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1.
The signaling domain of the erythropoietin receptor rescues prolactin receptor-mutant mammary epithelium
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Brisken C Socolovsky M Lodish HF Weinberg R 《Proceedings of the National Academy of Sciences of the United States of America》2002,99(22):14241-14245
The cytokine hormones prolactin and erythropoietin mediate tissue-specific developmental outcomes by activating their cognate receptors, prolactin receptor (PrlR) and erythropoietin receptor (EpoR), respectively. The EpoR is essential for red blood cell formation, whereas a principal function of PrlR is in the development of the mammary gland during pregnancy and lactation [Ormandy, C., et al. (1997) Genes Dev. 11, 167-178]. The instructive model of differentiation proposes that such distinct, cytokine-dependent developmental outcomes are a result of cytokine receptor-unique signals that bring about induction of lineage-specific genes. This view was challenged by our finding that an exogenously expressed PrlR could rescue EpoR(-/-) erythroid progenitors and mediate their differentiation into red blood cells. Together with similar findings in other hematopoietic lineages, this suggested that cytokine receptors do not play an instructive role in hematopoietic differentiation. Here, we show that these findings are not limited to the hematopoietic system but are of more general relevance to cytokine-dependent differentiation. We demonstrate that the developmental defect of PrlR(-/-) mammary epithelium is rescued by an exogenously expressed chimeric receptor (prl-EpoR) containing the PrlR extracellular domain joined to the EpoR transmembrane and intracellular domains. Like the wild-type PrlR, the prl-EpoR rescued alveologenesis and milk secretion in PrlR(-/-) mammary epithelium. These results suggest that, in cell types as unrelated as erythrocytes and mammary epithelial cells, cytokine receptors employ similar, generic signals that permit the expression of predetermined, tissue-specific differentiation programs. 相似文献
2.
Ligand-independent oligomerization of cell-surface erythropoietin receptor is mediated by the transmembrane domain
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Constantinescu SN Keren T Socolovsky M Nam H Henis YI Lodish HF 《Proceedings of the National Academy of Sciences of the United States of America》2001,98(8):4379-4384
Binding of erythropoietin (Epo) to the Epo receptor (EpoR) is crucial for production of mature red cells. Although it is well established that the Epo-bound EpoR is a dimer, it is not clear whether, in the absence of ligand, the intact EpoR is a monomer or oligomer. Using antibody-mediated immunofluorescence copatching (oligomerizing) of epitope-tagged receptors at the surface of live cells, we show herein that a major fraction of the full-length murine EpoR exists as preformed dimers/oligomers in BOSC cells, which are human embryo kidney 293T-derived cells. This observed oligomerization is specific because, under the same conditions, epitope-tagged EpoR did not oligomerize with several other tagged receptors (thrombopoietin receptor, transforming growth factor beta receptor type II, or prolactin receptor). Strikingly, the EpoR transmembrane (TM) domain but not the extracellular or intracellular domains enabled the prolactin receptor to copatch with EpoR. Preformed EpoR oligomers are not constitutively active and Epo binding was required to induce signaling. In contrast to tyrosine kinase receptors (e.g., insulin receptor), which cannot signal when their TM domain is replaced by the strongly dimerizing TM domain of glycophorin A, the EpoR could tolerate the replacement of its TM domain with that of glycophorin A and retained signaling. We propose a model in which TM domain-induced dimerization maintains unliganded EpoR in an inactive state that can readily be switched to an active state by physiologic levels of Epo. 相似文献
3.
Leonard JN Ghirlando R Askins J Bell JK Margulies DH Davies DR Segal DM 《Proceedings of the National Academy of Sciences of the United States of America》2008,105(1):258-263
Toll-like receptors (TLRs) initiate immune responses by recognizing pathogen-associated molecules, but the molecular basis for recognition is poorly understood. In particular, it is unclear how receptor-ligand interactions lead to the initiation of downstream signaling. Here, we describe the mechanism by which TLR3 recognizes its ligand, double-stranded RNA (dsRNA), and forms an active signaling complex. We show that dsRNA binds saturably, specifically, and reversibly to a defined ligand-binding site (or sites) on the TLR3 ectodomain (TLR3ecd). Binding affinities increase with both buffer acidity and ligand size. Purified TLR3ecd protein is exclusively monomeric in solution, but through a highly cooperative process, it forms dimers when bound to dsRNA, and multiple TLR3ecd dimers bind to long dsRNA strands. The smallest dsRNA oligonucleotides that form stable complexes with TLR3ecd (40-50 bp) each bind one TLR3ecd dimer, and these are also the smallest oligonucleotides that efficiently activate TLR3 in cells. We conclude that TLR3 assembles on dsRNA as stable dimers and that the minimal signaling unit is one TLR3 dimer. 相似文献
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5.
Thyroid hormone receptor alpha (TRalpha) and the oncoprotein v-erbA (a mutated form of TRalpha incapable of binding T3) bind as heterodimers with retinoid X receptor (RXR) to DNA sequences with different orientations of AGGTCA half sites. v-erbA can also form homodimers, whereas, TRalpha1 homodimerizes poorly. Therefore, in order to obtain a better understanding for the distinct homodimerization properties between TRalpha1 and v-erbA, we created chimeras between these two receptors and tested their abilities to homodimerize on direct and everted repeats (DRs, ERs). We found that the enhanced homodimerization properties of v-erbA compared to TRalpha1 map to isoleucine at position 339 in conjunction with serine at position 351 and alanine at position 358. Our data indicate that the methyl group in isoleucine at position 339 plays an important role in v-erbA homodimerization, particularly on ER 6. Functional studies with I339V+S351P+A358T, a v-erbA mutant unable to homodimerize but still able to heterodimerize with RXR on ERs and DRs, indicate that v-erbA-RXR heterodimers mediate the dominant negative activity of v-erbA on DRs. However, the repressor activity of this mutant is weaker than that of the wild type v-erbA on ERs, suggesting that v-erbA homodimers rather than v-erbA-RXR heterodimers mediate the potent dominant negative activity of v-erbA on ERs. 相似文献
6.
Grace CR Perrin MH Gulyas J Digruccio MR Cantle JP Rivier JE Vale WW Riek R 《Proceedings of the National Academy of Sciences of the United States of America》2007,104(12):4858-4863
The corticotropin releasing factor (CRF) family of ligands and their receptors coordinate endocrine, behavioral, autonomic, and metabolic responses to stress and play additional roles within the cardiovascular, gastrointestinal, and other systems. The actions of CRF and the related urocortins are mediated by activation of two receptors, CRF-R1 and CRF-R2, belonging to the B1 family of G protein-coupled receptors. The short-consensus-repeat fold (SCR) within the first extracellular domain (ECD1) of the CRF receptor(s) comprises the major ligand binding site and serves to dock a peptide ligand via its C-terminal segment, thus positioning the N-terminal segment to interact with the receptor's juxtamembrane domains to activate the receptor. Here we present the 3D NMR structure of ECD1 of CRF-R2beta in complex with astressin, a peptide antagonist. In the structure of the complex the C-terminal segment of astressin forms an amphipathic helix, whose entire hydrophobic face interacts with the short-consensus-repeat motif, covering a large intermolecular interface. In addition, the complex is characterized by intermolecular hydrogen bonds and a salt bridge. These interactions are quantitatively weighted by an analysis of the effects on the full-length receptor affinities using an Ala scan of CRF. These structural studies identify the major determinants for CRF ligand specificity and selectivity and support a two-step model for receptor activation. Furthermore, because of a proposed conservation of the fold for both the ECD1s and ligands, this structure can serve as a model for ligand recognition for the entire B1 receptor family. 相似文献
7.
The human erythropoietin receptor gene rescues erythropoiesis and developmental defects in the erythropoietin receptor null mouse. 总被引:10,自引:4,他引:6
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Erythropoietin and its receptor are required for definitive erythropoiesis and maturation of erythroid progenitor cells. Mice lacking the erythropoietin receptor exhibit severe anemia and die at about embryonic day 13.5. This phenotype can be rescued by the human erythropoietin receptor transgene. Animals expressing only the human erythropoietin receptor survived through adulthood with normal hematologic parameters and appeared to respond appropriately to induced anemic stress. In addition to restoration of erythropoiesis during development, the cardiac defect associated with embryos lacking the erythropoietin receptor was corrected and the increased apoptosis in fetal liver, heart, and brain in the erythropoietin receptor null phenotype was markedly reduced. These studies indicate that no species barrier exists between mouse and human erythropoietin receptor and that the human erythropoietin receptor transgene is able to provide specific expression in hematopoietic and other selected tissues to rescue erythropoiesis and other organ defects observed in the erythropoietin receptor null mouse. 相似文献
8.
Detection and isolation of the erythropoietin receptor using biotinylated erythropoietin 总被引:7,自引:1,他引:6
Procedures have been developed to label human erythropoietin (Ep) with biotin to detect and isolate the Ep-receptor. The labeling method used the abundant carbohydrate groups on Ep and resulted in biologically active biotin-Ep (b-Ep) containing 8 to 10 biotins per Ep molecule. Specific binding of b-Ep to cells from spleens of mice made anemic by phenylhydrazine injections was demonstrated using 125I-labeled streptavidin. B-Ep, together with fluorescently tagged streptavidin, was found to specifically detect Ep-receptor-bearing cells by flow cytometry. This was demonstrated in several ways. First, approximately 90% of nucleated spleen cells from phenylhydrazine-treated mice were clearly fluorescent after staining with b-Ep and streptavidin-phycoerythrin, whereas only background fluorescence was detected using spleen cells from untreated mice. In addition, Ep-receptors were detected on 5% to 10% of normal mouse bone marrow cells, and these cells could be identified as erythroid in nature by separating the cells into subpopulations based on light-scatter properties. Third, Ep-receptor expression was found to correlate positively with expression of transferrin receptors, confirming the erythroid nature of these cells. B-Ep was also used to isolate Ep-receptors from monkey COS cells transfected with the murine Ep-receptor cDNA. In these experiments a cell-surface-bound protein of approximately 65 Kd and an intracellular protein of approximately 60 Kd were isolated from these cells. The procedures described in this report for detecting Ep-receptor expressing cells and for isolating the Ep-receptor should be valuable for purifying erythroid cells from heterogeneous cell populations, for elucidating the structure of the Ep-receptor, and for studying the biological activities of Ep at the cellular and molecular level. 相似文献
9.
Multiple tyrosine residues in the cytosolic domain of the erythropoietin receptor promote activation of STAT5. 总被引:9,自引:0,他引:9
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U Klingmüller S Bergelson J G Hsiao H F Lodish 《Proceedings of the National Academy of Sciences of the United States of America》1996,93(16):8324-8328
Signaling through the erythropoietin receptor (EPO-R) is crucial for proliferation, differentiation, and survival of erythroid progenitor cells. EPO induces homodimerization of the EPO-R, triggering activation of the receptor-associated kinase JAK2 and activation of STAT5. By mutating the eight tyrosine residues in the cytosolic domain of the EPO-R, we show that either Y343 or Y401 is sufficient to mediate maximal activation of STAT5; tyrosine residues Y429 and Y431 can partially activate STAT5. Comparison of the sequences surrounding these tyrosines reveals YXXL as the probable motif specifying recruitment of STAT5 to the EPO-R. Expression of a mutant EPO-R lacking all eight tyrosine residues in the cytosolic domain supported a low but detectable level of EPO-induced STAT5 activation, indicating the existence of an alternative pathway for STAT5 activation independent of any tyrosine in the EPO-R. The kinetics of STAT5 activation and inactivation were the same, regardless of which tyrosine residue in the EPO-R mediated its activation or whether the alternative pathway was used. The ability of mutant EPO-Rs to activate STAT5 did not directly correlate with their mitogenic potential. 相似文献
10.
Activation of erythropoietin receptor in the absence of hormone by a peptide that binds to a domain different from the hormone binding site 总被引:3,自引:0,他引:3
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Tatjana Naranda Kenneth Wong R. Ilene Kaufman Avram Goldstein Lennart Olsson 《Proceedings of the National Academy of Sciences of the United States of America》1999,96(13):7569-7574
Applying a homology search method previously described, we identified a sequence in the extracellular dimerization site of the erythropoietin receptor, distant from the hormone binding site. A peptide identical to that sequence was synthesized. Remarkably, it activated receptor signaling in the absence of erythropoietin. Neither the peptide nor the hormone altered the affinity of the other for the receptor; thus, the peptide does not bind to the hormone binding site. The combined activation of signal transduction by hormone and peptide was strongly synergistic. In mice, the peptide acted like the hormone, protecting against the decrease in hematocrit caused by carboplatin. 相似文献
11.
Primary familial polycythaemia associated with a novel point mutation in the erythropoietin receptor 总被引:2,自引:0,他引:2
Tatsuo Furukawa Miwako Narita Minori Sakaue Tomio Otsuka Takashi Kuroha Masayoshi Masuko Takuaki Azegami Kenji Kishi Masuhiro Takahashi Jirou Utsumi Tadashi Koike & Yoshifusa Aizawa 《British journal of haematology》1997,99(1):222-227
To identify and treat patients at high risk of cytomegalovirus (CMV) pneumonia after bone marrow transplantation (BMT), we tested for CMV viraemia weekly, and performed broncho-alveolar lavage (BAL) on day 35 post-transplant in 63 recipients. 36 allogeneic BMT recipients were at a high risk of CMV pneumonia (25 CMV-seropositive recipients and 11 patients receiving marrow from a CMV-seropositive donor). Patients with a positive BAL or viraemia received a 14 d course of ganciclovir or foscarnet. CMV was detected in 29 (46%) of the 63 BMT recipients and excretion of CMV in blood and BAL was significantly linked. However, among the 29 patients who excreted the virus, only 10 (35%) shed CMV in blood and BAL at the same time; 19 patients (65%) had detectable CMV in blood (11 patients) or BAL (eight patients) only. Therefore, on the basis of viraemia or BAL alone, 21/29 patients (70%) and 18/29 patients (60%), respectively, would have received antiviral treatment. BAL increased the CMV detection rate by 13% (8/63 patients) relative to viraemia. With this strategy, the incidence of CMV pneumonia was reduced to 3% in allografted patients. Only two of the 19 autografted patients developed fatal CMV pneumonia. We avoided anti-CMV treatment in 54% of all the BMT recipients. In conclusion, CMV should be tested for in both blood and BAL fluid of BMT recipients at high risk of CMV pneumonia. 相似文献
12.
Estrogen regulates the function, growth, and proliferation of lactotropes in the pituitary. We report here that low-density lipoprotein (LDL) receptor (LDLR) gene expression and LDL uptake are strongly up-regulated by estrogen in pituitary somatolactotropic GH(3) cells. The uptake of LDL was significantly inhibited by the F-actin-severing drug, swinholide A, indicating that LDL uptake is dependent on the integrity of the cortical actin cytoskeleton in GH(3) cells. We examined whether the estrogen-inducible cytoskeletal linker protein, ezrin, interacts with the LDLR. The LDLR coimmunoprecipitated with ezrin, and fluorescently labeled LDL bound to regions of the cell membrane that colocalized with the active, phosphorylated form of ezrin (phosphoezrin). Evidence for a functional interaction between ezrin and the LDLR was obtained by transient transfection experiments using ezrin-green fluorescent protein (GFP) expression constructs. We observed that transient transfection of GH(3) cells with an ezrin N terminus-GFP dominant-negative construct prevented the uptake of LDL particles, whereas expression of GFP alone or an ezrin C terminus-GFP construct had no effect on LDL uptake. Transfection with the ezrin N terminus dominant- negative construct had no effect on the endocytosis of transferrin. Thus, estrogen stimulates the expression of ezrin and the LDLR in GH(3) cells, which interact physically and functionally to facilitate the endocytosis of LDL. We propose that the up-regulation and interaction of ezrin and the LDLR serves to augment the delivery of cholesterol and other lipids in support of the hypertrophic and proliferative response of cells to estrogen. 相似文献
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14.
Melanie J. Percy Mary Frances McMullin Anthony W. W. Roques Nigel B. Westwood Jay Acharya Anne E. Hughes Terence R. J. Lappin & Thomas C. Pearson 《British journal of haematology》1998,100(2):407-410
Familial erythrocytosis, associated with high haemoglobin levels and low serum erythropoietin (Epo), has been shown to co-segregate with a sequence repeat polymorphism at the 5' region of the erythropoietin receptor (EpoR) in a large Finnish family. We have investigated the cause of erythrocytosis in an English boy. Sequencing of the cytoplasmic region of the EpoR detected a de novo transition mutation of G to A at nucleotide 6002. This mutation resulted in the formation of a stop codon at amino acid 439 with the loss of 70 amino acids from the carboxy terminus. The mutation (G6002A) has arisen independently in a Finnish family and de novo in this English boy. Patients with unexplained erythrocytosis and low serum Epo levels should be investigated for EpoR mutations. 相似文献
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《Allergology international》2020,69(1):111-120
BackgroundPeriostin is a matricellular protein belonging to the fasciclin family, playing a role for the pathogenesis of allergic diseases by binding to integrins on cell surfaces. Serum periostin is elevated in various allergic diseases reflecting type 2 inflammation and tissue remodeling so that for allergic diseases, periostin is expected to be a novel biomarker for diagnosis, assessing severity or prognosis, and predicting responsiveness to treatments. We have previously shown that most serum periostin exists in the oligomeric form by intermolecular disulfide bonds.MethodsIn this study, we examined how periostin forms a complex in serum, whether the periostin complex in serum is functional, and whether the complex formation interferes with reactivity to anti-periostin Abs.ResultsWe found that periostin formed a complex with IgA1 at a 1:1 ratio. The periostin in the serum complex contained at least five different isoforms. However, IgA was not essential for the oligomeric formation of periostin in mouse serum or in IgA-lacking serum. The periostin-IgA complex in human serum was functional, sustaining the ability to bind to αVβ3 integrin on cell surfaces. Moreover, periostin formed the complex with IgA broadly, which interferes the binding of the Abs recognizing all of the domains except the R4 domain to periostin.ConclusionsPeriostin is a novel member of the IgA-associated molecules. These results are of great potential use to understand the pathological roles of periostin in allergic diseases and, from a practical standpoint, to develop diagnostics or therapeutic agents against periostin. 相似文献
18.
AIM: To investigate the expression of Erythropoietin (Epo) and its receptor (EpoR) in gastric adenocarcinoma (GAC) and the correlation with angiogenesis and clinicopathological features. METHODS: The expressions of Epo, EpoR and vascular endothelial growth factor (VEGF), as well as microvessel density were evaluated in 172 GAC biopsies by immunohistochemical staining. The correlations between these parameters and patient’s clinicopathological features were analyzed statistically. RESULTS: The proportion of ... 相似文献
19.
L Feldman R Heinzerling R P Hillam Y E Chern J G Frazier K L Davis A J Sytkowski 《Experimental hematology》1992,20(1):64-68
We have produced a series of monoclonal antibodies (MoAbs) to amino acid region 99-129 of human erythropoietin (Epo) that distinguish unique structural features within this putative receptor binding domain of the hormone. The MoAbs recognize denatured Epo with widely different sensitivities on a Western blot and differentially bind to native Epo in solution. In addition, three of the four MoAbs neutralize the biological activity of Epo in a concentration-dependent fashion in vitro. Neutralization was measured both by inhibition of Epo-induced differentiation in Rauscher murine erythroleukemia cells and by inhibition of Epo-induced proliferation in normal murine splenic erythroid precursors. Characterization of the structural epitopes recognized by each of these four reagent MoAbs should provide us with important information concerning the requirements for hormone-receptor interaction. 相似文献
20.
D Linnekin G A Evans A D''''Andrea W L Farrar 《Proceedings of the National Academy of Sciences of the United States of America》1992,89(14):6237-6241
We have examined the signal transduction mechanism of the hematopoietic growth factor erythropoietin (Epo). Epo stimulation of Ba/F3 cells transfected with the Epo receptor resulted in increases in tyrosine phosphorylation of proteins of 97, 75, and 55 kDa. Epo-induced increases in tyrosine phosphorylation of a 97-kDa protein were also detected within the Epo receptor complex, suggesting that a protein tyrosine kinase is associated with the Epo receptor. Protein tyrosine kinase activity was found within the Epo receptor complex and modulation of this activity was observed after treatment of cells with Epo. Furthermore, constitutively high amounts of protein kinase activity were observed in Epo receptor complexes isolated from autonomously growing cells coexpressing the Epo receptor and the leukemogenic glycoprotein gp55. The dominant phosphotyrosylprotein found associated with the Epo receptor was 97 kDa. An Epo receptor-associated protein of identical molecular mass was also found to bind ATP, a characteristic critical for protein kinases. Collectively, these data demonstrate that the Epo receptor is associated with protein tyrosine kinase activity and further suggest that a 97-kDa phosphotyrosylprotein associated with the Epo receptor is a protein tyrosine kinase involved in Epo-mediated signal transduction. 相似文献