RESUMEN
The adipokine Leptin activates its receptor LEP-R in the hypothalamus to regulate body weight and exerts additional pleiotropic functions in immunity, fertility and cancer. However, the structure and mechanism of Leptin-mediated LEP-R assemblies has remained unclear. Intriguingly, the signaling-competent isoform of LEP-R is only lowly abundant amid several inactive short LEP-R isoforms contributing to a mechanistic conundrum. Here we show by X-ray crystallography and cryo-EM that, in contrast to long-standing paradigms, Leptin induces type I cytokine receptor assemblies featuring 3:3 stoichiometry and demonstrate such Leptin-induced trimerization of LEP-R on living cells via single-molecule microscopy. In mediating these assemblies, Leptin undergoes drastic restructuring that activates its site III for binding to the Ig domain of an adjacent LEP-R. These interactions are abolished by mutations linked to obesity. Collectively, our study provides the structural and mechanistic framework for how evolutionarily conserved Leptin:LEP-R assemblies with 3:3 stoichiometry can engage distinct LEP-R isoforms to achieve signaling.
Asunto(s)
Adipoquinas , Leptina , Leptina/genética , Leptina/metabolismo , Leptina/farmacología , Isoformas de Proteínas/genética , Transducción de SeñalRESUMEN
The IL-17 family of cytokines and receptors have central roles in host defence against infection and development of inflammatory diseases1. The compositions and structures of functional IL-17 family ligand-receptor signalling assemblies remain unclear. IL-17E (also known as IL-25) is a key regulator of type 2 immune responses and driver of inflammatory diseases, such as allergic asthma, and requires both IL-17 receptor A (IL-17RA) and IL-17RB to elicit functional responses2. Here we studied IL-25-IL-17RB binary and IL-25-IL-17RB-IL-17RA ternary complexes using a combination of cryo-electron microscopy, single-molecule imaging and cell-based signalling approaches. The IL-25-IL-17RB-IL-17RA ternary signalling assembly is a C2-symmetric complex in which the IL-25-IL-17RB homodimer is flanked by two 'wing-like' IL-17RA co-receptors through a 'tip-to-tip' geometry that is the key receptor-receptor interaction required for initiation of signal transduction. IL-25 interacts solely with IL-17RB to allosterically promote the formation of the IL-17RB-IL-17RA tip-to-tip interface. The resulting large separation between the receptors at the membrane-proximal level may reflect proximity constraints imposed by the intracellular domains for signalling. Cryo-electron microscopy structures of IL-17A-IL-17RA and IL-17A-IL-17RA-IL-17RC complexes reveal that this tip-to-tip architecture is a key organizing principle of the IL-17 receptor family. Furthermore, these studies reveal dual actions for IL-17RA sharing among IL-17 cytokine complexes, by either directly engaging IL-17 cytokines or alternatively functioning as a co-receptor.
Asunto(s)
Interleucina-17 , Receptores de Interleucina-17 , Microscopía por Crioelectrón , Interleucina-17/química , Interleucina-17/metabolismo , Ligandos , Dominios Proteicos , Multimerización de Proteína , Receptores de Interleucina-17/química , Receptores de Interleucina-17/metabolismo , Receptores de Interleucina-17/ultraestructura , Transducción de Señal , Imagen Individual de MoléculaRESUMEN
Thrombopoietin (TPO) and its receptor, MPL, are the primary regulators of platelet production and critical for hematopoietic stem cell (HSC) maintenance. Since TPO was first cloned in 1994, the physiological and pathological roles of TPO and MPL have been well characterized, culminating in the first MPL agonists being approved for the treatment of chronic immune thrombocytopenia in 2008. Dysregulation of the TPO-MPL signaling axis contributes to the pathogenesis of hematological disorders: decreased expression or function results in severe thrombocytopenia progressing to bone marrow failure, while hyperactivation of MPL signaling, either by mutations in the receptor or associated Janus kinase 2 (JAK2), results in pathological myeloproliferation. Despite its importance, it was only recently that the long-running debate over the mechanism by which TPO binding activates MPL has been resolved. This review will cover key aspects of TPO and MPL structure and function and their importance in receptor activation, discuss how these are altered in hematological disorders and consider how a greater understanding could lead to the development of better-targeted and more efficacious therapies.
Asunto(s)
Plaquetas/metabolismo , Receptores de Trombopoyetina/metabolismo , Humanos , Transducción de SeñalRESUMEN
Cytokines elicit pleiotropic and non-redundant activities despite strong overlap in their usage of receptors, JAKs and STATs molecules. We use IL-6 and IL-27 to ask how two cytokines activating the same signaling pathway have different biological roles. We found that IL-27 induces more sustained STAT1 phosphorylation than IL-6, with the two cytokines inducing comparable levels of STAT3 phosphorylation. Mathematical and statistical modeling of IL-6 and IL-27 signaling identified STAT3 binding to GP130, and STAT1 binding to IL-27Rα, as the main dynamical processes contributing to sustained pSTAT1 levels by IL-27. Mutation of Tyr613 on IL-27Rα decreased IL-27-induced STAT1 phosphorylation by 80% but had limited effect on STAT3 phosphorgylation. Strong receptor/STAT coupling by IL-27 initiated a unique gene expression program, which required sustained STAT1 phosphorylation and IRF1 expression and was enriched in classical Interferon Stimulated Genes. Interestingly, the STAT/receptor coupling exhibited by IL-6/IL-27 was altered in patients with systemic lupus erythematosus (SLE). IL-6/IL-27 induced a more potent STAT1 activation in SLE patients than in healthy controls, which correlated with higher STAT1 expression in these patients. Partial inhibition of JAK activation by sub-saturating doses of Tofacitinib specifically lowered the levels of STAT1 activation by IL-6. Our data show that receptor and STATs concentrations critically contribute to shape cytokine responses and generate functional pleiotropy in health and disease.
Asunto(s)
Receptor gp130 de Citocinas/agonistas , Interleucina-27/farmacología , Interleucina-6/farmacología , Receptores de Interleucina/agonistas , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/metabolismo , Células TH1/efectos de los fármacos , Secuencias de Aminoácidos , Unión Competitiva , Estudios de Casos y Controles , Células Cultivadas , Receptor gp130 de Citocinas/genética , Receptor gp130 de Citocinas/metabolismo , Humanos , Factor 1 Regulador del Interferón/metabolismo , Interleucina-27/metabolismo , Interleucina-6/metabolismo , Cinética , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/metabolismo , Modelos Biológicos , Mutación , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Transducción de Señal , Células TH1/inmunología , Células TH1/metabolismoRESUMEN
Homodimeric class I cytokine receptors are assumed to exist as preformed dimers that are activated by ligand-induced conformational changes. We quantified the dimerization of three prototypic class I cytokine receptors in the plasma membrane of living cells by single-molecule fluorescence microscopy. Spatial and spatiotemporal correlation of individual receptor subunits showed ligand-induced dimerization and revealed that the associated Janus kinase 2 (JAK2) dimerizes through its pseudokinase domain. Oncogenic receptor and hyperactive JAK2 mutants promoted ligand-independent dimerization, highlighting the formation of receptor dimers as the switch responsible for signal activation. Atomistic modeling and molecular dynamics simulations based on a detailed energetic analysis of the interactions involved in dimerization yielded a mechanistic blueprint for homodimeric class I cytokine receptor activation and its dysregulation by individual mutations.
Asunto(s)
Carcinogénesis/genética , Membrana Celular/química , Janus Quinasa 2/química , Janus Quinasa 2/genética , Multimerización de Proteína , Receptores de Eritropoyetina/química , Receptores de Somatotropina/química , Receptores de Trombopoyetina/química , Sustitución de Aminoácidos/genética , Células HeLa , Humanos , Janus Quinasa 2/antagonistas & inhibidores , Ligandos , Microscopía Fluorescente , Modelos Moleculares , Mutación , Nitrilos , Fenilalanina/genética , Pirazoles/farmacología , Pirimidinas , Transducción de Señal , Imagen Individual de Molécula , Valina/genéticaRESUMEN
Cytokines activate signaling via assembly of cell surface receptors, but it is unclear whether modulation of cytokine-receptor binding parameters can modify biological outcomes. We have engineered IL-6 variants with different affinities to gp130 to investigate how cytokine receptor binding dwell-times influence functional selectivity. Engineered IL-6 variants showed a range of signaling amplitudes and induced biased signaling, with changes in receptor binding dwell-times affecting more profoundly STAT1 than STAT3 phosphorylation. We show that this differential signaling arises from defective translocation of ligand-gp130 complexes to the endosomal compartment and competitive STAT1/STAT3 binding to phospho-tyrosines in gp130, and results in unique patterns of STAT3 binding to chromatin. This leads to a graded gene expression response and differences in ex vivo differentiation of Th17, Th1 and Treg cells. These results provide a molecular understanding of signaling biased by cytokine receptors, and demonstrate that manipulation of signaling thresholds is a useful strategy to decouple cytokine functional pleiotropy.
Asunto(s)
Receptor gp130 de Citocinas/química , Interleucina-6/química , Factor de Transcripción STAT1/metabolismo , Linfocitos T Reguladores/metabolismo , Células TH1/metabolismo , Células Th17/metabolismo , Sitios de Unión , Diferenciación Celular , Clonación Molecular , Receptor gp130 de Citocinas/genética , Receptor gp130 de Citocinas/metabolismo , Endosomas/química , Endosomas/metabolismo , Expresión Génica , Células HeLa , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Cinética , Modelos Moleculares , Fosforilación , Cultivo Primario de Células , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Ingeniería de Proteínas/métodos , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunología , Células TH1/citología , Células TH1/inmunología , Células Th17/citología , Células Th17/inmunologíaRESUMEN
Although tunable signaling by G protein-coupled receptors can be exploited through medicinal chemistry, a comparable pharmacological approach has been lacking for the modulation of signaling through dimeric receptors, such as those for cytokines. We present a strategy to modulate cytokine receptor signaling output by use of a series of designed C2-symmetric cytokine mimetics, based on the designed ankyrin repeat protein (DARPin) scaffold, that can systematically control erythropoietin receptor (EpoR) dimerization orientation and distance between monomers. We sampled a range of EpoR geometries by varying intermonomer angle and distance, corroborated by several ligand-EpoR complex crystal structures. Across the range, we observed full, partial, and biased agonism as well as stage-selective effects on hematopoiesis. This surrogate ligand strategy opens access to pharmacological modulation of therapeutically important cytokine and growth factor receptor systems.