scholarly journals Epo receptor marks the spot

Blood ◽  
2019 ◽  
Vol 134 (5) ◽  
pp. 413-414 ◽  
Author(s):  
Robert F. Paulson
Keyword(s):  
Epo Receptor

scholarly journals Inhibition of receptor binding and neutralization of bioactivity by anti-erythropoietin monoclonal antibodies

Blood ◽  
1990 ◽  
Vol 75 (4) ◽  
pp. 874-880 ◽  
Author(s):  
AD D'Andrea ◽  
PJ Szklut ◽  
HF Lodish ◽  
EM Alderman
Keyword(s):  
Monoclonal Antibodies ◽  
Receptor Binding ◽  
Sodium Dodecyl ◽  
Dependent Manner ◽  
Epo Receptor ◽  
High Affinity ◽  
Group I ◽  
Dependent Cell ◽  
Group Ii ◽  
Murine Erythroleukemia

Abstract We have generated four high affinity monoclonal antibodies (MoAbs) to recombinant human erythropoietin (EPO). All four MoAbs immunoprecipitate radioiodinated native EPO, and the concentrations of MoAbs required for maximum binding range from 10 nmol/L to 100 nmol/L. Two MoAbs, designated Group I MoAbs, bind to an epitope within the N- terminal 20 amino acids of EPO and also immunoprecipitate sodium dodecyl sulfate (SDS)-denatured EPO. Two other MoAbs (Group II MoAbs) do not immunoprecipitate SDS-denatured EPO and do not bind to any of the eight endo C fragments of EPO. We first used murine erythroleukemia (MEL) cells to test the MoAbs for inhibition of EPO-receptor binding. MEL cells, although unresponsive to EPO, express 760 high affinity receptors for EPO per cell (Kd = 0.24 nmol/L). To assay our MoAbs, MEL cells were grown as monolayers on fibronectin-coated Petri dishes and incubated at 4 degrees C with radioiodinated EPO. Group I MoAbs do not inhibit binding of radioiodinated EPO to the MEL EPO-receptor, but Group II MoAbs do inhibit binding in a dose-dependent manner. We next examined the neutralization of EPO bioactivity by our MoAbs, using EPO- dependent cell line. Only Group II MoAbs inhibit a newly developed EPO- dependent cell growth, demonstrating that inhibition of EPO-receptor binding correlates with neutralization of EPO bioactivity.

Both proteasomes and lysosomes degrade the activated erythropoietin receptor

Blood ◽  
2005 ◽  
Vol 105 (2) ◽  
pp. 600-608 ◽  
Author(s):  
Pierre Walrafen ◽  
Frédérique Verdier ◽  
Zahra Kadri ◽  
Stany Chrétien ◽  
Catherine Lacombe ◽  
...  
Keyword(s):  
Cell Surface ◽  
Intracellular Signaling ◽  
Erythropoietin Receptor ◽  
Janus Kinase ◽  
Receptor Internalization ◽  
Down Regulation ◽  
Epo Receptor ◽  
Receptor Complexes ◽  
Regulation Mechanisms ◽  
Rapid Activation

AbstractActivation of the erythropoietin receptor (EpoR) after Epo binding is very transient because of the rapid activation of strong down-regulation mechanisms that quickly decrease Epo sensitivity of the cells. Among these down-regulation mechanisms, receptor internalization and degradation are probably the most efficient. Here, we show that the Epo receptor was rapidly ubiquitinated after ligand stimulation and that the C-terminal part of the Epo receptor was degraded by the proteasomes. Both ubiquitination and receptor degradation by the proteasomes occurred at the cell surface and required Janus kinase 2 (Jak2) activation. Moreover, Epo-EpoR complexes were rapidly internalized and targeted to the lysosomes for degradation. Neither Jak2 nor proteasome activities were required for internalization. In contrast, Jak2 activation was necessary for lysosome targeting of the Epo-EpoR complexes. Blocking Jak2 with the tyrphostin AG490 led to some recycling of internalized Epo-Epo receptor complexes to the cell surface. Thus, activated Epo receptors appear to be quickly degraded after ubiquitination by 2 proteolytic systems that proceed successively: the proteasomes remove part of the intracellular domain at the cell surface, and the lysosomes degrade the remaining part of the receptor-hormone complex. The efficiency of these processes probably explains the short duration of intracellular signaling activated by Epo.

scholarly journals Expression and modulation of specific, high affinity binding sites for erythropoietin on the human erythroleukemic cell line K562

Blood ◽  
1988 ◽  
Vol 71 (1) ◽  
pp. 104-109 ◽  
Author(s):  
JK Fraser ◽  
FK Lin ◽  
MV Berridge
Keyword(s):  
Cell Line ◽  
K562 Cell ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Receptor Expression ◽  
Target Cells ◽  
Epo Receptor ◽  
High Affinity ◽  
Erythroleukemic Cell ◽  
Erythroleukemic Cell Line

Erythroid differentiation is mediated by several interacting factors which include the glycoprotein hormone erythropoietin (Epo), interleukin-3 (IL-3) in the mouse, and erythroid-potentiating activity (EPA) in humans. Each of these factors binds to specific cell surface receptors on responsive target cells, but the way in which these factors interact to modulate erythropoiesis is unknown. In the present study, we used the human erythroleukemic cell line K562 to examine expression and regulation of the receptor for Epo using 125I-labeled, bioactive recombinant human Epo. K562 cells expressed low numbers of a single class of high-affinity Epo receptors corresponding to 4 to 6 receptors per K562 cell (KD = 270 to 290 pmol/L). Treatment of K562 cell cultures with medium conditioned by the EPA-secreting cell line U937 (U937CM) increased receptor expression 2.6 to 3.5-fold to 13 to 17 receptors/cell (KD = 260 to 300 pmol/L). That all of the Epo receptor- potentiating activity in U937CM was accounted for by EPA was shown by a similar increase in Epo receptor expression on K562 cells with recombinant EPA. The effect of U937CM on Epo receptors was reversed by culturing cells in inducer-free medium for 3 days. Medium conditioned by the 5637 cell line had no effect on Epo receptors on K562 cells. In methylcellulose culture, U937CM and Epo acted synergistically to increase erythroid differentiation of K562. Similarly, U937CM stimulated human cord blood CFU-E growth under conditions in which Epo was limiting or in excess. Increases in Epo receptor expression on K562 cells and on CFU-E in response to EPA may mediate the effects of Epo on these cells.

scholarly journals Epobis is a Nonerythropoietic and Neuroprotective Agonist of the Erythropoietin Receptor with Anti-Inflammatory and Memory Enhancing Effects

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Oksana Dmytriyeva ◽  
Stanislava Pankratova ◽  
Irina Korshunova ◽  
Peter S. Walmod
Keyword(s):  
Clinical Signs ◽  
Neuronal Cell ◽  
Erythropoietin Receptor ◽  
Autoimmune Encephalomyelitis ◽  
Erythroid Progenitor ◽  
Epo Receptor ◽  
Antioxidative Effects ◽  
Anti Inflammatory ◽  
Memory Enhancing

The cytokine erythropoietin (EPO) stimulates proliferation and differentiation of erythroid progenitor cells. Moreover, EPO has neuroprotective, anti-inflammatory, and antioxidative effects, but the use of EPO as a neuroprotective agent is hampered by its erythropoietic activity. We have recently designed the synthetic, dendrimeric peptide, Epobis, derived from the sequence of human EPO. This peptide binds the EPO receptor and promotes neuritogenesis and neuronal cell survival. Here we demonstrate that Epobisin vitropromotes neuritogenesis in primary motoneurons and has anti-inflammatory effects as demonstrated by its ability to decrease TNF release from activated AMJ2-C8 macrophages and rat primary microglia. When administered systemically Epobis is detectable in both plasma and cerebrospinal fluid, demonstrating that the peptide crosses the blood-brain barrier. Importantly, Epobis is not erythropoietic, but systemic administration of Epobis in rats delays the clinical signs of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, and the peptide has long-term, but not short-term, effects on working memory, detected as an improved social memory 3 days after administration. These data reveal Epobis to be a nonerythropoietic and neuroprotective EPO receptor agonist with anti-inflammatory and memory enhancing properties.

Erythropoietin: Physiology and Pharmacology Update

2003 ◽  
Vol 228 (1) ◽  
pp. 1-14 ◽  
Author(s):  
James W. Fisher
Keyword(s):  
Cell Proliferation ◽  
Renal Failure ◽  
Chronic Renal Failure ◽  
Receptor Activation ◽  
Chemotherapeutic Agents ◽  
Phospholipase A ◽  
Cell Surface Receptor ◽  
Erythroid Progenitor ◽  
Epo Receptor ◽  
Stimulation Of

This minireview is an update of a 1997 review on erythropoletin (EPO) in this journal (1). EPO is a 30,400-dalton glycoprotein that regulates red cell production. In the human, EPO is produced by peritubular cells in the kidneys of the adult and in hepatocytes in the fetus. Small amounts of extra-renal EPO are produced by the liver in adult human subjects. EPO binds to an erythroid progenitor cell surface receptor that includes a p66 chain, and, when activated, the p66 protein becomes dimerized. EPO receptor activation induces a JAK2 tyrosine kinase, which leads to tyrosine phosphorylation of the EPO receptor and several proteins. EPO receptor binding leads to intracellular activation of the Ras/mitogen-activated kinase pathway, which is involved with cell proliferation, phosphatidylinositol 3-kinase, and STATS 1, 3, 5A, and 5B transcriptional factors. EPO acts primarily to rescue erythroid cells from apoptosis (programmed cell death) to increase their survival. EPO acts synergistically with several growth factors (SCF, GM-CSF, 1L-3, and IGF-1) to cause maturation and proliferation of erythroid progenitor cells (primarily colony-forming unit-E). Oxygen-dependent regulation of EPO gene expression is postulated to be controlled by a hypoxia-inducible transcription factor (HIF-1α). Hypoxia-inducible EPO production is controlled by a 50-bp hypoxia-inducible enhancer that is approximately 120 bp 3' to the polyadenylation site. Hypoxia signal transduction pathways involve kinases A and C, phospholipase A2, and transcription factors ATF-1 and CREB-1. A model has been proposed for adenosine activation of EPO production that involves protein kinases A and C and the phospholipase A2 pathway. Other effects of EPO include a hematocrit-independent, vasoconstriction-dependent hypertension, increased endothelin production, upregulation of tissue renin, change in vascular tissue prostaglandins production, stimulation of angiogenesis, and stimulation of endothelial and vascular smooth muscle cell proliferation. Recombinant human EPO (rHuEPO) is currently being used to treat patients with anemias associated with chronic renal failure, AIDS patients with anemia due to treatment with zidovudine, nonmyeloid malignancies in patients treated with chemotherapeutic agents, perioperative surgical patients, and autologous blood donation. A novel erythropolesis-stimulating factor (NESP, darbepoetin) has been synthesized and when compared with rHuEPO, NESP has a higher carbohydrate content (52% vs 40%), a longer plasma half-life, the amino acid sequence differs from that of native human EPO at five positions, and has been reported to maintain hemoglobin levels just as effectively in patients with chronic renal failure as rHuEPO at less frequent dosing. The use of rHuEPO and darbepoetin to enhance athletic performance is officially banned by most sports-governing bodies because the excessive erythrocytosis can lead to increased thrombogenicity and can cause deep vein, coronary, and cerebral thromboses.

A Minimal Cytoplasmic Subdomain of the Erythropoietin Receptor Mediates Erythroid and Megakaryocytic Cell Development

Blood ◽  
1999 ◽  
Vol 94 (10) ◽  
pp. 3381-3387 ◽  
Author(s):  
Chris P. Miller ◽  
Zi Y. Liu ◽  
Constance T. Noguchi ◽  
Don M. Wojchowski
Keyword(s):  
Progenitor Cells ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Cell Development ◽  
Erythropoietin Receptor ◽  
Erythroid Progenitor ◽  
Epo Receptor ◽  
Receptor Complexes ◽  
Erythroid Progenitor Cells ◽  
Fetal Liver Cells

Signals provided by the erythropoietin (Epo) receptor are essential for the development of red blood cells, and at least 15 distinct signaling factors are now known to assemble within activated Epo receptor complexes. Despite this intriguing complexity, recent investigations in cell lines and retrovirally transduced murine fetal liver cells suggest that most of these factors and signals may be functionally nonessential. To test this hypothesis in erythroid progenitor cells derived from adult tissues, a truncated Epo receptor chimera (EE372) was expressed in transgenic mice using a GATA-1 gene-derived vector, and its capacity to support colony-forming unit-erythroid proliferation and development was analyzed. Expression at physiological levels was confirmed in erythroid progenitor cells expanded ex vivo, and this EE372 chimera was observed to support mitogenesis and red blood cell development at wild-type efficiencies both independently and in synergy with c-Kit. In addition, the activity of this minimal chimera in supporting megakaryocyte development was tested and, remarkably, was observed to approximate that of the endogenous receptor for thrombopoietin. Thus, the box 1 and 2 cytoplasmic subdomains of the Epo receptor, together with a tyrosine 343 site (each retained within EE372), appear to provide all of the signals necessary for the development of committed progenitor cells within both the erythroid and megakaryocytic lineages.

Erythropoietin Induces the Tyrosine Phosphorylation of GAB1 and Its Association With SHC, SHP2, SHIP, and Phosphatidylinositol 3-Kinase

Blood ◽  
1999 ◽  
Vol 93 (8) ◽  
pp. 2578-2585 ◽  
Author(s):  
Carinne Lecoq-Lafon ◽  
Frédérique Verdier ◽  
Serge Fichelson ◽  
Stany Chrétien ◽  
Sylvie Gisselbrecht ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Intracellular Signaling ◽  
Direct Consequence ◽  
Receptor Activation ◽  
Phosphatidylinositol 3 Kinase ◽  
Erythroid Progenitors ◽  
Epo Receptor ◽  
Gm Csf ◽  
Macrophage Colony ◽  
Phosphatidylinositol 3

Abstract Five tyrosine-phosphorylated proteins with molecular masses of 180, 145, 116, 100, and 70 kD are associated with phosphatidylinositol 3-kinase (PI 3-kinase) in erythropoietin (Epo)-stimulated UT-7 cells. The 180- and 70-kD proteins have been previously shown to be IRS2 and the Epo receptor. In this report, we show that the 116-kD protein is the IRS2-related molecular adapter, GAB1. Indeed, Epo induced the transient tyrosine phosphorylation of GAB1 in UT-7 cells. Both kinetics and Epo dose-response experiments showed that GAB1 tyrosine phosphorylation was a direct consequence of Epo receptor activation. After tyrosine phosphorylation, GAB1 associated with the PI 3-kinase, the phosphotyrosine phosphatase SHP2, the phosphatidylinositol 3,4,5 trisphosphate 5-phosphatase SHIP, and the molecular adapter SHC. GAB1 was also associated with the molecular adapter GRB2 in unstimulated cells, and this association dramatically increased after Epo stimulation. Thus, GAB1 could be a scaffold protein able to couple the Epo receptor activation with the stimulation of several intracellular signaling pathways. Epo-induced tyrosine phosphorylation of GAB1 was also observed in normal human erythroid progenitors isolated from cord blood. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and thrombopoietin (TPO) also induced the tyrosine phosphorylation of GAB1 in UT-7 cells, indicating that this molecule participates in the signal transduction of several cytokine receptors.

scholarly journals Mechanisms of Stem Cell Factor and Erythropoietin Proliferative Co-signaling in FDC2-ER Cells

Blood ◽  
1997 ◽  
Vol 90 (9) ◽  
pp. 3533-3545 ◽  
Author(s):  
Bhavana Joneja ◽  
Hong-Chi Chen ◽  
Dhaya Seshasayee ◽  
Amy L. Wrentmore ◽  
Don M. Wojchowski
Keyword(s):  
Stem Cell ◽  
Cell Lines ◽  
Stem Cell Factor ◽  
Progenitor Cell ◽  
Egf Receptor ◽  
Synergistic Effects ◽  
Ectopic Expression ◽  
Hematopoietic Progenitor Cell ◽  
Detectable Effect ◽  
Epo Receptor

Abstract Studies of hematopoietic progenitor cell development in vivo, ex vivo, and in factor-dependent cell lines have shown that c-kit promotes proliferation through synergistic effects with at least certain type 1 cytokine receptors, including the erythropoietin (Epo) receptor. Presently, c-kit is shown to efficiently support both mitogenesis and survival in the FDCP1 cell subline, FDC2. In this system, mitogenic synergy with c-kit was observed for ectopically expressed wild-type Epo receptors (wt-ER), an epidermal growth factor (EGF) receptor/Epo receptor chimera, and a highly truncated Epo receptor construct ER-Bx1. Thus, the Epo receptor cytoplasmic box 1 subdomain appears, at least in part, to mediate mitogenic synergy with c-kit. In studies of potential effectors of this response, Jak2 tyrosine phosphorylation was shown to be induced by Epo, but not by stem cell factor (SCF). In addition and in contrast to signaling in Mo7e and BM6 cell lines, in FDC2-ER cells SCF and Epo each were shown to rapidly activate Pim 1 gene expression. Recently, roles also have been suggested for the nuclear trans-factor GATA-1 in regulating progenitor cell proliferation. In FDC2-ER cells, the ectopic expression of GATA-1 had no detectable effect on Epo inhibition of apoptosis. However, GATA-1 expression did result in a selective and marked inhibition in mitogenic responsiveness to SCF and to a decrease in c-kit transcript expression. These studies of SCF and Epo signaling in FDC2–wt-ER cells serve to functionally map the ERB1 region as a c-kit–interactive domain, suggest that Pim1 might contribute to SCF and Epo mitogenic synergy and support the notion that SCF and Epo may act in opposing ways during red cell differentiation.

scholarly journals Mapping of the Active Site of Recombinant Human Erythropoietin

Blood ◽  
1997 ◽  
Vol 89 (2) ◽  
pp. 493-502 ◽  
Author(s):  
Steve Elliott ◽  
Tony Lorenzini ◽  
David Chang ◽  
Jack Barzilay ◽  
Evelyne Delorme
Keyword(s):  
Receptor Binding ◽  
Recombinant Human Erythropoietin ◽  
Electrostatic Interactions ◽  
Binding Activity ◽  
Amino Acid Residues ◽  
Epo Receptor ◽  
Human Erythropoietin ◽  
Biologic Activity ◽  
Identify Amino Acid

Abstract Recombinant human erythropoietin (rHuEPO) variants have been constructed to identify amino acid residues important for biological activity. Immunoassays were used to determine the effect of each mutation on rHuEPO folding. With this strategy, we could distinguish between mutations that affected bioactivity directly and those that affected bioactivity because the mutation altered rHuEPO conformation. Four regions were found to be important for bioactivity: amino acids 11 to 15, 44 to 51, 100 to 108, and 147 to 151. EPO variants could be divided into two groups according to the differential effects on EPO receptor binding activity and in vitro biologic activity. This suggests that rHuEPO has two separate receptor binding sites. Mutations in basic residues reduced the biologic activity, whereas mutations in acidic residues did not. This suggests that electrostatic interactions between rHuEPO and the human EPO receptor may involve positive charges on rHuEPO.

Characterization of three erythropoietin (Epo)-binding proteins in various human Epo-responsive cell lines and in cells transfected with human Epo-receptor cDNA [see comments]

Blood ◽  
1995 ◽  
Vol 85 (1) ◽  
pp. 106-114 ◽  
Author(s):  
T Takahashi ◽  
S Chiba ◽  
N Hirano ◽  
Y Yazaki ◽  
H Hirai
Keyword(s):  
Molecular Cloning ◽  
Cell Lines ◽  
Cross Linking ◽  
Specific Antibodies ◽  
Epo Receptor ◽  
Receptor Complexes ◽  
Receptor Cdna ◽  
Cloned Cdna ◽  
Responsive Cell

Molecular cloning of a cDNA for a mouse erythropoietin (Epo) receptor (EpoR) has facilitated the understanding of the structure of this receptor. However, there is, as yet, no explanation for the discrepancy between the protein recognized by specific antibodies against mouse EpoR and the unexpectedly larger species that can be cross-linked to labeled Epo. It is unclear whether the product of an unidentified gene is included in the EpoR complex. In the present study, we directly compared the cross-linking patterns for human EpoR that were endogenously expressed in three types of Epo-responsive cell, and that was artificially expressed in nonhematopoietic cells after transfection with cDNA for human EpoR. We observed that 85-kD and 105-kD proteins formed ligand-receptor complexes in all the human Epo-responsive cells and, furthermore, that the formation of a complex derived from the 70- kD protein was dependent on the level of expression of the cloned EpoR mRNA in these cells. By contrast, a prominent cross-linked band derived from the 70-kD protein and a weaker band derived from the 80- to 85-kD protein, but no band derived from the 105-kD protein, could be shown in the case of nonhematopoietic cells transfected with the human EpoR cDNA. These observations suggest that the cloned cDNA for human EpoR alone does not allow generation of the complete EpoR in nonhematopoietic cells and that the 105-kD Epo-binding protein may represent the product of an as yet unidentified gene that is expressed in hematopoietic cells.

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