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Sylvie Gisselbrecht - One of the best experts on this subject based on the ideXlab platform.
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proteasomes regulate Erythropoietin Receptor and signal transducer and activator of transcription 5 stat5 activation possible involvement of the ubiquitinated cis protein
Journal of Biological Chemistry, 1998Co-Authors: Frederique Verdier, Sylvie Gisselbrecht, Stany Chrétien, Catherine Lacombe, Odile Muller, Paule Varlet, Akihiko Yoshimura, Patrick MayeuxAbstract:Abstract Cis is an Src homology 2 domain-containing protein, which binds to the Erythropoietin Receptor and decreases Erythropoietin-stimulated cell proliferation. We show that Cis associates with the second tyrosine residue of the intracellular domain of the Erythropoietin Receptor (Tyr401). Two forms of Cis with molecular masses of 32 and 37 kDa were detected, and we demonstrate that the 37-kDa protein resulted from post-translational modifications of the 32-kDa form. Anti-ubiquitin antibodies recognized the 37-kDa form of Cis and the proteasome inhibitorsN-acetyl-leucyl-leucyl-norleucinal and lactacystin inhibited its degradation, showing that the 37-kDa form of Cis is a ubiquitinated protein, which seems to be rapidly degraded by the proteasome. In Erythropoietin-stimulated UT-7 cells, the activation of the Erythropoietin Receptor and signal transducer and activator of transcription 5 (STAT5) was transient and returned to basal levels after 30–60 min of Erythropoietin stimulation. In contrast, these proteins remained strongly phosphorylated, and STAT5 remained activated for at least 120 min in the presence of proteasome inhibitors. These experiments demonstrate that the proteasomes are involved in the down-regulation of the Erythropoietin Receptor activation signals. Because the proteasome inhibitors induced the accumulation of both the ubiquitinated form of Cis and the Cis-Erythropoietin Receptor complexes, our results suggest that the ubiquitinated form of Cis could be involved in the proteasome-mediated inactivation of the Erythropoietin Receptor.
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Identification of tyrosine residues within the intracellular domain of the Erythropoietin Receptor crucial for STAT5 activation.
The EMBO Journal, 1996Co-Authors: Stephanie Gobert, Isabelle Dusanter-fourt, Sylvie Gisselbrecht, Stany Chrétien, Fabrice Gouilleux, Christophe Pallard, Catherine Lacombe, Bernd Groner, O. Muller, Patrick MayeuxAbstract:Abstract FDCP-1 cells are hematopoietic progenitor cells which require interleukin-3 for survival and proliferation. FDCP-1 cells stably transfected with the murine Erythropoietin Receptor cDNA survive and proliferate in the presence of Erythropoietin. Erythropoietin induces the activation of the short forms (80 kDa) of STAT5 in the cells. Erythropoietin-induced activation of STAT5 was strongly reduced in cells expressing mutated variants of the Erythropoietin Receptors in which tyrosine residues in their intracellular domain have been eliminated. We determined that the Erythropoietin Receptor tyrosine residues 343 and 401 are independently necessary for STAT5 activation. The amino acid sequences surrounding these two tyrosine residues are very similar. Peptides comprising either phosphorylated Tyr343 or phosphorylated Tyr401, but not their unphosphorylated counterparts, inhibited the STAT5 activation. We propose that these two tyrosine residues of the Erythropoietin Receptor constitute docking sites for the STAT5 SH2 domain. The growth stimulus mediated by Erythropoietin was decreased in cells expressing Erythropoietin Receptors lacking both Tyr343 and Tyr401. This suggests that STAT5 activation could be involved in the growth control of FDCP-1 cells.
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Structure of the Erythropoietin Receptor.
Experimental Biology and Medicine, 1994Co-Authors: Patrick Mayeux, Sylvie Pallu, Stephanie Gobert, Catherine Lacombe, Sylvie GisselbrechtAbstract:Despite extensive studies, the structure of the Erythropoietin Receptor remains little understood. cDNAs encoding the human and murine Erythropoietin Receptors have been cloned and the structure of these proteins is discussed. Although the proteins encoded by these cDNAs play key roles in Erythropoietin binding and in Erythropoietin signal transduction, increasing evidence strongly suggests that the Erythropoietin Receptor is a multimeric complex. The murine Erythropoietin Receptor has been solubilized under mild conditions and the molecular mass of the native Receptor has been shown to be significantly higher than the molecular mass of the cloned chain. Cross-linking experiments have revealed the presence of three proteins covalently bound to Erythropoietin by the cross-linking reagents; however, only one of them seems to derive from the cloned chain. Moreover, functional evidence also suggests the presence of other Erythropoietin Receptor subunits.
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Erythropoietin induces the association of phosphatidylinositol 3′-kinase with a tyrosine-phosphorylated protein complex containing the Erythropoietin Receptor
FEBS Journal, 1993Co-Authors: Patrick Mayeux, Philippe Mauduit, Michèle Sabbah, Isabelle Dusanter-fourt, Brian J. Druker, Catherine Lacombe, William Vainchenker, O. Muller, S Fischer, Sylvie GisselbrechtAbstract:Stimulation of sensitive cells with Erythropoietin results in rapid induction of protein tyrosine phosphorylation. Other than tyrosine phosphorylation of one chain of the Erythropoietin Receptor, the identities of the remaining tyrosine-phosphorylated proteins are undefined. In this report, we demonstrate that the stimulation of the Erythropoietin-sensitive human UT7 cells by Erythropoietin rapidly resulted in the appearance of phosphatidylinositol 3-kinase activity in anti-phosphotyrosine immunoprecipitates. Erythropoietin action was rapid, detectable after as early as 1 min stimulation, transient, returning to control level after 30 min stimulation and was observed using the Erythropoietin concentrations able to stimulate the cell proliferation. Anti-(phosphatidylinositol 3-kinase) antibodies specifically immunoprecipitated 125I-Erythropoietin bound to its Receptor, strongly suggesting that phosphatidylinositol 3-kinase associated with a protein complex containing the activated Erythropoietin Receptor. To confirm this result, phosphatidylinositol 3-kinase was immunoprecipitated from Erythropoietin-stimulated cells using mild conditions followed by Western analysis using anti-phosphotyrosine antibodies. Five tyrosine phosphorylated proteins were revealed: the cloned chain of the Erythropoietin Receptor, the regulatory subunit of phosphatidylinositol 3-kinase and three unidentified proteins of 111, 97 and 64 kDa. None of these tyrosine phosphorylated proteins was detected in anti-(phosphatidylinositol 3-kinase) immunoprecipitates from unstimulated cells. Thus, our results show that phosphatidylinositol 3-kinase associates with a tyrosine-phosphorylated protein complex containing the activated Erythropoietin Receptor.
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Erythropoietin induces the association of phosphatidylinositol 3'-kinase with a tyrosine-phosphorylated protein complex containing the Erythropoietin Receptor.
European journal of biochemistry, 1993Co-Authors: Patrick Mayeux, Philippe Mauduit, Michèle Sabbah, Isabelle Dusanter-fourt, Brian J. Druker, Catherine Lacombe, William Vainchenker, S Fischer, Odile Muller, Sylvie GisselbrechtAbstract:Stimulation of sensitive cells with Erythropoietin results in rapid induction of protein tyrosine phosphorylation. Other than tyrosine phosphorylation of one chain of the Erythropoietin Receptor, the identities of the remaining tyrosine-phosphorylated proteins are undefined. In this report, we demonstrate that the stimulation of the Erythropoietin-sensitive human UT7 cells by Erythropoietin rapidly resulted in the appearance of phosphatidylinositol 3-kinase activity in anti-phosphotyrosine immunoprecipitates. Erythropoietin action was rapid, detectable after as early as 1 min stimulation, transient, returning to control level after 30 min stimulation and was observed using the Erythropoietin concentrations able to stimulate the cell proliferation. Anti-(phosphatidylinositol 3-kinase) antibodies specifically immunoprecipitated 125I-Erythropoietin bound to its Receptor, strongly suggesting that phosphatidylinositol 3-kinase associated with a protein complex containing the activated Erythropoietin Receptor. To confirm this result, phosphatidylinositol 3-kinase was immunoprecipitated from Erythropoietin-stimulated cells using mild conditions followed by Western analysis using anti-phosphotyrosine antibodies. Five tyrosine phosphorylated proteins were revealed: the cloned chain of the Erythropoietin Receptor, the regulatory subunit of phosphatidylinositol 3-kinase and three unidentified proteins of 111, 97 and 64 kDa. None of these tyrosine phosphorylated proteins was detected in anti-(phosphatidylinositol 3-kinase) immunoprecipitates from unstimulated cells. Thus, our results show that phosphatidylinositol 3-kinase associates with a tyrosine-phosphorylated protein complex containing the activated Erythropoietin Receptor.
Patrick Mayeux - One of the best experts on this subject based on the ideXlab platform.
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both proteasomes and lysosomes degrade the activated Erythropoietin Receptor
Blood, 2005Co-Authors: Pierre Walrafen, Stany Chrétien, Frederique Verdier, Zahra Kadri, C Lacombe, Patrick MayeuxAbstract:Activation 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.
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proteasomes regulate Erythropoietin Receptor and signal transducer and activator of transcription 5 stat5 activation possible involvement of the ubiquitinated cis protein
Journal of Biological Chemistry, 1998Co-Authors: Frederique Verdier, Sylvie Gisselbrecht, Stany Chrétien, Catherine Lacombe, Odile Muller, Paule Varlet, Akihiko Yoshimura, Patrick MayeuxAbstract:Abstract Cis is an Src homology 2 domain-containing protein, which binds to the Erythropoietin Receptor and decreases Erythropoietin-stimulated cell proliferation. We show that Cis associates with the second tyrosine residue of the intracellular domain of the Erythropoietin Receptor (Tyr401). Two forms of Cis with molecular masses of 32 and 37 kDa were detected, and we demonstrate that the 37-kDa protein resulted from post-translational modifications of the 32-kDa form. Anti-ubiquitin antibodies recognized the 37-kDa form of Cis and the proteasome inhibitorsN-acetyl-leucyl-leucyl-norleucinal and lactacystin inhibited its degradation, showing that the 37-kDa form of Cis is a ubiquitinated protein, which seems to be rapidly degraded by the proteasome. In Erythropoietin-stimulated UT-7 cells, the activation of the Erythropoietin Receptor and signal transducer and activator of transcription 5 (STAT5) was transient and returned to basal levels after 30–60 min of Erythropoietin stimulation. In contrast, these proteins remained strongly phosphorylated, and STAT5 remained activated for at least 120 min in the presence of proteasome inhibitors. These experiments demonstrate that the proteasomes are involved in the down-regulation of the Erythropoietin Receptor activation signals. Because the proteasome inhibitors induced the accumulation of both the ubiquitinated form of Cis and the Cis-Erythropoietin Receptor complexes, our results suggest that the ubiquitinated form of Cis could be involved in the proteasome-mediated inactivation of the Erythropoietin Receptor.
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Identification of tyrosine residues within the intracellular domain of the Erythropoietin Receptor crucial for STAT5 activation.
The EMBO Journal, 1996Co-Authors: Stephanie Gobert, Isabelle Dusanter-fourt, Sylvie Gisselbrecht, Stany Chrétien, Fabrice Gouilleux, Christophe Pallard, Catherine Lacombe, Bernd Groner, O. Muller, Patrick MayeuxAbstract:Abstract FDCP-1 cells are hematopoietic progenitor cells which require interleukin-3 for survival and proliferation. FDCP-1 cells stably transfected with the murine Erythropoietin Receptor cDNA survive and proliferate in the presence of Erythropoietin. Erythropoietin induces the activation of the short forms (80 kDa) of STAT5 in the cells. Erythropoietin-induced activation of STAT5 was strongly reduced in cells expressing mutated variants of the Erythropoietin Receptors in which tyrosine residues in their intracellular domain have been eliminated. We determined that the Erythropoietin Receptor tyrosine residues 343 and 401 are independently necessary for STAT5 activation. The amino acid sequences surrounding these two tyrosine residues are very similar. Peptides comprising either phosphorylated Tyr343 or phosphorylated Tyr401, but not their unphosphorylated counterparts, inhibited the STAT5 activation. We propose that these two tyrosine residues of the Erythropoietin Receptor constitute docking sites for the STAT5 SH2 domain. The growth stimulus mediated by Erythropoietin was decreased in cells expressing Erythropoietin Receptors lacking both Tyr343 and Tyr401. This suggests that STAT5 activation could be involved in the growth control of FDCP-1 cells.
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Structure of the Erythropoietin Receptor.
Experimental Biology and Medicine, 1994Co-Authors: Patrick Mayeux, Sylvie Pallu, Stephanie Gobert, Catherine Lacombe, Sylvie GisselbrechtAbstract:Despite extensive studies, the structure of the Erythropoietin Receptor remains little understood. cDNAs encoding the human and murine Erythropoietin Receptors have been cloned and the structure of these proteins is discussed. Although the proteins encoded by these cDNAs play key roles in Erythropoietin binding and in Erythropoietin signal transduction, increasing evidence strongly suggests that the Erythropoietin Receptor is a multimeric complex. The murine Erythropoietin Receptor has been solubilized under mild conditions and the molecular mass of the native Receptor has been shown to be significantly higher than the molecular mass of the cloned chain. Cross-linking experiments have revealed the presence of three proteins covalently bound to Erythropoietin by the cross-linking reagents; however, only one of them seems to derive from the cloned chain. Moreover, functional evidence also suggests the presence of other Erythropoietin Receptor subunits.
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Erythropoietin induces the association of phosphatidylinositol 3′-kinase with a tyrosine-phosphorylated protein complex containing the Erythropoietin Receptor
FEBS Journal, 1993Co-Authors: Patrick Mayeux, Philippe Mauduit, Michèle Sabbah, Isabelle Dusanter-fourt, Brian J. Druker, Catherine Lacombe, William Vainchenker, O. Muller, S Fischer, Sylvie GisselbrechtAbstract:Stimulation of sensitive cells with Erythropoietin results in rapid induction of protein tyrosine phosphorylation. Other than tyrosine phosphorylation of one chain of the Erythropoietin Receptor, the identities of the remaining tyrosine-phosphorylated proteins are undefined. In this report, we demonstrate that the stimulation of the Erythropoietin-sensitive human UT7 cells by Erythropoietin rapidly resulted in the appearance of phosphatidylinositol 3-kinase activity in anti-phosphotyrosine immunoprecipitates. Erythropoietin action was rapid, detectable after as early as 1 min stimulation, transient, returning to control level after 30 min stimulation and was observed using the Erythropoietin concentrations able to stimulate the cell proliferation. Anti-(phosphatidylinositol 3-kinase) antibodies specifically immunoprecipitated 125I-Erythropoietin bound to its Receptor, strongly suggesting that phosphatidylinositol 3-kinase associated with a protein complex containing the activated Erythropoietin Receptor. To confirm this result, phosphatidylinositol 3-kinase was immunoprecipitated from Erythropoietin-stimulated cells using mild conditions followed by Western analysis using anti-phosphotyrosine antibodies. Five tyrosine phosphorylated proteins were revealed: the cloned chain of the Erythropoietin Receptor, the regulatory subunit of phosphatidylinositol 3-kinase and three unidentified proteins of 111, 97 and 64 kDa. None of these tyrosine phosphorylated proteins was detected in anti-(phosphatidylinositol 3-kinase) immunoprecipitates from unstimulated cells. Thus, our results show that phosphatidylinositol 3-kinase associates with a tyrosine-phosphorylated protein complex containing the activated Erythropoietin Receptor.
Catherine Lacombe - One of the best experts on this subject based on the ideXlab platform.
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proteasomes regulate Erythropoietin Receptor and signal transducer and activator of transcription 5 stat5 activation possible involvement of the ubiquitinated cis protein
Journal of Biological Chemistry, 1998Co-Authors: Frederique Verdier, Sylvie Gisselbrecht, Stany Chrétien, Catherine Lacombe, Odile Muller, Paule Varlet, Akihiko Yoshimura, Patrick MayeuxAbstract:Abstract Cis is an Src homology 2 domain-containing protein, which binds to the Erythropoietin Receptor and decreases Erythropoietin-stimulated cell proliferation. We show that Cis associates with the second tyrosine residue of the intracellular domain of the Erythropoietin Receptor (Tyr401). Two forms of Cis with molecular masses of 32 and 37 kDa were detected, and we demonstrate that the 37-kDa protein resulted from post-translational modifications of the 32-kDa form. Anti-ubiquitin antibodies recognized the 37-kDa form of Cis and the proteasome inhibitorsN-acetyl-leucyl-leucyl-norleucinal and lactacystin inhibited its degradation, showing that the 37-kDa form of Cis is a ubiquitinated protein, which seems to be rapidly degraded by the proteasome. In Erythropoietin-stimulated UT-7 cells, the activation of the Erythropoietin Receptor and signal transducer and activator of transcription 5 (STAT5) was transient and returned to basal levels after 30–60 min of Erythropoietin stimulation. In contrast, these proteins remained strongly phosphorylated, and STAT5 remained activated for at least 120 min in the presence of proteasome inhibitors. These experiments demonstrate that the proteasomes are involved in the down-regulation of the Erythropoietin Receptor activation signals. Because the proteasome inhibitors induced the accumulation of both the ubiquitinated form of Cis and the Cis-Erythropoietin Receptor complexes, our results suggest that the ubiquitinated form of Cis could be involved in the proteasome-mediated inactivation of the Erythropoietin Receptor.
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Identification of tyrosine residues within the intracellular domain of the Erythropoietin Receptor crucial for STAT5 activation.
The EMBO Journal, 1996Co-Authors: Stephanie Gobert, Isabelle Dusanter-fourt, Sylvie Gisselbrecht, Stany Chrétien, Fabrice Gouilleux, Christophe Pallard, Catherine Lacombe, Bernd Groner, O. Muller, Patrick MayeuxAbstract:Abstract FDCP-1 cells are hematopoietic progenitor cells which require interleukin-3 for survival and proliferation. FDCP-1 cells stably transfected with the murine Erythropoietin Receptor cDNA survive and proliferate in the presence of Erythropoietin. Erythropoietin induces the activation of the short forms (80 kDa) of STAT5 in the cells. Erythropoietin-induced activation of STAT5 was strongly reduced in cells expressing mutated variants of the Erythropoietin Receptors in which tyrosine residues in their intracellular domain have been eliminated. We determined that the Erythropoietin Receptor tyrosine residues 343 and 401 are independently necessary for STAT5 activation. The amino acid sequences surrounding these two tyrosine residues are very similar. Peptides comprising either phosphorylated Tyr343 or phosphorylated Tyr401, but not their unphosphorylated counterparts, inhibited the STAT5 activation. We propose that these two tyrosine residues of the Erythropoietin Receptor constitute docking sites for the STAT5 SH2 domain. The growth stimulus mediated by Erythropoietin was decreased in cells expressing Erythropoietin Receptors lacking both Tyr343 and Tyr401. This suggests that STAT5 activation could be involved in the growth control of FDCP-1 cells.
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Structure of the Erythropoietin Receptor.
Experimental Biology and Medicine, 1994Co-Authors: Patrick Mayeux, Sylvie Pallu, Stephanie Gobert, Catherine Lacombe, Sylvie GisselbrechtAbstract:Despite extensive studies, the structure of the Erythropoietin Receptor remains little understood. cDNAs encoding the human and murine Erythropoietin Receptors have been cloned and the structure of these proteins is discussed. Although the proteins encoded by these cDNAs play key roles in Erythropoietin binding and in Erythropoietin signal transduction, increasing evidence strongly suggests that the Erythropoietin Receptor is a multimeric complex. The murine Erythropoietin Receptor has been solubilized under mild conditions and the molecular mass of the native Receptor has been shown to be significantly higher than the molecular mass of the cloned chain. Cross-linking experiments have revealed the presence of three proteins covalently bound to Erythropoietin by the cross-linking reagents; however, only one of them seems to derive from the cloned chain. Moreover, functional evidence also suggests the presence of other Erythropoietin Receptor subunits.
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Erythropoietin induces the association of phosphatidylinositol 3′-kinase with a tyrosine-phosphorylated protein complex containing the Erythropoietin Receptor
FEBS Journal, 1993Co-Authors: Patrick Mayeux, Philippe Mauduit, Michèle Sabbah, Isabelle Dusanter-fourt, Brian J. Druker, Catherine Lacombe, William Vainchenker, O. Muller, S Fischer, Sylvie GisselbrechtAbstract:Stimulation of sensitive cells with Erythropoietin results in rapid induction of protein tyrosine phosphorylation. Other than tyrosine phosphorylation of one chain of the Erythropoietin Receptor, the identities of the remaining tyrosine-phosphorylated proteins are undefined. In this report, we demonstrate that the stimulation of the Erythropoietin-sensitive human UT7 cells by Erythropoietin rapidly resulted in the appearance of phosphatidylinositol 3-kinase activity in anti-phosphotyrosine immunoprecipitates. Erythropoietin action was rapid, detectable after as early as 1 min stimulation, transient, returning to control level after 30 min stimulation and was observed using the Erythropoietin concentrations able to stimulate the cell proliferation. Anti-(phosphatidylinositol 3-kinase) antibodies specifically immunoprecipitated 125I-Erythropoietin bound to its Receptor, strongly suggesting that phosphatidylinositol 3-kinase associated with a protein complex containing the activated Erythropoietin Receptor. To confirm this result, phosphatidylinositol 3-kinase was immunoprecipitated from Erythropoietin-stimulated cells using mild conditions followed by Western analysis using anti-phosphotyrosine antibodies. Five tyrosine phosphorylated proteins were revealed: the cloned chain of the Erythropoietin Receptor, the regulatory subunit of phosphatidylinositol 3-kinase and three unidentified proteins of 111, 97 and 64 kDa. None of these tyrosine phosphorylated proteins was detected in anti-(phosphatidylinositol 3-kinase) immunoprecipitates from unstimulated cells. Thus, our results show that phosphatidylinositol 3-kinase associates with a tyrosine-phosphorylated protein complex containing the activated Erythropoietin Receptor.
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Erythropoietin induces the association of phosphatidylinositol 3'-kinase with a tyrosine-phosphorylated protein complex containing the Erythropoietin Receptor.
European journal of biochemistry, 1993Co-Authors: Patrick Mayeux, Philippe Mauduit, Michèle Sabbah, Isabelle Dusanter-fourt, Brian J. Druker, Catherine Lacombe, William Vainchenker, S Fischer, Odile Muller, Sylvie GisselbrechtAbstract:Stimulation of sensitive cells with Erythropoietin results in rapid induction of protein tyrosine phosphorylation. Other than tyrosine phosphorylation of one chain of the Erythropoietin Receptor, the identities of the remaining tyrosine-phosphorylated proteins are undefined. In this report, we demonstrate that the stimulation of the Erythropoietin-sensitive human UT7 cells by Erythropoietin rapidly resulted in the appearance of phosphatidylinositol 3-kinase activity in anti-phosphotyrosine immunoprecipitates. Erythropoietin action was rapid, detectable after as early as 1 min stimulation, transient, returning to control level after 30 min stimulation and was observed using the Erythropoietin concentrations able to stimulate the cell proliferation. Anti-(phosphatidylinositol 3-kinase) antibodies specifically immunoprecipitated 125I-Erythropoietin bound to its Receptor, strongly suggesting that phosphatidylinositol 3-kinase associated with a protein complex containing the activated Erythropoietin Receptor. To confirm this result, phosphatidylinositol 3-kinase was immunoprecipitated from Erythropoietin-stimulated cells using mild conditions followed by Western analysis using anti-phosphotyrosine antibodies. Five tyrosine phosphorylated proteins were revealed: the cloned chain of the Erythropoietin Receptor, the regulatory subunit of phosphatidylinositol 3-kinase and three unidentified proteins of 111, 97 and 64 kDa. None of these tyrosine phosphorylated proteins was detected in anti-(phosphatidylinositol 3-kinase) immunoprecipitates from unstimulated cells. Thus, our results show that phosphatidylinositol 3-kinase associates with a tyrosine-phosphorylated protein complex containing the activated Erythropoietin Receptor.
Ryuzo Sasaki - One of the best experts on this subject based on the ideXlab platform.
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Erythropoietin and Erythropoietin-Receptor producing cells demonstrated by in situ hybridization in mouse visceral yolk sacs.
Anatomical Science International, 2002Co-Authors: Yoshiko Yasuda, Seiji Masuda, Masaya Nagao, Yoshihiko Fujita, Masaki Okano, Ryuzo SasakiAbstract:We have previously demonstrated that mRNAs for Erythropoietin and the Erythropoietin Receptor temporarily express on the visceral yolk sacs on days 9–11 of gestation in mice. In order to investigate the sites of expression, we performed in situ hybridization on visceral yolk sacs. Visceral yolk sacs from 10-day-old mice embryos were frozen in liquid nitrogen, and processed for cryosections. Sections were hybridized with a 35S-labeled RNA probe complementary to mRNA coding for Erythropoietin or Erythropoietin Receptor. Erythropoietin mRNA was detectable in 57.6% of the endodermal epithelial cells, while Erythropoietin-Receptor mRNA was discerned in 90.8% of the endodermal cells and mesodermal cells, including hemocyteblasts. Moreover, Erythropoietin protein was detectable in 52.8% of the endodermal epithelial cells, and on the surface of hemocyteblasts and mesothelial cells. Erythropoietin-Receptor protein was discernible in 87.2% of the endodermal cells and in the corresponding mesodermal cells to those where Erythropoietin protein was expressed by immunohistochemical examinations. The results indicate that Erythropoietin-synthesizing cells are located in half of the endodermal epithelial cells, while the majority of cells in the visceral yolk sac are Erythropoietin-Receptor-producing cells, indicating that almost all cell population in the visceral yolk sac is Erythropoietin-responding cells via both autocrine and paracrine routes.
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Erythropoietin Receptor is expressed in rat hippocampal and cerebral cortical neurons and Erythropoietin prevents in vitro glutamate induced neuronal death
Neuroscience, 1996Co-Authors: Emi Morishita, Masaya Nagao, Seiji Masuda, Yoshiko Yasuda, Ryuzo SasakiAbstract:Abstract Recently, Erythropoietin has been shown to be produced by astrocytes and its production is hypoxia-inducible. In the present study, we demonstrated, using a reverse transcription–polymerase chain reaction assay and immunostaining of the cells, that the Erythropoietin Receptor was expressed in cultured hippocampal and cerebral cortical neurons of day 19 rat embryo. Erythropoietin protected the cultured neurons from glutamate neurotoxicity. Neurons cultured for seven to 10 days were exposed to glutamate for 15 min and after culture for a further 24 h in the absence of glutamate the neuron survival was assayed. Significant protection was observed with Erythropoietin from 3 pM (c. 100 pg/ml) in a dose-dependent manner. The protection was completely reversed by co-application of a soluble Erythropoietin Receptor, an extracellular domain capable of binding with Erythropoietin. For exhibition of the neuroprotective effect, exposure of neurons to Erythropoietin approximately 8 h prior to exposure to glutamate was required. Experiments with the inhibitors indicated that RNA and protein syntheses were necessary for the protection. However, exposure to Erythropoietin for a short period (5 min or less) was sufficient to elicit the protective effect. The protective effect of Erythropoietin was blocked by the simultaneous addition of EGTA. These findings and the previous finding that Erythropoietin induces a rapid and transient increase in intracellular Ca2+ concentration in neuronal cells suggest that Erythropoietin plays a neuroprotective role in brain injury caused by hypoxia or ischemia and that Erythropoietin-induced Ca2+ influx from outside of the cells is a critical initial event yielding an enhanced resistance of the neurons to glutamate toxicity.
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Erythropoietin Receptor is expressed in rat hippocampal and cerebral cortical neurons and Erythropoietin prevents in vitro glutamate induced neuronal death
Neuroscience, 1996Co-Authors: Emi Morishita, Masaya Nagao, Seiji Masuda, Yoshiko Yasuda, Ryuzo SasakiAbstract:Abstract Recently, Erythropoietin has been shown to be produced by astrocytes and its production is hypoxia-inducible. In the present study, we demonstrated, using a reverse transcription–polymerase chain reaction assay and immunostaining of the cells, that the Erythropoietin Receptor was expressed in cultured hippocampal and cerebral cortical neurons of day 19 rat embryo. Erythropoietin protected the cultured neurons from glutamate neurotoxicity. Neurons cultured for seven to 10 days were exposed to glutamate for 15 min and after culture for a further 24 h in the absence of glutamate the neuron survival was assayed. Significant protection was observed with Erythropoietin from 3 pM (c. 100 pg/ml) in a dose-dependent manner. The protection was completely reversed by co-application of a soluble Erythropoietin Receptor, an extracellular domain capable of binding with Erythropoietin. For exhibition of the neuroprotective effect, exposure of neurons to Erythropoietin approximately 8 h prior to exposure to glutamate was required. Experiments with the inhibitors indicated that RNA and protein syntheses were necessary for the protection. However, exposure to Erythropoietin for a short period (5 min or less) was sufficient to elicit the protective effect. The protective effect of Erythropoietin was blocked by the simultaneous addition of EGTA. These findings and the previous finding that Erythropoietin induces a rapid and transient increase in intracellular Ca2+ concentration in neuronal cells suggest that Erythropoietin plays a neuroprotective role in brain injury caused by hypoxia or ischemia and that Erythropoietin-induced Ca2+ influx from outside of the cells is a critical initial event yielding an enhanced resistance of the neurons to glutamate toxicity.
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Anti-Erythropoietin Receptor monoclonal antibody: epitope mapping, quantification of the soluble Receptor, and detection of the solubilized transmembrane Receptor and the Receptor-expressing cells
Blood, 1996Co-Authors: Emi Morishita, Seiji Masuda, Masaya Nagao, Hiroshi Narita, Motoyuki Nishida, Noriyoshi Kawashima, Kenji Yamagishi, Hajime Hatta, Ryuzo SasakiAbstract:A hybridoma cell line producing the monoclonal antibody against Erythropoietin Receptor (EpoR) was established using the soluble ectodomain of mouse Erythropoietin Receptor (sEpoR) as an antigen. The monoclonal antibody termed 1G3 bound to the denatured sEpoR. Epitope mapping with peptide library revealed that 1G3 recognized the amino terminal region including the hexapeptide (positions 6 to 11; LeuProAspProLysPhe). The amino acid sequence in this hexapeptide was identical in mice, rats, and humans, and therefore 1G3 bound to EpoR from all of these sources. Using 1G3, we evaluated sEpoR by a sandwich enzyme-linked immunoassay, and EpoR in the solubilized membrane preparation was detected by Western blotting. The cells expressing EpoR were identified with immunochemical staining. We confirmed the presence of EpoR in a neuronal cell line and PC12 cells, and EpoR was expressed in primary cultured hippocampal neurons.
Nicole Casadevall - One of the best experts on this subject based on the ideXlab platform.
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New pathogenic mechanisms induced by germline Erythropoietin Receptor mutations in primary erythrocytosis.
Haematologica, 2017Co-Authors: Florence Pasquier, Frederique Verdier, Caroline Marty, Thomas Balligand, Sarah Grosjean, Vitalina Gryshkova, Hana Raslova, Stefan N. Constantinescu, Nicole Casadevall, William VainchenkerAbstract:Primary familial and congenital polycythemia is characterized by Erythropoietin hypersensitivity of erythroid progenitors due to germline nonsense or frameshift mutations in the Erythropoietin Receptor gene. All mutations so far described lead to the truncation of the C-terminal Receptor sequence that contains negative regulatory domains. Their removal is presented as sufficient to cause the Erythropoietin hypersensitivity phenotype. Here we provide evidence for a new mechanism whereby the presence of novel sequences generated by frameshift mutations is required for the phenotype rather than just extensive truncation resulting from nonsense mutations. We show that the Erythropoietin hypersensitivity induced by a new Erythropoietin Receptor mutant, p.Gln434Profs*11, could not be explained by the loss of negative signaling and of the internalization domains, but rather by the appearance of a new C-terminal tail. The latter, by increasing Erythropoietin Receptor dimerization, stability and cell-surface localization, causes pre-activation of Erythropoietin Receptor and JAK2, constitutive signaling and hypersensitivity to Erythropoietin. Similar results were obtained with another mutant, p.Pro438Metfs*6, which shares the same last five amino acid residues (MDTVP) with Erythropoietin Receptor p.Gln434Profs*11, confirming the involvement of the new peptide sequence in the Erythropoietin hypersensitivity phenotype. These results suggest a new mechanism that might be common to Erythropoietin Receptor frameshift mutations. In summary, we show that primary familial and congenital polycythemia is more complex than expected since distinct mechanisms are involved in the Erythropoietin hypersensitivity phenotype, according to the type of Erythropoietin Receptor mutation.
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a peptide based Erythropoietin Receptor agonist for pure red cell aplasia
The New England Journal of Medicine, 2009Co-Authors: Iain C Macdougall, Nicole Casadevall, Jerome Rossert, Richard Stead, Annemarie Duliege, Marc Froissart, Kaiuwe EckardtAbstract:Background We investigated whether a novel, synthetic, peptide-based Erythropoietin-Receptor agonist (Hematide, Affymax) can stimulate erythropoiesis in patients with anemia that is caused by antiErythropoietin antibodies. Methods In this open-label, single-group trial, we enrolled patients with chronic kidney disease who had pure red-cell aplasia or hypoplasia due to antiErythropoietin antibodies and treated them with a synthetic peptide-based Erythropoietin-Receptor agonist. The agonist was administered by subcutaneous injection at an initial dose of 0.05 mg per kilogram of body weight every 4 weeks. The primary end point was a hemoglobin concentration above 11 g per deciliter without the need for transfusions. Results We treated 14 patients with the peptide agonist for a median of 28 months. The median hemoglobin concentration increased from 9.0 g per deciliter (with transfusion support in the case of 12 patients) before treatment to 11.4 g per deciliter at the time of the last administration of the ag...
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A Peptide-Based Erythropoietin-Receptor Agonist for Pure Red-Cell Aplasia
The New England journal of medicine, 2009Co-Authors: Iain C Macdougall, Nicole Casadevall, Jerome Rossert, Richard Stead, Annemarie Duliege, Marc Froissart, Kaiuwe EckardtAbstract:We investigated whether a novel, synthetic, peptide-based Erythropoietin-Receptor agonist (Hematide, Affymax) can stimulate erythropoiesis in patients with anemia that is caused by antiErythropoietin antibodies. In this open-label, single-group trial, we enrolled patients with chronic kidney disease who had pure red-cell aplasia or hypoplasia due to antiErythropoietin antibodies and treated them with a synthetic peptide-based Erythropoietin-Receptor agonist. The agonist was administered by subcutaneous injection at an initial dose of 0.05 mg per kilogram of body weight every 4 weeks. The primary end point was a hemoglobin concentration above 11 g per deciliter without the need for transfusions. We treated 14 patients with the peptide agonist for a median of 28 months. The median hemoglobin concentration increased from 9.0 g per deciliter (with transfusion support in the case of 12 patients) before treatment to 11.4 g per deciliter at the time of the last administration of the agonist; transfusion requirements diminished within 12 weeks after the first dose, after which 13 of the 14 patients no longer required regular transfusions. Peak reticulocyte counts increased from a median of 10x10(9) per liter before treatment to peak counts of greater than 100x10(9) per liter. The level of antiErythropoietin antibodies declined over the course of the study and became undetectable in six patients. One patient who initially responded to treatment had a diminished hematologic response a few months later despite increased doses of the agonist and required transfusions again; this patient was found to have antibodies against the agonist. One patient died 4 months after the last dose of the agonist, and a grade 3 or 4 adverse event occurred in seven other patients during the study period. This novel agonist of the Erythropoietin Receptor can correct anemia in patients with pure red-cell aplasia caused by antiErythropoietin antibodies. (ClinicalTrials.gov number, NCT00314795.). Copyright 2009 Massachusetts Medical Society.
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Structure of the murine Erythropoietin Receptor complex. Characterization of the Erythropoietin cross-linked proteins.
The Journal of biological chemistry, 1991Co-Authors: Patrick Mayeux, Stany Chrétien, Nicole Casadevall, Claire Lacombe, I. Dusanter, Sylvie GisselbrechtAbstract:The structure of the murine Erythropoietin Receptor was studied using antibodies against the intracellular part of the cloned Erythropoietin Receptor chain. These antibodies precipitated Erythropoietin-Receptor complexes from Triton X-100-solubilized cells. When the complexes were cross-linked by disuccinimidyl suberate, the 85- and 100-kDa Erythropoietin-cross-linked proteins previously described were immunoprecipitated. However, these proteins were not precipitated when the complexes were denatured and reduced before immunoprecipitation. Using 1-ethyl 3-(3-dimethylaminopropyl)carbodiimide, we observed Erythropoietin cross-linking with a protein of 66 kDa in addition to the 100- and 85-kDa proteins. Only the 66-kDa Erythropoietin-cross-linked protein was immunoprecipitated by anti-Receptor antibodies after denaturation and reduction of the complex. Thus, our results suggest that the 85- and 100-kDa proteins previously evidenced by cross-linking are associated with the cloned chain of the Receptor to form a multimeric complex but these proteins seem immunologically unrelated to the cloned chain. We observed that reducing the length of molecules able to cross-link amino groups decreased the efficiency of cross-linking with the 100-kDa protein and only the 85-kDa protein was cross-linked with Erythropoietin using 1,5-difluoro-2,4-dinitrobenzene. These results suggest that the 85- and 100-kDa proteins occupate slightly different positions relative to the Erythropoietin molecule bound to the Receptor.
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Multimeric structure of the membrane Erythropoietin Receptor of murine erythroleukemia cells (Friend cells). Cross-linking of Erythropoietin with the spleen focus-forming virus envelope protein.
The Journal of biological chemistry, 1991Co-Authors: Nicole Casadevall, Sylvie Gisselbrecht, Odile Muller, Claire Lacombe, P MayeuxAbstract:Abstract In erythroleukemia cells infected with the polycythemia strain of the Friend virus complex, Erythropoietin could be cross-linked mainly to a protein of 63 kDa when using disuccinimidyl suberate. In contrast, Erythropoietin in other erythroleukemia cells cross-linked to two proteins of 85 and 100 kDa. When native Erythropoietin Receptor complexes were immunoprecipitated, the 63-kDa Erythropoietin-cross-linked protein could be precipitated both by antibodies directed against the intracellular part of the cloned chain of the Erythropoietin Receptor and by antibodies directed against the envelope proteins of the Friend virus. However, after denaturation of the complexes, the 63-kDa protein was only precipitated by antibodies directed against the envelope proteins of the Friend virus. Enzymatic deglycosylation confirmed that Erythropoietin was cross-linked with the envelope protein of the defective virus and bidimensional diagonal gel electrophoresis analyses showed that some of the Erythropoietin cross-linked envelope proteins were dimerized by disulfide bonds. Thus, the main Erythropoietin-Receptor complex in the plasma membrane of these cells consisted of a molecule of the cloned chain of the Erythropoietin Receptor noncovalently associated with one or two disulfide-bonded molecule(s) of the envelope protein of the defective virus. Moreover, our results also showed that the viral envelope protein associated with the cloned chain of the Erythropoietin Receptor at a site distinct from the Erythropoietin binding site.