RESUMEN
Cytokines regulate immune responses by binding to cell surface receptors, including the common subunit beta (ßc), which mediates signaling for GM-CSF, IL-3, and IL-5. Despite known roles in inflammation, the structural basis of IL-5 receptor activation remains unclear. We present the cryo-EM structure of the human IL-5 ternary receptor complex, revealing architectural principles for IL-5, GM-CSF, and IL-3. In mammalian cell culture, single-molecule imaging confirms hexameric IL-5 complex formation on cell surfaces. Engineered chimeric receptors show that IL-5 signaling, as well as IL-3 and GM-CSF, can occur through receptor heterodimerization, obviating the need for higher-order assemblies of ßc dimers. These findings provide insights into IL-5 and ßc receptor family signaling mechanisms, aiding in the development of therapies for diseases involving deranged ßc signaling.
Asunto(s)
Microscopía por Crioelectrón , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Interleucina-3 , Multimerización de Proteína , Receptores de Interleucina-5 , Transducción de Señal , Humanos , Sitios de Unión , Subunidad beta Común de los Receptores de Citocinas/metabolismo , Subunidad beta Común de los Receptores de Citocinas/genética , Subunidad beta Común de los Receptores de Citocinas/química , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/química , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Células HEK293 , Interleucina-3/metabolismo , Interleucina-3/química , Interleucina-3/genética , Interleucina-5/metabolismo , Modelos Moleculares , Unión Proteica , Receptores de Interleucina-5/metabolismo , Receptores de Interleucina-5/genética , Receptores de Interleucina-5/química , Imagen Individual de Molécula , Relación Estructura-ActividadRESUMEN
Erythropoietin (EPO) has protective effects in several tissues and could be used for therapeutic purposes, but the doses of EPO that can be beneficial in case of hypoxic-ischemic conditions due to overinduced erythropoiesis could be detrimental in treated patients. Carbamylation of erythropoietin maintains the tissue-protective effects of EPO but without erythropoietic effects. Carbamylated EPO (CEPO) is listed in WADA Prohibited List in class S2 as "Innate repair receptor agonists." The CEPO was synthesized using the method described previously. Digestion with endoproteinase Lys-C was used to distinguish rhEPO from CEPO. The digested samples containing recombinant EPO, urinary EPO (uEPO), or CEPO were analyzed by the SAR-PAGE method (sarcosyl polyacrylamide gel electrophoresis-PAGE). Endoproteinase Lys-C breaks the peptide chains of lysine. Lysine residues, converted to homocitrulline by carbamylation, cannot be cleaved by endoproteinase Lys-C. Therefore, the CEPO protein chain remained unchanged in contrast to rhEPO and uEPO, which allows for easily differentiation of them.
Asunto(s)
Doping en los Deportes/prevención & control , Eritropoyetina/análogos & derivados , Detección de Abuso de Sustancias/métodos , Secuencia de Aminoácidos , Subunidad beta Común de los Receptores de Citocinas/química , Subunidad beta Común de los Receptores de Citocinas/genética , Subunidad beta Común de los Receptores de Citocinas/metabolismo , Doping en los Deportes/métodos , Eritropoyetina/química , Eritropoyetina/genética , Eritropoyetina/orina , Humanos , Receptores de Eritropoyetina/química , Receptores de Eritropoyetina/genética , Receptores de Eritropoyetina/metabolismo , Detección de Abuso de Sustancias/normasRESUMEN
Early acting cytokines and growth factors such as those of the CD131 ßc subunit, may offer an alternative method to the current use of antibiotics and chemicals such as anthelmintics in maintaining Porcine (Po) health. Thus far, the recombinant Po (rPo) Granulocyte-macrophage colony-stimulating factor (GM-CSF), rPo interleukin-3 (IL-3) and rPo interleukin-5 (IL-5) proteins have been identified and cloned and the biological activity of each cytokine has been confirmed in vitro, however, in vivo immune system regulation and hematopoietic stem cell (HSC) augmentation are regulated by numerous cytokines and cellular signals within the bone marrow (BM) niche. In order to quantify the use of recombinant cytokines in augmenting the immune response, it is necessary to determine the stages of hematopoiesis induced by each cytokine and possible areas of synergy requiring further investigation. Here we used the chemotherapeutic agent 5-fluorouracil (5-FU), to chemically induce a state of myelosuppression in young pigs. This allowed for the monitoring of both the autologous BM reconstitution and recombinant cytokine induced BM repopulation, precursor cell proliferation and cellular differentiation. The recombinant cytokines PoGM-CSF, PoIL-3 and PoIL-5 were administered by intramuscular injections (i.m.) following confirmation of 5-FU induced leukocytopenia. Blood and BM samples were collected and then analysed for cell composition. Statistically significant results were observed in several blood cell populations including eosinophils for animals treated with rPoIL-5, rPoGM-CSF and basophils for animals treated with rPoIL-3. BM analysis of CD90+ and CD172a+ cells confirmed myelosuppression in week one with significant results observed between rPoIL-3 and the 5-FU control group in week two and for the rPoGM-CSF group in week three. These results have demonstrated the effects of each of these rPo cytokines within the hematopoietic processes of the pig and may demonstrate similar outcomes in other mammalian models including human.
Asunto(s)
Subunidad beta Común de los Receptores de Citocinas/metabolismo , Citocinas/inmunología , Sus scrofa/inmunología , Animales , Antígenos CD/metabolismo , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Subunidad beta Común de los Receptores de Citocinas/química , Citocinas/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Hematopoyesis/efectos de los fármacos , Hematopoyesis/inmunología , Inmunización/métodos , Inmunización/veterinaria , Interleucina-3/inmunología , Interleucina-3/farmacología , Interleucina-5/inmunología , Interleucina-5/farmacología , Subunidades de Proteína , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacología , Antígenos Thy-1/metabolismoRESUMEN
In many families of cell surface receptors, a single transmembrane (TM) α-helix separates ecto- and cytosolic domains. A defined coupling of ecto- and TM domains must be essential to allosteric receptor regulation but remains little understood. Here, we characterize the linker structure, dynamics, and resulting ecto-TM domain coupling of integrin αIIb in model constructs and relate it to other integrin α subunits by mutagenesis. Cellular integrin activation assays subsequently validate the findings in intact receptors. Our results indicate a flexible yet carefully tuned ecto-TM coupling that modulates the signaling threshold of integrin receptors. Interestingly, a proline at the N-terminal TM helix border, termed NBP, is critical to linker flexibility in integrins. NBP is further predicted in 21% of human single-pass TM proteins and validated in cytokine receptors by the TM domain structure of the cytokine receptor common subunit ß and its P441A-substituted variant. Thus, NBP is a conserved uncoupling motif of the ecto-TM domain transition and the degree of ecto-TM domain coupling represents an important parameter in the allosteric regulation of diverse cell surface receptors.
Asunto(s)
Subunidad beta Común de los Receptores de Citocinas/química , Cadenas beta de Integrinas/química , Regulación Alostérica/fisiología , Animales , Células CHO , Cricetinae , Cricetulus , Subunidad beta Común de los Receptores de Citocinas/genética , Subunidad beta Común de los Receptores de Citocinas/metabolismo , Humanos , Cadenas beta de Integrinas/genética , Cadenas beta de Integrinas/metabolismo , Dominios Proteicos , Estructura Secundaria de ProteínaRESUMEN
The ß common-signaling cytokines interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-5 stimulate pro-inflammatory activities of haematopoietic cells via a receptor complex incorporating cytokine-specific α and shared ß common (ßc, CD131) receptor. Evidence from animal models and recent clinical trials demonstrate that these cytokines are critical mediators of the pathogenesis of inflammatory airway disease such as asthma. However, no therapeutic agents, other than steroids, that specifically and effectively target inflammation mediated by all 3 of these cytokines exist. We employed phage display technology to identify and optimize a novel, human monoclonal antibody (CSL311) that binds to a unique epitope that is specific to the cytokine-binding site of the human ßc receptor. The binding epitope of CSL311 on the ßc receptor was defined by X-ray crystallography and site-directed mutagenesis. CSL311 has picomolar binding affinity for the human ßc receptor, and at therapeutic concentrations is a highly potent antagonist of the combined activities of IL-3, GM-CSF and IL-5 on primary eosinophil survival in vitro. Importantly, CSL311 inhibited the survival of inflammatory cells present in induced sputum from human allergic asthmatic subjects undergoing allergen bronchoprovocation. Due to its high potency and ability to simultaneously suppress the activity of all 3 ß common cytokines, CSL311 may provide a new strategy for the treatment of chronic inflammatory diseases where the human ßc receptor is central to pathogenesis. The coordinates for the ßc/CSL311 Fab complex structure have been deposited with the RCSB Protein Data Bank (PDB 5DWU).
Asunto(s)
Anticuerpos Monoclonales de Origen Murino , Subunidad beta Común de los Receptores de Citocinas , Epítopos , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Interleucina-3 , Interleucina-5 , Animales , Anticuerpos Monoclonales de Origen Murino/química , Anticuerpos Monoclonales de Origen Murino/inmunología , Anticuerpos Monoclonales de Origen Murino/uso terapéutico , Complejo Antígeno-Anticuerpo/química , Complejo Antígeno-Anticuerpo/inmunología , Asma/tratamiento farmacológico , Asma/inmunología , Asma/patología , Cristalografía por Rayos X , Subunidad beta Común de los Receptores de Citocinas/química , Subunidad beta Común de los Receptores de Citocinas/inmunología , Eosinófilos/inmunología , Eosinófilos/patología , Epítopos/química , Epítopos/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/antagonistas & inhibidores , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Humanos , Interleucina-3/antagonistas & inhibidores , Interleucina-3/inmunología , Interleucina-5/antagonistas & inhibidores , Interleucina-5/inmunología , RatonesRESUMEN
Erythropoietin (EPO), the key factor for erythropoiesis, also protects macrophage foam cells from lipid accumulation, yet the definitive mechanisms are not fully understood. ß common receptor (ßCR) plays a crucial role in the nonhematopoietic effects of EPO. In the current study, we investigated the role of ßCR in EPO-mediated protection in macrophages against oxidized low-density lipoprotein- (oxLDL-) induced deregulation of lipid metabolism and inflammation. Here, we show that ßCR expression was mainly in foamy macrophages of atherosclerotic aortas from apolipoprotein E-deficient mice. Results of confocal microscopy and immunoprecipitation analyses revealed that ßCR was colocalized and interacted with EPO receptor (EPOR) in macrophages. Inhibition of ßCR activation by neutralizing antibody or small interfering RNA (siRNA) abolished the EPO-conferred protection in oxLDL-induced lipid accumulation. Furthermore, EPO-promoted cholesterol efflux and upregulation of ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 were prevented by pretreatment with ßCR neutralizing antibody or ßCR siRNA. Additionally, blockage of ßCR abrogated the EPO-conferred anti-inflammatory action on oxLDL-induced production of macrophage inflammatory protein-2. Collectively, our findings suggest that ßCR may play an important role in the beneficial effects of EPO against oxLDL-elicited dysfunction of macrophage foam cells.
Asunto(s)
Subunidad beta Común de los Receptores de Citocinas/fisiología , Eritropoyetina/farmacología , Metabolismo de los Lípidos , Lipoproteínas LDL/farmacología , Macrófagos/metabolismo , Animales , Células Cultivadas , Colesterol/metabolismo , Subunidad beta Común de los Receptores de Citocinas/química , Inflamación/etiología , Ratones , Multimerización de ProteínaRESUMEN
Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) and IL-5 are members of a small family of cytokines that share a beta receptor subunit (ßc). These cytokines regulate the growth, differentiation, migration and effector function activities of many hematopoietic cells in bone marrow, blood and sites of inflammation. Excessive or aberrant signaling can result in chronic inflammatory conditions and myeloid leukemias. The crystal structures of the GM-CSF ternary complex, the IL-5 binary complex and the very recent IL-3 receptor alpha subunit build upon decades of structure-function studies, giving new insights into cytokine-receptor specificity and signal transduction. Selective modulation of receptor function is now a real possibility and the structures of the ßc receptor family are being used to discover novel and disease-specific therapeutics.
Asunto(s)
Subunidad beta Común de los Receptores de Citocinas , Citocinas , Transducción de Señal/inmunología , Animales , Subunidad beta Común de los Receptores de Citocinas/química , Subunidad beta Común de los Receptores de Citocinas/inmunología , Citocinas/química , Citocinas/inmunología , Humanos , Estructura Cuaternaria de Proteína , Relación Estructura-ActividadRESUMEN
Tissue-protective properties of erythropoietin (EPO) have let to the discovery of an alternative EPO signaling via an EPO-R/CD131 receptor complex which can now be specifically targeted through pharmaceutically designed short sequence peptides such as ARA290. However, little is still known about specific functions of alternative EPO signaling in defined cell populations. In this study, we investigated effects of signaling through EPO-R/CD131 complex on cellular stress responses and pro-inflammatory activation in different mesenchymal-derived phenotypes. We show that anti-apoptotic, anti-inflammatory effects of ARA290 and EPO coincide with the externalization of CD131 receptor component as an immediate response to cellular stress. In addition, alternative EPO signaling strongly modulated transcriptional, translational, or metabolic responses after stressor removal. Specifically, we saw that ARA290 was able to overcome a TNFα-mediated inhibition of transcription factor activation related to cell stress responses, most notably of serum response factor (SRF), heat shock transcription factor protein 1 (HSF1), and activator protein 1 (AP1). We conclude that alternative EPO signaling acts as a modulator of pro-inflammatory signaling pathways and likely plays a role in restoring tissue homeostasis. Key message: Erythropoietin (EPO) triggers an alternative pathway via heteroreceptor EPO/CD131. ARA290 peptide specifically binds EPO/CD131 but not the canonical EPO/EPO receptor. Oxidative stress and inflammation promote cell surface expression of CD131. ARA290 prevents tumor necrosis factor-mediated inhibition of stress-related genes. Alternative EPO signaling modulates inflammation and promotes tissue homeostasis.
Asunto(s)
Subunidad beta Común de los Receptores de Citocinas/metabolismo , Eritropoyetina/metabolismo , Células Madre Mesenquimatosas/metabolismo , Receptores de Eritropoyetina/metabolismo , Estrés Fisiológico , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular , Membrana Celular/metabolismo , Análisis por Conglomerados , Subunidad beta Común de los Receptores de Citocinas/química , Citocinas/metabolismo , Eritropoyetina/farmacología , Expresión Génica , Perfilación de la Expresión Génica , Inmunofenotipificación , Mediadores de Inflamación/metabolismo , Masculino , Ratones , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Estrés Oxidativo , Fosforilación , Unión Proteica , Multimerización de Proteína , Receptores de Eritropoyetina/química , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/metabolismo , TranscriptomaRESUMEN
Interleukin-3 (IL-3) is a cytokine secreted by mast cells and activated T-cells known to be an important regulator of differentiation, survival, proliferation and activation of a range of haemopoietic lineages. The effects of IL-3 on target cells are mediated by a transmembrane receptor system composed of a cytokine-specific α-subunit and a ß-subunit, the principal signalling entity. In the mouse, two ß-subunits have co-evolved: a common ß-subunit (ßc) shared between IL-3 and the related cytokines IL-5 and granulocyte/macrophage colony-stimulating factor (GM-CSF); and an IL-3-specific ß-subunit (ßIL-3). ßIL-3 differs from ßc in its specificity for IL-3 and its capacity to bind IL-3 directly in the absence of an α-subunit, and, in the absence of structural information, the basis for these properties has remained enigmatic. In the present study, we have solved the crystal structure of the ßIL-3 ectodomain at 3.45 Å (1 Å=0.1 nm) resolution. This structure provides the first evidence that ßIL-3 adopts an arch-shaped intertwined homodimer with similar topology to the paralogous ßc structure. In contrast with apo-ßc, however, the ligand-binding interface of ßIL-3 appears to pre-exist in a conformation receptive to IL-3 engagement. Molecular modelling of the IL-3-ßIL-3 interface, in conjunction with previous mutational studies, suggests that divergent evolution of both ßIL-3 and IL-3 underlies their unique capacity for direct interaction and specificity.
Asunto(s)
Subunidad beta Común de los Receptores de Citocinas/química , Interleucina-3/química , Animales , Cristalografía por Rayos X , Subunidad beta Común de los Receptores de Citocinas/metabolismo , Evolución Molecular , Interleucina-3/metabolismo , Ratones , Modelos Moleculares , Conformación Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/químicaRESUMEN
The cytokine interleukin-3 (IL-3) is a critical regulator of inflammation and immune responses in mammals. IL-3 exerts its effects on target cells via receptors comprising an IL-3-specific alpha-subunit and common beta-subunit (beta c; shared with IL-5 and granulocyte-macrophage colony-stimulating factor) or a beta-subunit that specifically binds IL-3 (beta(IL-3); present in mice but not humans). We recently identified two splice variants of the alpha-subunit of the IL-3 receptor (IL-3R alpha) that are relevant to hematopoietic progenitor cell differentiation or proliferation: the full length ("SP1" isoform) and a novel isoform (denoted "SP2") lacking the N-terminal Ig-like domain. Although our studies demonstrated that each mouse IL-3 (mIL-3) R alpha isoform can direct mIL-3 binding to two distinct sites on the beta(IL-3) subunit, it has remained unclear which residues in mIL-3 itself are critical to the two modes of beta(IL-3) recognition and whether the human IL-3R alpha SP1 and SP2 orthologs similarly instruct human IL-3 binding to two distinct sites on the human beta c subunit. Herein, we describe the identification of residues clustering around the highly conserved A-helix residue, Glu(23), in the mIL-3 A- and C-helices as critical for receptor binding and growth stimulation via the beta(IL-3) and mIL-3R alpha SP2 subunits, whereas an overlapping cluster was required for binding and activation of beta(IL-3) in the presence of mIL-3R alpha SP1. Similarly, our studies of human IL-3 indicate that two different modes of beta c binding are utilized in the presence of the hIL-3R alpha SP1 or SP2 isoforms, suggesting a possible conserved mechanism by which the relative orientations of receptor subunits are modulated to achieve distinct signaling outcomes.
Asunto(s)
Subunidad beta Común de los Receptores de Citocinas/metabolismo , Epítopos/metabolismo , Interleucina-3/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Proliferación Celular , Subunidad beta Común de los Receptores de Citocinas/química , Análisis Mutacional de ADN , Activación Enzimática , Ácido Glutámico/metabolismo , Humanos , Interleucina-3/química , Subunidad alfa del Receptor de Interleucina-3/metabolismo , Janus Quinasa 2/metabolismo , Sistema de Señalización de MAP Quinasas , Ratones , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismoRESUMEN
Erythropoietin (EPO) is a well-known therapeutic protein employed widely in the treatment of anemia. Over the past decade, abundant evidence has shown that in addition to its systemic role in the regulation of plasma pO(2) by modulating erythrocyte numbers, EPO is also a cytoprotective molecule made locally in response to injury or metabolic stress. Many studies have shown beneficial effects of EPO administration in reducing damage caused by ischemia-reperfusion, trauma, cytotoxicity, infection and inflammation in a variety of organs and tissues. Notably, the receptor mediating the nonerythropoietic effects of EPO differs from the one responsible for hematopoiesis. The tissue-protective receptor exhibits a lower affinity for EPO and is a heteromer consisting of EPO receptor monomers in association with the common receptor that is also employed by granulocyte macrophage colony-stimulating factor, interleukin 3, and interleukin 5. This heteromeric receptor is expressed immediately following injury, whereas EPO production is delayed. Thus, early administration of EPO can dramatically reduce the deleterious components of the local inflammatory cascade. However, a high dose of EPO is required and this also stimulates the bone marrow to produce highly reactive platelets and activates the vascular endothelium into a prothrombotic state. To circumvent these undesirable effects, the EPO molecule has been successfully altered to selectively eliminate erythropoietic and prothrombotic potencies, while preserving tissue-protective activities. Very recently, small peptide mimetics have been developed that recapitulate the tissue-protective activities of EPO. Nonerythropoietic tissue-protective molecules hold high promise in a wide variety of acute and chronic diseases.
Asunto(s)
Subunidad beta Común de los Receptores de Citocinas/fisiología , Hematínicos/uso terapéutico , Receptores de Eritropoyetina/fisiología , Estrés Fisiológico/efectos de los fármacos , Heridas y Lesiones/tratamiento farmacológico , Secuencia de Aminoácidos , Animales , Subunidad beta Común de los Receptores de Citocinas/química , Subunidad beta Común de los Receptores de Citocinas/efectos de los fármacos , Citocinas/metabolismo , Diseño de Fármacos , Eritropoyetina/administración & dosificación , Eritropoyetina/efectos adversos , Eritropoyetina/química , Eritropoyetina/farmacocinética , Eritropoyetina/fisiología , Eritropoyetina/uso terapéutico , Retroalimentación Fisiológica , Hematínicos/administración & dosificación , Hematínicos/efectos adversos , Hematínicos/química , Hematínicos/farmacología , Hematopoyesis/efectos de los fármacos , Humanos , Inflamación/prevención & control , Mediadores de Inflamación/metabolismo , Janus Quinasa 2/fisiología , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Multimerización de Proteína , Receptores de Eritropoyetina/química , Receptores de Eritropoyetina/efectos de los fármacos , Proteínas Recombinantes , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/fisiopatología , Daño por Reperfusión/prevención & control , Factores de Transcripción STAT/fisiología , Heridas y Lesiones/inmunología , Heridas y Lesiones/fisiopatologíaRESUMEN
GM-CSF (granulocyte/macrophage colony-stimulating factor) is an important mediator of inducible haemopoiesis and inflammation, and has a critical role in the function of alveolar macrophages. Its clinical applications include the mobilization of haemopoietic progenitors, and a role as an immune stimulant and vaccine adjuvant in cancer patients. GM-CSF signals via a specific alpha receptor (GM-CSFRalpha) and the shared hbetac (human common beta-subunit). The present study has investigated the role of the Ig-like domain of GM-CSFRalpha in GM-CSF binding and signalling. Deletion of the Ig-like domain abolished direct GM-CSF binding and decreased growth signalling in the presence of hbetac. To locate the specific residues in the Ig-like domain of GM-CSFRalpha involved in GM-CSF binding, a structural alignment was made with a related receptor, IL-13Ralpha1 (interleukin-13 receptor alpha1), whose structure and mode of interaction with its ligand has recently been elucidated. Mutagenesis of candidate residues in the predicted region of interaction identified Val51 and Cys60 as having critical roles in binding to the alpha receptor, with Arg54 and Leu55 also being important. High-affinity binding in the presence of hbetac was strongly affected by mutation of Cys60 and was also reduced by mutation of Val51, Arg54 and Leu55. Of the four key residues, growth signalling was most severely affected by mutation of Cys60. The results indicate a previously unrecognized role for the Ig-like domain, and in particular Cys60, of GM-CSFRalpha in the binding of GM-CSF and subsequent activation of cellular signalling.
Asunto(s)
Citocinas/metabolismo , Receptores de Citocinas/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Células COS , Línea Celular , Proliferación Celular , Chlorocebus aethiops , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Subunidad beta Común de los Receptores de Citocinas/química , Subunidad beta Común de los Receptores de Citocinas/genética , Subunidad beta Común de los Receptores de Citocinas/metabolismo , Citocinas/genética , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/química , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Subunidad alfa1 del Receptor de Interleucina-13/química , Subunidad alfa1 del Receptor de Interleucina-13/genética , Subunidad alfa1 del Receptor de Interleucina-13/metabolismo , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Estructura Terciaria de Proteína , Receptores de Citocinas/genética , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/química , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Homología de Secuencia de AminoácidoRESUMEN
Recent structural information for complexes of cytokine receptor ectodomains bound to their ligands has significantly expanded our understanding of the macromolecular topology and ligand recognition mechanisms used by our three principal shared cytokine signaling receptors-gp130, gamma(c), and beta(c). The gp130 family receptors intricately coordinate three structurally unique cytokine-binding sites on their four-helix bundle cytokine ligands to assemble multimeric signaling complexes. These organizing principles serve as topological blueprints for the entire gp130 family of cytokines. Novel structures of gamma(c) and beta(c) complexes show us new twists, such as the use of a nonstandard sushi-type alpha receptors for IL-2 and IL-15 in assembling quaternary gamma(c) signaling complexes and an antiparallel interlocked dimer in the GM-CSF signaling complex with beta(c). Unlike gp130, which appears to recognize vastly different cytokine surfaces in chemically unique fashions for each ligand, the gamma(c)-dependent cytokines appear to seek out some semblance of a knobs-in-holes shape recognition code in order to engage gamma(c) in related fashions. We discuss the structural similarities and differences between these three shared cytokine receptors, as well as the implications for transmembrane signaling.
Asunto(s)
Receptor gp130 de Citocinas/química , Receptor gp130 de Citocinas/metabolismo , Citocinas/metabolismo , Animales , Sitios de Unión , Subunidad beta Común de los Receptores de Citocinas/química , Subunidad beta Común de los Receptores de Citocinas/metabolismo , Citocinas/química , Humanos , Subunidad gamma Común de Receptores de Interleucina/química , Subunidad gamma Común de Receptores de Interleucina/metabolismo , Ligandos , Unión Proteica , Conformación Proteica , Transducción de SeñalRESUMEN
Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3 and IL-5 are related cytokines that play key roles in regulating the differentiation, proliferation, survival and activation of myeloid blood cells. The cell surface receptors for these cytokines are composed of cytokine-specific alpha-subunits and a common beta-receptor (betac), a shared subunit that is essential for receptor signaling in response to GM-CSF, IL-3 and IL-5. Previous studies have reached conflicting conclusions as to whether N-glycosylation of the betac-subunit is necessary for functional GM-CSF, IL-3 and IL-5 receptors. We sought to clarify whether betac N-glycosylation plays a role in receptor function, since all structural studies of human betac to date have utilized recombinant protein lacking N-glycosylation at Asn(328). Here, by eliminating individual N-glycans in human betac and the related murine homolog, beta(IL-3), we demonstrate unequivocally that ligand-binding and receptor activation are not critically dependent on individual N-glycosylation sites within the beta-subunit although the data do not preclude the possibility that N-glycans may exert some sort of fine control. These studies support the biological relevance of the X-ray crystal structures of the human betac domain 4 and the complete ectodomain, both of which lack N-glycosylation at Asn(328).
Asunto(s)
Subunidad beta Común de los Receptores de Citocinas/fisiología , Polisacáridos/fisiología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Receptores de Interleucina-3/fisiología , Receptores de Interleucina-5/fisiología , Animales , Células COS , Chlorocebus aethiops , Subunidad beta Común de los Receptores de Citocinas/química , Subunidad beta Común de los Receptores de Citocinas/genética , Humanos , Interleucina-3/metabolismo , Interleucina-5/metabolismo , Ratones , Mutagénesis Sitio-Dirigida , Polisacáridos/química , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/química , Receptores de Interleucina-3/química , Receptores de Interleucina-5/químicaRESUMEN
Interleukin (IL)-5 exerts hematopoietic functions through binding to the IL-5 receptor subunits, alpha and betac. Specific assembly steps of full-length subunits as they occur in cell membranes, ultimately leading to receptor activation, are not well understood. We tracked the oligomerization of IL-5 receptor subunits using fluorescence resonance energy transfer (FRET) imaging. Full-length IL-5Ralpha and betac were expressed in Phoenix cells as chimeric proteins fused to enhanced cyan or yellow fluorescent protein (CFP or YFP, respectively). A time- and dose-dependent increase in FRET signal between IL-5Ralpha-CFP and betac-YFP was observed in response to IL-5, indicative of heteromeric receptor alpha-betac subunit interaction. This response was inhibited by AF17121, a peptide antagonist of IL-5Ralpha. Substantial FRET signals with betac-CFP and betac-YFP co-expressed in the absence of IL-5Ralpha demonstrated that betac subunits exist as preformed homo-oligomers. IL-5 had no effect on this betac-alone FRET signal. Interestingly, the addition of IL-5 to cells co-expressing betac-CFP, betac-YFP, and nontagged IL-5Ralpha led to further increase in FRET efficiency. Observation of preformed betac oligomers fits with the view that this form can lead to rapid cellular responses upon IL-5 stimulation. The IL-5-induced effects on betac assembly in the presence of nontagged IL-5Ralpha provide direct evidence that IL-5 can cause higher order rearrangements of betac homo-oligomers. These results suggest that IL-5 and perhaps other betac cytokines (IL-3 and granulocyte/macrophage colony-stimulating factor) trigger cellular responses by the sequential binding of cytokine ligand to the specificity receptor (subunit alpha), followed by binding of the ligand-subunit alpha complex to, and consequent rearrangement of, a ground state form of betac oligomers.
Asunto(s)
Subunidad beta Común de los Receptores de Citocinas/química , Subunidad beta Común de los Receptores de Citocinas/metabolismo , Subunidad alfa del Receptor de Interleucina-5/química , Subunidad alfa del Receptor de Interleucina-5/metabolismo , Línea Celular , Cristalografía por Rayos X , Transferencia Resonante de Energía de Fluorescencia , Expresión Génica , Humanos , Interleucina-5/metabolismo , Modelos Moleculares , Mutación/genética , Unión Proteica , Estructura Terciaria de Proteína , Factores de Tiempo , TransgenesRESUMEN
Tyrosine and serine phosphorylation of the common beta chain (beta(c)) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors is widely viewed as a general mechanism that provides positive inputs by coupling the receptor to signaling pathways that stimulate several cellular functions. We show here that despite the known action of Tyr577 in beta(c) to recruit Shc-PI-3 kinase (PI3K) pathway members, Tyr577 plays, surprisingly, a negative regulatory role in cell function, and that this is mediated, at least in part, through the uncoupling of SH2-containing inositol 5'-phosphatase (SHIP) from beta(c). Fetal liver cells from beta(c)/beta(IL-3)(-/-) mice expressing human GM-CSF receptor alpha chain and beta(c) Tyr577Phe mutant showed enhanced colony formation and expansion of progenitor cells in response to GM-CSF. Dissection of these activities revealed that basal survival was increased, as well as cytokine-stimulated proliferation. As expected, the recruitment and activation of Shc was abolished, but interestingly, Gab-2 and Akt phosphorylation increased. Significantly, the activation of PI3K was enhanced and prolonged, accompanied by loss of SHIP activity. These results reveal a previously unrecognized negative signaling role for Tyr577 in beta(c) and demonstrate that uncoupling Shc from cytokine receptors enhances PI3K signaling as well as survival and proliferation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Subunidad beta Común de los Receptores de Citocinas/química , Subunidad beta Común de los Receptores de Citocinas/fisiología , Hematopoyesis/genética , Animales , Sitios de Unión , Células de la Médula Ósea/metabolismo , Supervivencia Celular/genética , Células Cultivadas , Subunidad beta Común de los Receptores de Citocinas/genética , Subunidad beta Común de los Receptores de Citocinas/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Inositol Polifosfato 5-Fosfatasas , Hígado/embriología , Hígado/metabolismo , Ratones , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Mutación Puntual , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Transducción GenéticaRESUMEN
The cytokines, interleukin-3 (IL-3), interleukin-5 (IL-5), and granulocyte-macrophage colony stimulating factor (GM-CSF), are polypeptide growth factors that exhibit overlapping activities in the regulation of hematopoietic cells. They appear to be primarily involved in inducible hematopoiesis in response to infections and are involved in the pathogenesis of allergic and inflammatory diseases and possibly in leukemia. The X-ray structure of the beta common (betac) receptor ectodomain has given new insights into the structural biology of signaling by IL-3, IL-5, and GM-CSF. This receptor is shared between the three ligands and functions together with three ligand-specific alpha-subunits. The structure shows betac is an intertwined homodimer in which each chain contains four domains with approximate fibronectin type-III topology. The two betac-subunits that compose the homodimer are interlocked by virtue of the swapping of beta-strands between domain 1 of one subunit and domain 3 of the other subunit. Site-directed mutagenesis has shown that the interface between domains 1 and 4 in this unique structure forms the functional epitope. This epitope is similar to those of other members of the cytokine class I receptor family but is novel in that it is formed by two different receptor chains. The chapter also reviews knowledge on the closely related mouse beta(IL-3) receptor and on the alpha-subunit-ligand interactions. The knowledge on the two beta receptors is placed in context with advances in understanding of the structural biology of other members of the cytokine class I receptor family.
Asunto(s)
Subunidad beta Común de los Receptores de Citocinas/química , Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Interleucina-3/fisiología , Interleucina-5/fisiología , Transducción de Señal/fisiología , Animales , Cristalografía por Rayos X , Factor Estimulante de Colonias de Granulocitos y Macrófagos/química , Humanos , Interleucina-3/química , Interleucina-5/química , Ratones , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
The human interleukin-3 receptor (hIL-3R) consists of a unique alpha subunit (hIL-3Ralpha) and a common beta subunit (betac). Binding of IL-3 to IL-3R activates Janus kinases JAK1 and JAK2. Our previously study showed that JAK2 and JAK1 were constitutively associated with the hIL-3Ralpha and betac subunits, respectively. In this study, we further demonstrate that JAK2 binds to the intracellular domain of hIL-3Ralpha and JAK1 binds to the Box 1 and Box 2 motifs of betac using GST-hIL-3R fusion proteins in pull-down assays. JAK1 mutational analysis revealed that its JH7-3 domains bound directly to the Box 1 and Box 2 motifs of betac. We further examined the role of JAK1 JH7-3 domains in JAK1 and JAK2-mediated signaling using the CDJAKs fusion proteins, which consisted of a CD16 extracellular domain, a CD7 transmembrane domain, and either JAK1 (CDJAK1), JAK2 (CDJAK2), or JAK1-JH7-3 domains (CDJAK1-JH7-3) as intracellular domains. Anti-CD16 antibody crosslinking of wild type fusion proteins CDJAK1 with CDJAK2 could mimic IL-3 signaling, however, the crosslinking of fusion proteins CDJAK1-JH7-3 with CDJAK2 failed to activate downstream proteins. These results suggest that the JAK1-JH7-3 domains are required for betac interaction and abolish wild type JAK1 and JAK2-mediated signaling.