Characterization of murine erythropoietin receptor genes

We have isolated and characterized the murine genomic and complementary DNAs encoding erythropoietin (Epo) receptor from Epo-responsive and unresponsive mouse erythroleukemia cells. Two classes of Epo receptor cDNAs were isolated from Epo-responsive cells. One is a 55,000 Mr membrane-bound Epo receptor, and the other is a 29,000 Mr soluble Epo receptor lacking the transmembrane and cytoplasmic domains. As a result of alternative splicing, two insert sequences containing termination codons are produced, and the encoded polypeptide diverges four amino acids upstream from the transmembrane domain, adding 20 new amino acids before terminating. Amino acid sequence of the Epo receptor cDNA isolated from Epo-responsive cells was identical with that of Epo-unresponsive cells, indicating that Epo-responsiveness does not depend upon the primary structure of the Epo receptor (binding) protein. Analysis of 6.6 x 10(3) base-pairs (kb) genomic DNA segments covering complete Epo receptor gene and promoter regions revealed that potential regulatory elements (NF-E1, GF-1 or Eryf 1) for erythroid-specific and differentiation stage-specific gene expression are located in the promoter and 3' noncoding regions.     

Maintenance by erythropoietin of viability and maturation of murine erythroid precursor cells

Erythroblasts isolated from the spleens of mice infected with the anemia-inducing strain of Friend virus (FVA cells)-are erythropoietin (EP)-sensitive cells at the late colony forming unit-erythroid (CFU-E) and cluster forming unit stages of differentiation (Koury et al., J. Cell. Physiol. 121:526-532, 1984). We investigate here the EP requirements of FVA cells in vitro for viability, proliferation, and maturation. By delaying the addition of EP to FVA cell cultures or by withdrawing EP at early times of culture, the subsequent viability, cell numbers, and maturation were diminished. The longer the delay in EP addition or the earlier the EP withdrawal, the more diminished these parameters were when compared to cultures which contained EP throughout the 48 h of differentiation. FVA cells had a period of EP requirement in vitro that lasted for only 24 h or less after the initiation of culture. During these crucial first 24 h, EP induced an increase in the synthesis of all size classes of RNA. Protein synthesis was maintained at a stable level in cells cultured with EP, but it declined in cells cultured without it. In contrast, the synthesis rate of DNA and the content of DNA per cell were not affected by the presence of EP in the culture. However, FVA cells cultured without EP had progressive accumulation of small sized DNA due to breakage of higher molecular weight DNA. The rate of DNA breakdown was sufficient to prevent DNA accumulation and thus it probably plays a role in the abortion of cell proliferation. No such breakage was found in cells cultured with EP. Our results indicate that EP exerts an effect on FVA cells in culture which is reflected in their viability, cell number, and maturation. This effect is not mediated by a stimulation of the rate of DNA synthesis, but is accompanied by stimulation of overall RNA synthesis and maintenance of protein synthesis.     

The erythropoietin receptor of rat erythroid progenitor lens. Characterization and affinity cross-linkage

Commercially available 125I-labeled erythropoietin, obtained by genetic engineering from a human gene, was used to characterize receptors for this hormone on the cell surface of rat erythroid progenitor cells. A low number of high affinity binding sites (487 +/- 32 sites/cell, Kd = 167 +/- 14 pm) were found. Nonerythroid cells and erythrocytes did not exhibit specific binding. The high affinity binding was reversible and displaced by unlabeled erythropoietin, but not by other hormones and growth factors. After incubation at 37 degrees C, nearly 35% of the specifically bound erythropoietin seemed to be internalized, as judged by resistance to acidic buffer treatment. Thus, binding showed characteristics of a hormone-receptor association. 125I-Erythropoietin-labeled cells were treated with the bifunctional reagent dissucinimidyl suberate. Analysis of the cellular extracts by polyacrylamide gel electrophoresis under denaturing and reducing conditions revealed that erythropoietin can be cross-linked to two molecules of 94 and 78 kDa, respectively. Both labeled bands disappeared when the cells were labeled in the presence of an excess of unlabeled erythropoietin. Under nonreducing conditions, a cross-linked band of 230-255 kDa was observed. The relationships between these bands are discussed.     

Structure of the murine erythropoietin receptor complex. Characterization of the erythropoietin cross-linked proteins.

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.     

Characterization of erythropoietin receptor on erythropoietin-unresponsive mouse erythroleukemia cells

A membrane receptor for erythropoietin was identified in various erythropoietin-unresponsive mouse erythroleukemia cells. Scatchard analyses of the binding of human 125I-labeled erythropoietin to T3C1-2-0, K-1, GM86 and 707 cells showed the presence of a single class of binding sites with apparent Kd values of 0.27-0.78 nM, which are slightly higher than those of erythropoietin-responsive cells. The number of binding sites varied from 110 to 930 per cell. Crosslinking of 125I-erythropoietin to its binding sites with disuccinimidyl suberate revealed the existence of a single binding protein with molecular mass of 63 kDa. No binding sites with higher molecular mass, as observed in erythropoietin-responsive cells, were detected, nor was any specific binding observed to the non-erythroid hematopoietic cell or to the human erythroleukemia cells examined.     

Glycosylation of the murine erythropoietin receptor

Murine erythropoietin-responsive Rauscher Red 5-1.5 cells were used to determine the contribution of glycosylation to the size and function of the erythropoietin receptor. The half life of the receptors was determined to be 4 h. The number of receptors was not significantly decreased in cells treated for 48 h with inhibitors of glycosylation (tunicamycin, glucosamine or swainsonine) and their affinity was slightly enhanced in tunicamycin- or glucosamine-treated cells. Erythropoietin was cross-linked with two proteins of 104 and 86 kDa. Their molecular masses were not significantly reduced in cells treated with the glycosylation inhibitors. When immunoprecipitated cross-linked receptors were digested with endoglycosidases, the molecular masses of both proteins were only slightly modified giving values of 100 and 82 kDa. Thus we can conclude that the proteins cross-linked to erythropoietin are very weakly glycosylated.     

Characterization of cellular receptors for erythroid differentiation factor on murine erythroleukemia cells

Erythroid differentiation factor (EDF), which is structurally related to transforming growth factor-beta family and induces differentiation of murine erythroleukemia cell clone F5-5, has been labeled with 125I to characterize its interaction with cellular receptors. Binding of 125I-EDF to F5-5 cells is time- and temperature-dependent, specific, saturable, and reversible. Transforming growth factor-beta 1 has no significant effects on growth of F5-5 cells and binding of 125I-EDF to F5-5 cells. Scatchard analysis of the binding data indicated that F5-5 cells have a single class of binding sites (3,200/cell) with an apparent Kd of 3.1 X 10(-10) M. Affinity cross-linking experiments demonstrated three radiolabeled components of 140,000, 76,000, and 67,000 daltons under both reducing and nonreducing conditions. Labeling of these three components has been inhibited by incubation of the cells with excess unlabeled EDF. These results imply molecular weights of 115,000, 51,000, and 42,000 for the EDF receptors on this cell line.     

Mechanism of erythropoietin action on the erythroid progenitor cells induced from murine erythroleukemia cells (TSA8)

Erythropoietin is a well-known erythroid differentiation and growth factor, but the mechanism of its action is not well understood. In this work, we have examined its mechanism of action on the erythropoietin-responsive murine erythroleukemia cells (TSA8). TSA8 cells become responsive to erythropoietin after induction with DMSO. Stimulatory effects on erythropoietin response are observed with the addition of compounds affecting the cAMP level such as forskolin, phosphodiesterase inhibitor and cholera toxin only in the presence of erythropoietin. cAMP analogues themselves show no stimulatory effect on TSA8 cells, nor does erythropoietin increase cAMP level in the cells. Thus, it is suggested that cAMP does not act as a direct second messenger for signal transduction through erythropoietin receptors, but as a stimulator of the erythropoietin receptor pathway and/or as a second messenger in combination with the receptor pathway. The mechanism for acquisition of responsiveness to growth and differentiation factors of progenitor cells is discussed.     

Expression cloning of the murine erythropoietin receptor

Two independent cDNA clones encoding the erythropoietin receptor (EPO-R) were isolated from a pXM expression library made from uninduced murine erythroleukemia (MEL) cells. The clones were identified by screening COS cell transfectants for binding and uptake of radioiodinated recombinant human erythropoietin. As inferred from the cDNA sequence, the murine erythropoietin receptor is a 507 amino acid polypeptide with a single membrane-spanning domain. It shows no similarities to known proteins or nucleic acid sequences in the data bases. Although the MEL cell EPO-R has a single affinity with a dissociation constant of approximately 240 pM, the EPO-R cDNA, expressed in COS cells, generates both a high-affinity (30 pM) and a low-affinity (210 pM) receptor.     

Identification of erythropoietin receptors on fetal liver erythroid cells

Erythropoietin (EPO) has a central role in the growth and development of erythroid cells. Using a biologically active radioiodinated derivative, EPO receptors were identified on fetal mouse liver cells mostly consisting of erythroid cells. 125I-EPO was cross-linked to two receptors forms with apparent molecular masses of 110 and 95 kilodaltons, respectively and both having similar affinity toward EPO.     

Characterization of new multimeric erythropoietin receptor agonists

In addition to its natural ligand, the receptor for erythropoietin can be activated by small peptides known as erythropoietin mimetic peptides (EMPs). Although EMPs are less potent than the natural ligand, EMP dimers, consisting of two EMPs joined via a linker, have been shown to exhibit significantly improved activity compared to the corresponding monomers, with potency approaching that of the native hormone. In this study, we used a panel of novel EMP dimers to explore the effects of linker length and EMP attachment site on potency. The EC50 values obtained in an EPO-dependent proliferation assay indicated that, as has been shown with similar molecules, EMP dimerization can lead to increases in potency of more than 2 orders of magnitude. We found that both C-terminal and N-terminal attachment of the linker to EMP was tolerated, and that, with the exception of the shortest linker, all of the linker lengths tested provided a similar increase in potency. In follow-up work devised to explore the potential benefit of contacting additional cell surface EPO receptors, we designed a tetrameric template consisting of lysine-based dimers joined via commercial PEG linkers of various molecular weights. Evaluation of the resulting molecules indicated a clear effect of PEG linker size on activity, while the "dimer of dimer" with the shortest linker exhibited 10-fold lower potency than the corresponding dimer, the longest tetramer increased potency by fivefold. We discuss the implications of these results for the further development of EMP multimers.     

Quantitation of specific binding of erythropoietin to human erythroid colony-forming cells

Highly purified human erythroid colony-forming cells (ECFC), which consist predominately of colony-forming units-erythroid (CFU-E), were prepared from human blood and used to study the binding and processing of erythropoietin (Ep). When radioiodinated human recombinant Ep (125I-rEp) was incubated with these cells, binding was specific and saturable. Specific binding was directly proportional to cell concentration and did not occur with other human cells. Saturation of specific binding at 3 degrees C occurred at 1 nM (3.9/U/ml), and Scatchard analysis revealed two classes of binding sites on the cell surface. Of a total of 1,050 binding sites per ECFC, one-fifth had a Kd of 0.10 nM, while the remainder had a Kd of 0.57 nM. Specific binding was twofold greater at 37 degrees C than at 3 degrees C, and removal of surface-bound Ep with acid indicated that 125I-rEp was internalized into the cells after incubation at 37 degrees C. Further incubation at this temperature showed a decline of cellular radioactivity, with a release of small molecular weight degradation fragments into the medium. These studies demonstrate two classes of receptors for Ep on normal human ECFC. Internalization and degradation of EP occur, and the biologic effect of the hormone is produced by a small number of Ep molecules, as demonstrated in murine erythroid progenitor cells.     

Binding and receptor-mediated endocytosis of erythropoietin in Friend virus-infected erythroid cells

The binding of labeled erythropoietin (EP) to cell surface receptors and subsequent processing of the hormone within the cell was studied in erythroid cells procured from the spleens of mice infected with the anemia strain of Friend virus. These immature erythroid cells respond to EP in culture to differentiate into reticulocytes and erythrocytes. Radiolabeled EP (both iodinated and tritiated) binds to 800-1000 cell surface receptors on these cells at 4 degrees C. Using 125I-EP, we found that 300 of these cell surface receptors have a higher affinity for EP (Kd = 0.09 nM) than the remaining receptors (Kd = 0.57 nM). The number of molecules of EP bound per cell increased about 2-fold when binding was carried out at 37 degrees C. Treatment of the cell surface with pronase or removal of surface-bound EP with a low pH wash revealed that radiolabeled EP is internalized by the cells at 37 degrees C. Pulse chase experiments showed that degradation products of radiolabeled EP are released into the medium with a corresponding loss of label from the interior of the cell. Inhibitors of lysosomal function greatly reduced this degradation of 125I-EP. Since 180 of the 300 high affinity receptors and very few of the low affinity receptors are occupied at the concentration of EP which elicits the maximum biological response in these cells, we suggest that interaction of EP with the high affinity receptors are necessary for the full biological effect of the hormone. A different murine erythroleukemia cell line which does not differentiate in response to EP was found to have only the lower affinity binding sites for the hormone.     

Combined action of c-kit and erythropoietin on erythroid progenitor cells.

Mutations at the murine dominant-white spotting locus (W) (c-kit) affect various aspects of hematopoiesis. We have made antibodies against c-Kit with the synthetic peptides deduced from the murine c-kit gene and examined the role of c-Kit in erythropoiesis. The antibody inhibited the stromal cell-dependent large colony formation of the erythroid progenitors. In the culture of erythropoietin-responsive erythroid progenitors of the anemia-inducing Friend virus-infected mouse spleen, the antibody inhibited only proliferation, but not differentiation of the progenitor cells. The inhibition was effective only at the early phase (within 6 hours after erythropoietin addition) before the cells start to proliferate induced by erythropoietin. During the early phase, erythropoietin down-regulated c-kit gene expression. These results suggest a mechanism of combined action of c-Kit with erythropoietin on the lineage-restricted erythroid progenitor cells.     

Partial purification and characterization of erythropoietin receptors from erythroid progenitor cells

We have partially purified and characterized erythropoietin (Epo) receptors of erythroid progenitor cells which were obtained from the spleens of anemia-inducing Friend virus infected mice. Membrane proteins of splenic erythroid progenitor cells were solubilized with 1% Triton X-102. Upon chromatography on DEAE-Sephacel anion-exchange columns, two distinct Epo receptor peak fractions referred to as Peak I and Peak II were identified by 125I-Epo binding assays using the polyethylene glycol precipitation method. The Peak I and Peak II samples were then individually chromatographed on an S-Sepharose column. The S-Sepharose-purified Peak I and Peak II samples were crosslinked with 125I-Epo in the presence and absence of excess unlabeled Epo by disuccinimidyl suberate treatment, and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. Both Peak I and Peak II samples showed a radiolabeled peptide with a Mr 135K and the labeling was blocked by excess unlabeled Epo. Since the Mr of Epo is about 35K, Epo receptor peptide has a Mr approximately 100K. To determine whether Epo stimulates autophosphorylation of the receptors, the S-Sepharose-purified Peak I and Peak II samples were incubated with or without Epo, and then briefly incubated in the presence of [gamma-32P]ATP and Mn2+. The tyrosine residue phosphorylated protein was isolated by an immunochemical technique, and then analyzed by SDS-PAGE and autoradiography. The result showed that Epo stimulates phosphorylation of a 100-kDa peptide.     

Immunofluorescent detection of erythrocyte sialoglycoprotein antigens on murine erythroid cells

A sialoglycoprotein fraction isolated from murine (DBA/2) erythrocytic ghosts (see companion article, Sarris and Palade, 1982, J. Cell. Biol. 93:583-590) was used to raise antibodies in rabbits. By immune-IgG (serum)-[125I] protein A overlays, the antibodies were found to react positively with the four sialoglycoprotein monomers (gp-2.1, gp-2.2, gp- 3.1, and gp-3.2) of the original fraction, with the sialoglycoproteins detected in erythrocytic ghosts (gp-2.1 and gp-3.1), with a diffuse component (probably a macroglycolipid) trailing around gp-3.1 in SDS polyacrylamide gel electrophoretograms of solubilized ghosts, and with a minor sialoglycoprotein hidden under this trail. IgG's isolated from immune and nonimmune rabbit sera were conjugated to tetramethylrhodamine isothiocyanate and used to survey, by fluorescence microscopy, the distribution of the cognate antigens on the three different erythroid lines known to succeed each other during the life span of the mouse. In the peripheral blood of the adult, the antibodies recognized only mature erythrocytes; they did not crossreact with either platelets, monocytes, or different types of granulocytes. In the spleen of adult anemic mice, the antibodies reacted weakly with proerythroblasts and strongly with all types of erythroblasts. In enucleating erythroblasts, antigens were preferentially segregated on the cell membrane of the nascent reticulocyte. In the 10-day-old embryo, antigens were already present on the primitive nucleated erythrocytes (produced by the blood islets of the yolk sack), and in the 14-d fetus they were found on all hepatic erythroblasts and derived non-nucleated erythrocytes. A positive immunoreaction was also obtained on Friend erythroleukemic cells, before or after induction by dimethyl sulfoxide. Nonimmune serum, or nonimmune IgGs gave negative reactions in all cases. The antibodies were species-specific: they did not crossreact with either human or rat erythrocytes.     

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.

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.     

Comparative analysis of the effects of the unpurified preparations of murine erythropoietin and human recombinant erythropoietin on erythroid and granulocyte-macrophage progenitor cells in semi-solid mouse bone marrow cultures]

Effects of unpurified murine erythropoietin and unpurified human recombinant erythropoietin on the growth of erythroid--BFU-E and granulocyte--macrophage progenitor cells--CFU--GM from the mouse bone marrow were compared using a methylcellulose culture system. Average erythropoietin titers for murine serum and supernatant human recombinant erythropoietin were 16 U/ml and 33 U/ml, respectively. The maximal stimulation was observed at 1-2 U/ml culture recombinant erythropoietin and 0.5 U/ml culture murine erythropoietin. Murine erythropoietin was more effective then human one. Murine and human recombinant erythropoietin had no significant effect on the number of CFU-GM colonies. But human recombinant erythropoietin could be preferentially used when studying the mechanism of erythropoiesis in man and animals because there were erythropoiesis inhibitors in mouse serum.     

A constitutively activated erythropoietin receptor stimulates proliferation and contributes to transformation of multipotent, committed nonerythroid and erythroid progenitor cells.

If the env gene of spleen focus-forming virus (SFFV) is replaced by a cDNA encoding a constitutively active form of the erythropoietin receptor, EPO-R(R129C), the resultant recombinant virus, SFFVcEPO-R, induces transient thrombocytosis and erythrocytosis in infected mice. Clonogenic progenitor cell assays of cells from the bone marrow and spleens of these infected mice suggest that EPO-R(R129C) can stimulate proliferation of committed megakaryocytic and erythroid progenitors as well as nonerythroid multipotent progenitors. From the spleens of SFFVcEPO-R-infected mice, eight multiphenotypic immortal cell lines were isolated and characterized. These included primitive erythroid, lymphoid, and monocytic cells. Some expressed proteins characteristic of more than one lineage. All cell lines resulting from SFFVcEPO-R infection contained a mutant form of the p53 gene. However, in contrast to infection by SFFV, activation of PU.1 gene expression, by retroviral integration, was not observed. One cell line had integrated a provirus upstream of the fli-1 gene, in a location typically seen in erythroleukemic cells generated by Friend murine leukemia virus infection. This event led to increased expression of fli-1 in this cell line. Thus, infection by SFFVcEPO-R can induce proliferation and lead to transformation of nonerythroid as well as very immature erythroid progenitor cells. The sites of proviral integration in clonal cell lines are distinct from those in SFFV-derived lines.     

Rapid activation by erythropoietin of protein kinase C in nuclei of erythroid progenitor cells

The glycoprotein hormone erythropoietin (Ep) regulates the proliferation and differentiation of erythroid progenitor cells by a signal transduction system which is not well understood. It has recently been reported that prolactin, a mitogen and trophic hormone for liver, will activate a nuclear protein kinase C in hepatocytes. As similarities exist in the actions of Ep and prolactin in their target cells, we tested the hypothesis that Ep could activate protein kinase C in nuclei isolated from erythroid progenitor cells. In a pure population of such nuclei, Ep induced a rapid, time- and dose-dependent increase in phosphorylation of endogenous nuclear substrate which could be blocked by inhibitors of protein kinase C or by antibody to Ep. Other known activators of protein kinase C were also effective in this system. These findings show that Ep may exert its effects by a novel signalling pathway, the activation of a nuclear protein kinase C.