Expression of a constitutively active erythropoietin receptor in primary hematopoietic progenitors abrogates erythropoietin dependence and enhances erythroid colony-forming unit, erythroid burst-forming unit, and granulocyte/macrophage progenitor growth.

Pharr, Pamela N.; Hankins, David; Hofbauer, Ann; Lodish, Harvey F.; Longmore, Gregory D.

doi:10.1073/pnas.90.3.938

Cited by 56 publications

(32 citation statements)

References 38 publications

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“…Previous studies have shown that dimerization of the EPOR stimulates the signals leading to erythroid cell survival, proliferation, and differentiation (8,10,11,44). Crystallographic structural analysis of the EPOR extracellular region complexed to a peptide agonist demonstrated directly the dimeric nature of the receptor, and biochemical experiments have shown that EPO promotes dimerization of the EPOR extracellular region (9,12,52).…”

Section: Discussionmentioning

confidence: 99%

Oligomerization and Scaffolding Functions of the Erythropoietin Receptor Cytoplasmic Tail

Watowich¹,

Liu²,

Xie³

et al. 1999

Journal of Biological Chemistry

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Signal transduction by the erythropoietin receptor (EPOR) is activated by ligand-mediated receptor homodimerization. However, the relationship between extracellular and intracellular domain oligomerization remains poorly understood. To assess the requirements for dimerization of receptor cytoplasmic sequences for signaling, we overexpressed mutant EPORs in combination with wild-type (WT) EPOR to drive formation of heterodimeric (i.e. WT-mutant) receptor complexes. Dimerization of the membrane-proximal portion of the EPOR cytoplasmic region was found to be critical for the initiation of mitogenic signaling. However, dimerization of the entire EPOR cytoplasmic region was not required. To examine this process more closely, we generated chimeras between the intracellular and transmembrane portions of the EPOR and the extracellular domains of the interleukin-2 receptor ␤ and ␥ c chains. These chimeras allowed us to assess more precisely the signaling role of each receptor chain because only heterodimers of WT and mutant receptor chimeras form in the presence of interleukin-2. Coexpression studies demonstrated that a functional receptor complex requires the membrane-proximal region of each receptor subunit in the oligomer to permit activation of JAK2 but only one membrane-distal tail to activate STAT5 and to support cell proliferation. Thus, this study defines key relationships involved in the assembly and activation of the EPOR signal transduction complex which may be applicable to other homodimeric cytokine receptors. Erythropoietin (EPO)1 is essential for the survival, proliferation, and terminal differentiation of erythroid progenitor cells (1-3). The erythropoietin receptor (EPOR) belongs to the cytokine receptor superfamily and as such contains the structural motifs of four cysteine residues with canonical spacing and the sequence WSXWS in the extracellular domain (4, 5). Although protein tyrosine phosphorylation is required for cytokine signaling (6), the cytoplasmic regions of these receptors lack intrinsic enzymatic activity. Signal transduction instead is mediated by cytoplasmic protein tyrosine kinases of the JAK family (7). These kinases are constitutively associated with receptor chains; residues in the membrane-proximal region of the EPOR cytoplasmic tail are required for this association. Ligand binding results in homodimerization of EPOR chains, which is critical for the initiation of intracellular signaling (8 -12).The role of homodimerization in the initiation of signaling is highlighted by constitutively active variants of the EPOR chain (10, 13). In the three mutants described to date, R129C, E132C, and E133C, amino acids in the extracellular domain are replaced by cysteines, leading to the formation of intermolecular disulfide bridges and constitutive signaling. In contrast, a number of other cytokine receptors signal through the heterodimerization of asymmetric receptor chains. For example, interleukin-2 (IL-2) binds and heterodimerizes the IL-2R␤ and ␥ c receptor chains (for review, see Refs. 14...

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Section: Discussionmentioning

confidence: 99%

Oligomerization and Scaffolding Functions of the Erythropoietin Receptor Cytoplasmic Tail

Watowich¹,

Liu²,

Xie³

et al. 1999

Journal of Biological Chemistry

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“…The contrasting inductive, or instructive, hypothesis attributes to growth factors a direct role in cell differentiation, predicting that growth factor receptors transduce signals that uniquely specify the differentiation outcome of a progenitor (15)(16)(17). A number of hybrid hypotheses have also been proposed, where, for example, committed progenitors arise stochastically, but their subsequent differentiation and acquisition of the mature phenotype are uniquely induced by lineage-specific growth factors (18).Although Epo is essential for the production of red blood cells, it is not thought to play a role in the generation of committed erythroid progenitors from pluripotent progenitors: expression of recombinant EpoR does not bias the lineage commitment of pluripotent hematopoietic progenitors (19,20), and normal numbers of committed erythroid BFU-e and CFU-e progenitors are found in the fetal livers of EpoR Ϫ/Ϫ mutant mice (21). However, there is a unique requirement for EpoR activation during the subsequent proliferation and terminal differentiation of committed erythroid progenitors: the EpoR Ϫ/Ϫ CFU-e and BFU-e progenitors fail to give rise to mature erythrocytes unless EpoR is expressed by retroviral infection (21); and in vitro, other growth factors only partially substitute for .…”

mentioning

confidence: 99%

“…Although Epo is essential for the production of red blood cells, it is not thought to play a role in the generation of committed erythroid progenitors from pluripotent progenitors: expression of recombinant EpoR does not bias the lineage commitment of pluripotent hematopoietic progenitors (19,20), and normal numbers of committed erythroid BFU-e and CFU-e progenitors are found in the fetal livers of EpoR Ϫ/Ϫ mutant mice (21). However, there is a unique requirement for EpoR activation during the subsequent proliferation and terminal differentiation of committed erythroid progenitors: the EpoR Ϫ/Ϫ CFU-e and BFU-e progenitors fail to give rise to mature erythrocytes unless EpoR is expressed by retroviral infection (21); and in vitro, other growth factors only partially substitute for .…”

mentioning

confidence: 99%

The Prolactin Receptor and Severely Truncated Erythropoietin Receptors Support Differentiation of Erythroid Progenitors

Socolovsky

Dusanter‐Fourt

Lodish

1997

Journal of Biological Chemistry

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The EpoR 1 belongs to a large family of cytokine receptors, many of which are required for the proliferation and differentiation of hematopoietic as well as other cell types (4 -7). Throughout life, eight different hematopoietic lineages arise from pluripotent stem cells in the bone marrow (8, 9). The exact role of growth factors in this process is not clear and has been described broadly by two alternative hypotheses. The stochastic hypothesis suggests that commitment of a progenitor to a particular lineage is a stochastic event, subsequent to which cell differentiation proceeds along a predetermined program; growth factors are merely required to ensure the survival and proliferation of committed progenitors (10 -14). The contrasting inductive, or instructive, hypothesis attributes to growth factors a direct role in cell differentiation, predicting that growth factor receptors transduce signals that uniquely specify the differentiation outcome of a progenitor (15)(16)(17). A number of hybrid hypotheses have also been proposed, where, for example, committed progenitors arise stochastically, but their subsequent differentiation and acquisition of the mature phenotype are uniquely induced by lineage-specific growth factors (18).Although Epo is essential for the production of red blood cells, it is not thought to play a role in the generation of committed erythroid progenitors from pluripotent progenitors: expression of recombinant EpoR does not bias the lineage commitment of pluripotent hematopoietic progenitors (19,20), and normal numbers of committed erythroid BFU-e and CFU-e progenitors are found in the fetal livers of EpoR Ϫ/Ϫ mutant mice (21). However, there is a unique requirement for EpoR activation during the subsequent proliferation and terminal differentiation of committed erythroid progenitors: the EpoR Ϫ/Ϫ CFU-e and BFU-e progenitors fail to give rise to mature erythrocytes unless EpoR is expressed by retroviral infection (21); and in vitro, other growth factors only partially substitute for . It is not known whether EpoR activation is essential at this stage of erythroid differentiation because of an EpoR-unique instructive signal or whether it is simply required for functions that are not unique to EpoR, such as its proliferative and anti-apoptotic effects.Some evidence for the capability of EpoR to promote the erythroid phenotype comes from the ability of Epo to induce surface expression of glycophorin (25) and transcription of the ␤-globin gene (26, 27) in pre-B Ba/F3 cells expressing a transfected EpoR. However, the uncertain lineage commitment of many cell lines and their incomplete differentiation response makes them less suitable for the study of signaling in differentiation. EpoR-mediated signaling for proliferation can be studied in a number of interleukin-3-dependent cell lines, where heterologous expression of EpoR allows Epo to support cell proliferation (1, 2). Only the membrane proximal ϳ120 amino acids is essential for this function (1-3). Similarly truncated mutants of other cytokine r...

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“…(21) R129C also supports Epo-independent erythropoiesis in ex vivo cultures. (25) Collectively, these results demonstrate that R129C signaling is dominant to the endogenous WT EpoR. Therefore, use of R129C-containing EpoR mutants is a powerful method to analyze EpoR signaling requirements in a wild-type genetic background.…”

Section: Fetal Liver Cfu-e Development Is Supported By Epors That Actmentioning

confidence: 95%

“…This approach has been used previously to study EpoR functions, as it enables rapid analysis of receptor mutants in primary erythroid cells. (16,21,22,25,26) ER343-S3, ER401-S3, and ER343/401-S3 support CFU-E development from fetal liver erythroid progenitors. Furthermore, mice infected with recombinant retroviruses develop erythrocytosis and splenomegaly, demonstrating that these receptor isoforms support erythropoiesis in the adult animal.…”

Section: Discussionmentioning

confidence: 99%

Erythropoietin Receptors That Signal Through Stat5 or Stat3 Support Fetal Liver and Adult Erythropoiesis: Lack of Specificity of Stat Signals During Red Blood Cell Development

Watowich¹,

Mikami

Busche

et al. 2000

Journal of Interferon & Cytokine Research

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Erythropoietin (Epo) is essential for formation of mature red blood cells (RBC). However, the function of Epo receptor (EpoR)-dependent signaling pathways in the regulation of erythropoiesis remains unclear. To determine whether specific Stat signals are required for RBC development, we changed the Stat signaling specificity of the EpoR. The wild-type EpoR activates only Stat5. Thus, we substituted the major Stat5 binding sites (residues 343 and 401) in the EpoR cytoplasmic region with the Stat3 binding/activation motif from gp130. We demonstrated that activated EpoRs containing a single substitution stimulate Stat5 and Stat3, whereas an EpoR with both substitutions stimulates Stat3 but not Stat5. We then determined the ability of these receptors to support fetal liver and adult erythropoiesis. Our results show that erythropoiesis is stimulated by EpoRs that activate Stat5, both Stat5 and Stat3, or Stat3 in place of Stat5. These findings demonstrate that the specificity of EpoR Stat signaling is not essential for RBC development.

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Expression of a constitutively active erythropoietin receptor in primary hematopoietic progenitors abrogates erythropoietin dependence and enhances erythroid colony-forming unit, erythroid burst-forming unit, and granulocyte/macrophage progenitor growth.

Abstract: We tested the ability of a constitutively activated erythropoietin receptor [EpoR(R129C)

Cited by 56 publications

References 38 publications

Oligomerization and Scaffolding Functions of the Erythropoietin Receptor Cytoplasmic Tail

Oligomerization and Scaffolding Functions of the Erythropoietin Receptor Cytoplasmic Tail

The Prolactin Receptor and Severely Truncated Erythropoietin Receptors Support Differentiation of Erythroid Progenitors

Erythropoietin Receptors That Signal Through Stat5 or Stat3 Support Fetal Liver and Adult Erythropoiesis: Lack of Specificity of Stat Signals During Red Blood Cell Development

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