Developmental changes in erythropoietin receptor expression of fetal mouse liver.

Erythropoietin (EPO) stimulates proliferation and differentiation of late erythroid precursor cells (CFU-E) and thereby determines the rate of erythropoiesis. Liver is the major erythropoietic site in a fetus. We dealt with developmental changes in CFU-E and EPO receptor (EPO-R) of fetal mouse liver. The affinity of the EPO-R to EPO was unchanged during fetal development. The population size of CFU-E, the number of EPO-R per liver cell, and EPO-R mRNA decreased as gestation proceeded, in a pattern indicating that the expression of EPO-R on erythroid precursor cells in fetal mouse liver is governed mostly by the process of mRNA production.     

Murine pluripotent hematopoietic progenitors constitutively expressing a normal erythropoietin receptor proliferate in response to erythropoietin without preferential erythroid cell differentiation.

Erythropoietin (EPO) is a prime regulator of the growth and differentiation of erythroid blood cells. The EPO receptor (EPO-R) is expressed in late erythroid progenitors (mature BFU-E and CFU-E), and EPO induces proliferation and differentiation of these cells. By introducing, with a retroviral vector, a normal EPO-R cDNA into murine adult bone marrow cells, we showed that EPO is also able to induce proliferation in pluripotent progenitor cells. After 7 days of coculture with virus-producing cells, bone marrow cells were plated in methylcellulose culture in the presence of EPO, interleukin-3, or Steel factor alone or in combination. In the presence of EPO alone, EPO-R virus-infected bone marrow cells gave rise to mixed colonies comprising erythrocytes, granulocytes, macrophages and megakaryocytes. The addition of interleukin-3 or Steel factor to methylcellulose cultures containing EPO did not significantly modify the number of mixed colonies. The cells which generate these mixed colonies have a high proliferative potential as shown by the size and the ability of the mixed colonies to give rise to secondary colonies. Thus, it appears that EPO has the same effect on EPO-R-expressing multipotent cell proliferation as would a combination of several growth factors. Finally, our results demonstrate that inducing pluripotent progenitor cells to proliferate via the EPO signaling pathway has no major influence on their commitment.     

Diagnostic value of thyrotropin releasing hormone test in 129 patients with suspected tumoral hyperprolactinemia

In 129 hyperprolactinemic (PRL > or = 100 ng/mL) and 100 normoprolactinemic patients (PRL 0-25 ng/mL), delta max. PRL (the difference between maximal prolactin (PRL) after thyrotropin releasing hormone (TRH) injection and basal value) was compared with basal PRL and computed tomography (CT) of the sellar region. In 122 hyperprolactinemic patients delta max. PRL was < 100%, while tumor was found in 106 of them. In the remainder seven hyperprolactinemic patients delta max. PRL was > or = 100% and CT showed no tumor. A significant difference in delta max. PRL between hyperprolactinemic patients without and those with verified adenoma was found and showed a significant negative correlation with basal PRL. Between 122 hyperprolactinemic patients with delta max. PRL < 100%, mean basal PRL and duration of clinical symptoms were significantly lower in 16 patients with normal CT compared to 106 patients with tumor. All normoprolactinemic patients showed delta max. PRL > or = 100% and no tumor on CT. PRL stimulation disturbance precedes tumor visualization and represents a decisive diagnostic parameter in hyperprolactinemic patients with no tumor signs.     

Disturbed prolactin responses to dopamine-related substances in patients with acromegaly and hyperprolactinemia

We undertook this study, because conflicting data were reported about the dopaminergic regulation of prolactin (PRL) secretion in patients with acromegaly and hyperprolactinemia. In order to clarify the dopaminergic regulation of PRL secretion in patients with acromegaly and hyperprolactinemia, the effects of nomifensine, a central dopamine agonist, FK 33-824, a centrally antidopaminergically acting agent, and domperidone, a peripheral dopamine antagonist, on plasma PRL in these patients were studied. The results were compared with those observed in normal subjects and hyperprolactinemic patients, with or without a pituitary tumor. Nomifensine did not lower the PRL levels and FK 33-824 did not raise the PRL levels in acromegalic patients. In hyperprolactinemic patients, nomifensine did not lower the PRL levels and FK 33-824 failed to raise the PRL levels. Domperidone did not increase PRL in about a third of acromegalic patients, while TRH increased PRL in the all normoprolactinemic acromegalic patients. These results suggest that in acromegalic patients there may be a disturbance in dopamine related neurotransmission and that such disorders also seem to be present in patients with hyperprolactinemia, with or without a pituitary tumor.     

Prolactin lowering effect of amphetamine in normoprolactinemic subjects and in physiological and pathological hyperprolactinemia

The effect on plasma prolactin (PRL) of d-amphetamine (Amph) was studied in normo- and hyperprolactinemic subjects. In normoprolactinemic women Amph failed to lower plasma PRL levels when infused intravenously over 1 h at the dose of 7.5 mg, but induced at the dose of 15.0 mg a modest inhibition of plasma PRL (maximum PRL inhibition 20 +/- 4.5% at 45 min). Likewise, in puerperal women Amph at the dose of 7.5 mg did not decrease significantly plasma PRL levels but it was active in this respect (maximum inhibition 37 +/- 10% at 120 min) at the dose of 15.0 mg. In subjects with presumptive evidence of a PRL-secreting adenoma, Amph at either the 7.5 mg or the 15.0 mg dose failed to alter baseline PRL levels. These results indicate that Amph is a poor PRL suppressor in either normo- or hyperprolactinemic subjects. It is proposed that this may be due to the drug's ability to effect release of dopamine mainly from a non-granular pool of the amine.     

Identification of JAK protein tyrosine kinases as signaling molecules for prolactin. Functional analysis of prolactin receptor and prolactin-erythropoietin receptor chimera expressed in lymphoid cells.

The mechanism of action of prolactin (PRL) was studied in murine lymphoid BAF-3 cells transfected with either the long form of the PRL receptor (PRL-R), or a chimeric receptor consisting of the extracellular domain of the PRL-R and the transmembrane and intracellular domain of the erythropoietin receptor (PRL/EPO-R). PRL sustained normal and long-term proliferation of BAF-3 cells expressing either the PRL-R or the hybrid PRL/EPO-R. Upon [125I]PRL cross-linking, both types of BAF-3 transfectants were shown to express two [125I]PRL cross-linked species differing in size by 20 kDa. These cross-linked complexes, after denaturation, were recognized by antibody against the PRL-R, indicating that they contain the transfected receptor. PRL induced rapid and transient tyrosine phosphorylation of both the PRL-R and the PRL/EPO-R in BAF-3 transfectants. Furthermore, PRL induced rapid tyrosine phosphorylation of the Janus kinase 2 (JAK2) which was already physically associated with the PRL-R or the PRL/EPO-R in the absence of ligand. JAK1 was also associated with PRL-R and PRL/EPO-R in the absence of ligand. However, only in BAF-3 cells expressing the PRL-R does PRL induce rapid and transient tyrosine phosphorylation of JAK1. These results demonstrate that JAK protein tyrosine kinases couple PRL binding to tyrosine phosphorylation and proliferation.     

The erythropoietin receptor transmembrane region is necessary for activation by the Friend spleen focus-forming virus gp55 glycoprotein.

The erythropoietin receptor (EPO-R), a member of the cytokine receptor superfamily, can be activated by binding either erythropoietin (EPO) or gp55, the Friend spleen focus-forming virus glycoprotein. The highly specific interaction between gp55 and EPO-R triggers cell proliferation and thereby causes the first stage of Friend virus-induced erythroleukemia. We have generated functional chimeric receptors containing regions of the EPO-R and the interleukin-3 receptor (AIC2A polypeptide), a related cytokine receptor which does not interact with gp55. All chimeric receptors were expressed at similar levels, had similar binding affinities for EPO, and conferred EPO-dependent cell growth. Only those chimeric receptors which contained the EPO-R transmembrane region were activated by gp55. These results demonstrate that the transmembrane region of the EPO-R is critical for activation by gp55. In addition, analysis of a soluble, secreted EPO-R and cysteine point mutants of the EPO-R show that the extracytoplasmic region of the EPO-R specifically interacts with gp55.     

Cytosolic lysine residues enhance anterograde transport and activation of the erythropoietin receptor

Lysine residues are key residues in many cellular processes, in part due to their ability to accept a wide variety of post-translational modifications. In the present study, we identify the EPO-R [EPO (erythropoietin) receptor] cytosolic lysine residues as enhancers of receptor function. EPO-R drives survival, proliferation and differentiation of erythroid progenitor cells via binding of its ligand EPO. We mutated the five EPO-R cytosolic lysine residues to arginine residues (5KR EPO-R), eliminating putative lysine-dependent modifications. Overexpressed 5KR EPO-R displayed impaired ubiquitination and improved stability compared with wt (wild-type) EPO-R. Unexpectedly, fusion proteins consisting of VSVGtsO45 (vesicular stomatitis virus glycoprotein temperature-sensitive folding mutant) with wt or 5KR EPO-R cytosolic domains demonstrated delayed glycan maturation kinetics upon substitution of the lysine residues. Moreover, VSVG-wt EPO-R, but not VSVG-5KR EPO-R, displayed endoplasmic reticulum-associated ubiquitination. Despite similar cell-surface EPO-binding levels of both receptors and the lack of EPO-induced ubiquitination by 5KR EPO-R, the lysine-less mutant produced weaker receptor activation and signalling than the wt receptor. We thus propose that EPO-R cytosolic lysine residues enhance receptor function, most probably through ubiquitination and/or other post-translational modifications.     

Amino acid residues 268-276 of the erythropoietin receptor contain an endocytosis motif and are required for erythropoietin-mediated proliferation

Erythropoietin (EPO) promotes viability, proliferation and differentiation of mammalian erythroid progenitor cells via its specific cell surface receptor (EPO-R). We have previously shown that truncated EPO-Rs containing 267 amino acids or less were defective in internalization of (125)I-EPO, whereas internalization via a receptor derivative containing 276 amino acids was unaffected, thus directing focus to the nine amino acid residues FEGLFTTHK at positions 268-276 [Levin, Cohen, Supino, Yoshimura, Watowich, Neumann, FEBS Lett. 427 (1998) 164-170]. Here, a panel of EPO-R mutants was generated to determine the role of these residues in EPO endocytosis, down regulation of cell surface receptors and EPO-mediated signaling. While linking amino acid residues 268-276 to a truncated EPO-R (Delta+9 EPO-R) conferred both ligand uptake and ligand-independent down regulation of the respective receptor from the cell surface, Phe 272 was crucial for EPO endocytosis but not for ligand-independent down regulation. Additional receptor motifs probably play a role in EPO endocytosis and receptor down-regulation, as these processes were not adversely impaired in Delta268-276 EPO-R. A central role of residues 268-276, in particular Phe, was demonstrated by the inability of Delta268-276 and F268,272A EPO-Rs to support EPO-mediated signal transduction.     

Long-term maintenance of multilineal hemopoiesis in collagen gel culture.

We examined the long-term maintenance of multilineal hemopoiesis in a collagen gel culture of mouse bone marrow cells. When cells were inoculated into the gel, stromal cells formed foci that were composed of sinusoidlike capillary structures, fibroblastic cells, adipocytes and macrophages. Many small hemopoietic foci similar to granulocyte-macrophage colonies (CFU-GM) appeared within a week and disappeared after two weeks. Several large hemopoietic foci appeared after two to three weeks of culture, without a second challenge of marrow cells. These large hemopoietic foci were composed mainly of myeloid cells. Megakaryocytes and mast cells were also observed. When erythropoietin (EPO) was added to the culture at the beginning, the erythroid focus appeared after 3 weeks and the number of megakaryocytes was greater than that in the culture without EPO. However, when EPO was added to the cultures after 6 or 12 weeks, erythroid cells appeared after 1 week and the number of megakaryocytes increased. This hemopoiesis lasted more than 6 months.     

Production and ligand-binding characteristics of the soluble form of murine erythropoietin receptor.

A recombinant soluble form (sEPO-R) of erythropoietin (EPO) receptor (EPO-R) was produced by Chinese hamster ovary cells and isolated in high yield with the EPO-fixed gel. Ligand binding assays were done using three methods; precipitation of sEPO-R radiolabeled EPO complex and competition of sEPO-R for the binding of radiolabeled EPO with the cellular EPO-R. The results showed a Kd of 17 nM which was much lower than those for cellular EPO-R. One N-glycosylation site exists in sEPO-R but the glycosylation did not affect the binding affinity to EPO. A complex with a molecular size that corresponded to a 1:1 complex of EPO and sEPO-R was detected.     

The cytoplasmic region of the erythropoietin receptor contains nonoverlapping positive and negative growth-regulatory domains.

The erythropoietin (EPO) receptor (EPO-R), a member of a large cytokine receptor superfamily, has a 236-amino-acid cytoplasmic region which contains no obvious tyrosine kinase or other catalytic domain. In order to delineate the linear functional domains of the cytoplasmic tail, we generated truncated mutant cDNAs which were transfected into a murine interleukin-3-dependent cell line, Ba/F3, and the EPO-dependent growth characteristics of the stable transfectants were assayed. We identified two unique domains of the cytoplasmic tail. A membrane-proximal positive signal transduction domain of less than or equal to 103 amino acids, in a region highly similar to the interleukin-2 receptor beta chain, was sufficient for EPO-mediated signal transduction. A carboxy-terminal negative-control domain, a serine-rich region of approximately 40 amino acids, increased the EPO requirement for the Ba/F3 transfectants without altering EPO-R cell surface expression, affinity for EPO, receptor oligosaccharide processing, or receptor endocytosis. Truncation of this negative-control domain allowed the Ba/F3 transfectants to grow maximally in only 1 pM EPO, 1/10 the concentration required for growth of cells expressing the wild-type EPO-R. All truncated EPO-R mutants which retained the transmembrane region of the EPO-R polypeptide bound to the gp55 envelope protein of Friend spleen focus-forming virus. Only the functional EPO-R mutants were activated by the gp55, however, suggesting that gp55- and EPO-mediated signaling occur via a similar mechanism.     

A transplanted NPVY sequence in the cytosolic domain of the erythropoietin receptor enhances maturation

Activation of the EPO-R [EPO (erythropoietin) receptor] by its ligand EPO promotes erythropoiesis. Low cell surface EPO-R levels are traditionally attributed to inefficient folding mediated by the receptor extracellular domain. In the present study, we addressed the role of the EPO-R intracellular domain in exit from the ER (endoplasmic reticulum) and surface expression. A fusion protein between the thermo-reversible folding mutant of VSVG (vesicular-stomatitis-virus glycoprotein) (VSVGtsO45) and the EPO-R cytosolic domain [VSVG-WT (wild-type)] displayed delayed intracellular trafficking as compared with the parental VSVGtsO45, suggesting that the EPO-R cytosolic domain can hamper ER exit. Although NPXY-based motifs were originally associated with clathrin binding and endocytosis, they may also function in other contexts of the secretory pathway. A fusion protein between VSVGtsO45 and the cytosolic portion of EPO-R containing an NPVY insert (VSVG-NPVY) displayed enhanced glycan maturation and surface expression as compared with VSVG-WT. Notably, the NPVY insert also conferred improved maturation and augmented cell surface EPO-R. Our findings highlight three major concepts: (i) the EPO-R cytosolic domain is involved in ER exit of the receptor. (ii) Sequence motifs that participate in endocytosis can also modulate transport along the secretory pathway. (iii) VSVG-fusion proteins may be employed to screen for intracellular sequences that regulate transport.     

The SH2 inositol 5-phosphatase Ship1 is recruited in an SH2-dependent manner to the erythropoietin receptor

Ship1 (SH2 inositol 5-phosphatase 1) has been shown to be a target of tyrosine phosphorylation downstream of cytokine and immunoregulatory receptors. In addition to its catalytic activity on phosphatidylinositol substrates, it can serve as an adaptor protein in binding Shc and Grb2. Erythropoietin (EPO), the primary regulator of erythropoiesis, has been shown to activate the tyrosine phosphorylation of Shc, resulting in recruitment of Grb2. However, the mechanism by which the erythropoietin receptor (EPO-R) recruits Shc remains unknown. EPO activates the tyrosine phosphorylation of Ship1, resulting in the interdependent recruitment of Shc and Grb2. Ship1 is recruited to the EPO-R in an SH2-dependent manner. Utilizing a panel of EPO-R deletion and tyrosine mutants, we have discovered remarkable redundancy in Ship1 recruitment. EPO-R Tyr(401) appears to be a major site of Ship1 binding; however, Tyr(429) and Tyr(431) can also serve to recruit Ship1. In addition, we have shown that EPO stimulates the formation of a ternary complex consisting of Ship1, Shc, and Grb2. Ship1 may modulate several discrete signal transduction pathways. EPO-dependent activation of ERK1/2 and protein kinase B (PKB)/Akt was examined utilizing a panel of EPO-R deletion mutants. Activation of ERK1/2 was observed in EPO-RDelta99, which retains only the most proximal tyrosine, Tyr(343). In contrast, EPO-dependent PKB activation was observed in EPO-RDelta43, but not in EPO-RDelta99. It appears that EPO-dependent PKB activation is downstream of a region that indirectly couples to phosphatidylinositol 3-kinase.     

Catecholamines and pituitary function. VI. Effect of different dopamine doses on TRH-induced prolactin release in women with pathological hyperprolactinemia

The present study was designed to examine the effect of low-dose dopamine (DA) infusion rates (0.02 and 0.1 microgram/kg X min) on both basal and TRH-stimulated prolactin release in normal and hyperprolactinemic individuals. Sixteen normally menstruating women in the early follicular phase of a cycle and 23 hyperprolactinemic patients were studied. 0.1 microgram/kg X min DA was infused in 8 normal women and 15 patients with pathological hyperprolactinemia, while 8 normal controls and 8 patients received 0.02 microgram/kg X min DA TRH (200 micrograms, i.v.) was administered alone and at the 180th min of the 5-hour DA infusion in all controls and patients. A significant reduction in serum PRL levels, which was similar in normal women (-59.5 +/- 4.0%, mean +/- SE) and hyperprolactinemic patients (-48.2 +/- 5.5) was observed in response to 0.1 microgram/kg X min DA. In normal cycling women DA infusion significantly (P less than 0.02) reduced the PRL response to TRH with respect to the basal TRH test (delta PRL 45.0 +/- 7.0 vs. 77.9 +/- 15.4 ng/ml). On the contrary, the PRL response to TRH was significantly higher during 0.1 microgram/kg X min DA than in basal conditions in hyperprolactinemic patients, both in absolute (delta PRL 91.8 +/- 17.6 vs. 38.4 +/- 6.8, P less than 0.03) and per cent (198.5 +/- 67.6 vs. 32.1 +/- 7.5, P less than 0.02) values. A normal PRL response to TRH, arbitrarily defined as an increase greater than 100% of baseline, was restored in 11 out of 15 previously unresponsive hyperprolactinemic patients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in kidney tissue and effects of erythropoietin after acute heart failure

Impairment of cardiac function causes renal damage. Renal failure after heart failure is attributed to hemodynamic derangement including reduced renal perfusion and increased venous pressure. One mechanism involves apoptosis and is defined as cardiorenal syndrome type 1. Erythropoietin (EPO) is a cytokine that induces erythropoiesis under hypoxic conditions. Hypoxia inducible factor 1 alpha (HIF-1α) plays a regulatory role in cellular response to hypoxia. Protective effects of EPO on heart, kidney and nervous system are unrelated to red blood cell production. We investigated early changes in and effects of EPO on renal tissues of rats with myocardial infarction by morphology and immunohistochemistry. Coronary artery ligation was used to induce myocardial infarction in Wistar rats. Group 1 comprised sham operated rats; groups 2, 3 and 4 included rats after coronary artery ligation that were sacrificed 6 h after ligation and that were treated with saline, 5,000 U/kg EPO or 10,000 U/kg EPO, respectively; group 5 included rats sacrificed 1 h after ligation. Group 2 showed increased renal tubule damage. Significantly less tubule damage was observed in EPO treated groups. EPO and EPO receptor (EPO-R) immunostaining intensities increased slightly for group 5 and became more intense for group 2. EPO and EPO-R immunostaining was observed in the interstitial area, glomerular cells and tubule epithelial cells of EPO treated groups. HIF-1α immunostaining was observed in collecting tubules in the medulla only in group 2. Caspase-3 immunostaining is an indicator of apoptosis. Caspase-3 staining intensity decreased in renal medulla of EPO treated groups. EPO treatment may exert a protective effect on the renal tissues of patients with cardiorenal syndrome.     

The SH2B1 adaptor protein associates with a proximal region of the erythropoietin receptor

Gene targeting experiments have shown that the cytokine erythropoietin (EPO), its cognate erythropoietin receptor (EPO-R), and associated Janus tyrosine kinase, JAK2, are all essential for erythropoiesis. Structural-functional and murine knock-in experiments have suggested that EPO-R Tyr-343 is important in EPO-mediated mitogenesis. Although Stat5 binds to EPO-R phosphotyrosine 343, the initial Stat5-deficient mice did not have profound erythroid abnormalities suggesting that additional Src homology 2 (SH2) domain-containing effectors may bind to EPO-R Tyr-343 and couple to downstream signaling pathways. We have utilized cloning of ligand target (COLT) screening to demonstrate that EPO-R Tyr(P)-343 and Tyr(P)-401 bind to the SH2 domain-containing adaptor protein SH2B1β. Immunoprecipitation and in vitro mixing experiments reveal that EPO-R binds to SH2B1 in an SH2 domain-dependent manner and that the sequence that confers SH2B1 binding to the EPO-R is pYXXL. Previous studies have shown that SH2B1 binds directly to JAK2, but we show that in hematopoietic cells, SH2B1β preferentially associates with the EPO-R. SH2B1 is capable of constitutive association with EPO-R, which is necessary for its optimal SH2-dependent recruitment to EPO-R-Tyr(P)-343/Tyr(P)-401. We also demonstrate that SH2B1 is responsive to EPO stimulation and becomes phosphorylated, most likely on serines/threonines, in an EPO dose- and time-dependent manner. In the absence of SH2B1, we observe enhanced activation of signaling pathways downstream of the EPO-R, indicating that SH2B1 is a negative regulator of EPO signaling.     

A cytosolic domain of the erythropoietin receptor contributes to endoplasmic reticulum-associated degradation.

The erythropoietin receptor (EPO-R) is the cellular target for erythropoietin (EPO), the primary hormone that mediates the proliferation of immature erythroblasts and their differentiation into mature erythrocytes. Unusual features of the EPO-R are its short half-life (t(1/2) 1-2 h), its degradation via multiple pathways and the fact that less than 1% of total cellular EPO-R molecules are found on the cell surface. The contribution of EPO-R structural determinants to the regulation of its intracellular metabolism is still unclear. The epidermal growth factor receptor (EGF-R), unlike the EPO-R, is efficiently transported to the cell surface and displays a much longer metabolic half-life. To determine which EPO-R cytosolic domains are involved in intracellular degradation, we studied chimeric receptor molecules constructed of EGF-R extracellular and transmembrane parts, linked to the full length or truncated cytosolic part of the EPO-R. The chimeras were expressed in transiently transfected COS 7 cells and stably expressed in Ba/F3 cells. Our experiments indicate that the cytosolic part of the EPO-R contains determinants that mark it for rapid degradation, in association with the endoplasmic reticulum (ER). This degradation was insensitive to brefeldin A and was inhibited by specific proteasomal inhibitors. A truncated EGF-R/EPO-R chimera containing only 50 amino acids of the EPO-R membrane-proximal cytosolic part was also rapidly degraded suggesting that these 50 amino acids are involved in receptor degradation.     

A dominant negative erythropoietin (EPO) receptor inhibits EPO-dependent growth and blocks F-gp55-dependent transformation.

The erythropoietin receptor (EPO-R), a member of the cytokine receptor superfamily, can be activated to signal cell growth by binding either EPO or F-gp55, the Friend spleen focus-forming virus glycoprotein. Activation by F-gp55 results in constitutive EPO-R signalling and the first stage of Friend virus-induced erythroleukemia. We have generated a truncated form of the EPO-R polypeptide [EPO-R(T)] which lacks the critical cytoplasmic signal-transducing domain of the EPO-R required for EPO- or F-gp55-induced cell growth. EPO-R(T) specifically inhibited the EPO-dependent growth of EPO-R-expressing Ba/F3 cells without changing the interleukin-3-dependent growth of these cells. In addition, Ba/F3 cells that coexpressed wild-type EPO-R and EPO-R(T) were resistant to transformation by F-gp55 despite efficient expression of the F-gp55 transforming oncoprotein in infected cells. EPO-R(T) inhibited the EPO-dependent tyrosine phosphorylation of wild-type EPO-R, the tyrosine kinase (JAK2), and the SH2 adaptor protein (Shc). In conclusion, the EPO-R(T) polypeptide is a dominant negative polypeptide which specifically interferes with the early stages of EPO-R-mediated signal transduction and which prevents Friend virus transformation of erythroblasts.