvested energy in the violet- or blue-light range to the green-light absorbing retinal chromophore. Our data suggest that these antennas may have a substantial effect on rhodopsin phototrophy in the world's lakes, seas and oceans. However, the functional implications of our findings are yet to be discovered.
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Organismos Aquáticos , Processos Fototróficos , Bombas de Próton , Rodopsinas Microbianas , Organismos Aquáticos/metabolismo , Organismos Aquáticos/efeitos da radiação , Bactérias/metabolismo , Bactérias/efeitos da radiação , Carotenoides/metabolismo , Cor , Cianobactérias/metabolismo , Cianobactérias/efeitos da radiação , Processos Heterotróficos/efeitos da radiação , Luz , Oceanos e Mares , Processos Fototróficos/efeitos da radiação , Bombas de Próton/metabolismo , Bombas de Próton/efeitos da radiação , Rodopsinas Microbianas/metabolismo , Rodopsinas Microbianas/efeitos da radiação , Zeaxantinas/metabolismo , Zeaxantinas/efeitos da radiação , Luteína/metabolismo , Luteína/efeitos da radiação , Metagenoma , LagosRESUMO
Lysophosphatidic acid receptor 1 (LPA1) is one of the six G protein-coupled receptors activated by the bioactive lipid, lysophosphatidic acid (LPA). LPA1 is a drug target for various diseases, including cancer, inflammation, and neuropathic pain. Notably, LPA1 agonists have potential therapeutic value for obesity and urinary incontinence. Here, we report a cryo-electron microscopy structure of the active human LPA1-Gi complex bound to ONO-0740556, an LPA analog with more potent activity against LPA1. Our structure elucidated the details of the agonist binding mode and receptor activation mechanism mediated by rearrangements of transmembrane segment 7 and the central hydrophobic core. A structural comparison of LPA1 and other phylogenetically-related lipid-sensing GPCRs identified the structural determinants for lipid preference of LPA1. Moreover, we characterized the structural polymorphisms at the receptor-G-protein interface, which potentially reflect the G-protein dissociation process. Our study provides insights into the detailed mechanism of LPA1 binding to agonists and paves the way toward the design of drug-like agonists targeting LPA1.
Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Neuralgia , Receptores de Ácidos Lisofosfatídicos , Microscopia Crioeletrônica , Humanos , Receptores de Ácidos Lisofosfatídicos/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
The ß3-adrenergic receptor (ß3AR) is the most essential drug target for overactive bladder and has therapeutic potentials for the treatments of type 2 diabetes and obesity. Here, we report the cryo-electron microscopy structures of the ß3AR-Gs signaling complexes with the selective agonist, solabegron and the nonselective agonist, isoproterenol. Comparison of the isoproterenol-, mirabegron-, and solabegron-bound ß3AR structures revealed that the extracellular loop 2 changes its conformation depending on the bound agonist and plays an essential role in solabegron binding. Moreover, ß3AR has an intrinsically narrow exosite, regardless of the agonist type. This structural feature clearly explains why ß3AR prefers mirabegron and solabegron, as the narrow exosite is suitable for binding with agonists with elongated shapes. Our study deepens the understanding of the binding characteristics of ß3AR agonists and may pave the way for developing ß3AR-selective drugs.
Assuntos
Agonistas de Receptores Adrenérgicos beta 3 , Diabetes Mellitus Tipo 2 , Agonistas de Receptores Adrenérgicos beta 3/farmacologia , Compostos de Anilina , Benzoatos , Compostos de Bifenilo , Microscopia Crioeletrônica , Diabetes Mellitus Tipo 2/tratamento farmacológico , Humanos , Isoproterenol/farmacologia , Receptores Adrenérgicos beta 3/metabolismoRESUMO
Engineering G-protein-coupled receptors (GPCRs) for improved stability or altered function is of great interest, as GPCRs consist of the largest protein family, are involved in many important signaling pathways, and thus, are one of the major drug targets. Here, we report the development of a high-throughput screening method for GPCRs using a reconstituted in vitro transcription-translation (IVTT) system. Human endothelin receptor type-B (ETBR), a class A GPCR that binds endothelin-1 (ET-1), a 21-residue peptide hormone, was synthesized in the presence of nanodisc (ND) composed of a phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG). The ET-1 binding of ETBR was significantly reduced or was undetectable when other phospholipids were used for ND preparation. However, when functional ETBR purified from Sf9 cells was reconstituted into NDs, ET-1 binding was observed with two different phospholipids tested, including POPG. These results suggest that POPG likely supports the folding of ETBR into its functional form in the IVTT system. Using the same conditions as ETBR, whose three-dimensional structure has been solved, human endothelin receptor type-A (ETAR), whose three-dimensional structure remains unsolved, was also synthesized in its functional form. By adding POPG-ND to the IVTT system, both ETAR and ETBR were successfully subjected to ribosome display, a method of in vitro directed evolution that facilitates the screening of up to 1012 mutants. Finally, using a mock library, we showed that ribosome display can be applied for gene screening of ETBR, suggesting that high-throughput screening and directed evolution of GPCRs is possible in vitro.
Assuntos
Sistema Livre de Células , Endotelina-1 , Engenharia de Proteínas , Receptor de Endotelina A , Humanos , Fosfolipídeos , Engenharia de Proteínas/métodos , Receptor de Endotelina A/biossíntese , RibossomosRESUMO
The ß3 -adrenergic receptor (ß3 -AR) is found in several tissues such as adipose tissue and urinary bladder. It is a therapeutic target because it plays a role in thermogenesis, lipolysis, and bladder relaxation. Two ß3 -AR agonists are used clinically: mirabegron 1 and vibegron 2, which are indicated for overactive bladder syndrome. However, these drugs show adverse effects, including increased blood pressure in mirabegron patients. Hence, new ß3 -AR agonists are needed as starting points for drug development. Previous pharmacophore modeling studies of the ß3 -AR did not involve experimental inâ vitro validation. Therefore, this study aimed to conduct prospective virtual screening and confirm the biological activity of virtual hits. Ligand-based pharmacophore modeling was performed since no 3D structure of human ß3 -AR is yet available. A dataset consisting of ß3 -AR agonists was prepared to build and validate the pharmacophore models. The best model was employed for prospective virtual screening, followed by physicochemical property filtering and a docking evaluation. To confirm the activity of the virtual hits, an inâ vitro assay was conducted, measuring cAMP levels at the cloned ß3 -AR. Out of 35 tested compounds, 4â compounds were active in CHO-K1 cells expressing the human ß3 -AR, and 8â compounds were active in CHO-K1 cells expressing the mouse ß3 -AR.
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Tecido Adiposo , Agonistas Adrenérgicos beta , Agonistas Adrenérgicos beta/uso terapêutico , Animais , Células CHO , Cricetinae , Cricetulus , Camundongos , Estudos ProspectivosRESUMO
Melatonin receptors (MT1 and MT2) transduce inhibitory signaling by melatonin (N-acetyl-5-methoxytryptamine), which is associated with sleep induction and circadian rhythm modulation. Although recently reported crystal structures of ligand-bound MT1 and MT2 elucidated the basis of ligand entry and recognition, the ligand-induced MT1 rearrangement leading to Gi-coupling remains unclear. Here we report a cryo-EM structure of the human MT1-Gi signaling complex at 3.3 Å resolution, revealing melatonin-induced conformational changes propagated to the G-protein-coupling interface during activation. In contrast to other Gi-coupled receptors, MT1 exhibits a large outward movement of TM6, which is considered a specific feature of Gs-coupled receptors. Structural comparison of Gi and Gs complexes demonstrated conformational diversity of the C-terminal entry of the Gi protein, suggesting loose and variable interactions at the end of the α5 helix of Gi protein. These notions, together with our biochemical and computational analyses, highlight variable binding modes of Gαi and provide the basis for the selectivity of G-protein signaling.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Melatonina/metabolismo , Receptor MT1 de Melatonina/metabolismo , Microscopia Crioeletrônica , Humanos , Proteínas de Membrana/metabolismo , Estrutura Quaternária de Proteína , Transdução de Sinais/fisiologia , Relação Estrutura-AtividadeRESUMO
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.
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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
Schizorhodopsins (SzRs), a new rhodopsin family identified in Asgard archaea, are phylogenetically located at an intermediate position between type-1 microbial rhodopsins and heliorhodopsins. SzRs work as light-driven inward H+ pumps as xenorhodopsins in bacteria. Although E81 plays an essential role in inward H+ release, the H+ is not metastably trapped in such a putative H+ acceptor, unlike the other H+ pumps. It remains elusive why SzR exhibits different kinetic behaviors in H+ release. Here, we report the crystal structure of SzR AM_5_00977 at 2.1 Å resolution. The SzR structure superimposes well on that of bacteriorhodopsin rather than heliorhodopsin, suggesting that SzRs are classified with type-1 rhodopsins. The structure-based mutagenesis study demonstrated that the residues N100 and V103 around the ß-ionone ring are essential for color tuning in SzRs. The cytoplasmic parts of transmembrane helices 2, 6, and 7 are shorter than those in the other microbial rhodopsins, and thus E81 is located near the cytosol and easily exposed to the solvent by light-induced structural change. We propose a model of untrapped inward H+ release; H+ is released through the water-mediated transport network from the retinal Schiff base to the cytosol by the side of E81. Moreover, most residues on the H+ transport pathway are not conserved between SzRs and xenorhodopsins, suggesting that they have entirely different inward H+ release mechanisms.
Assuntos
Bombas de Próton/química , Rodopsinas Microbianas/química , Sítios de Ligação , Escherichia coli , Conformação ProteicaRESUMO
Channelrhodopsins (ChRs) are microbial light-gated ion channels utilized in optogenetics to control neural activity with light . Light absorption causes retinal chromophore isomerization and subsequent protein conformational changes visualized as optically distinguished intermediates, coupled with channel opening and closing. However, the detailed molecular events underlying channel gating remain unknown. We performed time-resolved serial femtosecond crystallographic analyses of ChR by using an X-ray free electron laser, which revealed conformational changes following photoactivation. The isomerized retinal adopts a twisted conformation and shifts toward the putative internal proton donor residues, consequently inducing an outward shift of TM3, as well as a local deformation in TM7. These early conformational changes in the pore-forming helices should be the triggers that lead to opening of the ion conducting pore.
Assuntos
Proteínas de Algas/genética , Channelrhodopsins/genética , Chlamydomonas reinhardtii/genética , Proteínas de Algas/química , Proteínas de Algas/metabolismo , Sequência de Aminoácidos , Channelrhodopsins/química , Channelrhodopsins/metabolismo , Chlamydomonas reinhardtii/metabolismo , Cristalografia , Isomerismo , Conformação Proteica , Estrutura Secundária de Proteína , Alinhamento de SequênciaRESUMO
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.