Cell Surface Erythropoietin Receptor Research Articles

A dimeric peptide with erythropoiesis-stimulating activity uniquely affects erythropoietin receptor ligation and cell surface expression.

Erythropoiesis-stimulating agents (ESAs) that exert long-acting antianemia effects have been developed recently, but their mechanisms are poorly understood. Analyses reveal unique erythropoietin receptor (EPOR)-binding properties for one such ESA, the synthetic EPOR agonist peginesatide. Compared with recombinant human EPO and darbepoietin, peginesatide exhibited a slow on rate, but sustained EPOR residency and resistant displacement. In EPO-dependent human erythroid progenitor UT7epo cells, culture in peginesatide unexpectedly upmodulated endogenous cell surface EPOR levels with parallel increases in full-length EPOR-68K levels. These unique properties are suggested to contribute to the durable activity of this (and perhaps additional) dimeric peptide hematopoietic growth factor receptor agonist.

Functional EpoR pathway utilization is not detected in primary tumor cells isolated from human breast, non-small cell lung, colorectal, and ovarian tumor tissues.

Several clinical trials in oncology have reported increased mortality or disease progression associated with erythropoiesis-stimulating agents. One hypothesis proposes that erythropoiesis-stimulating agents directly stimulate tumor proliferation and/or survival through cell-surface receptors. To test this hypothesis and examine if human tumors utilize the erythropoietin receptor pathway, the response of tumor cells to human recombinant erythropoietin was investigated in disaggregated tumor cells obtained from 186 patients with colorectal, breast, lung, ovarian, head and neck, and other tumors. A cocktail of well characterized tumor growth factors (EGF, HGF, and IGF-1) were analyzed in parallel as a positive control to determine whether freshly-isolated tumor cells were able to respond to growth factor activation ex vivo. Exposing tumor cells to the growth factor cocktail resulted in stimulation of survival and proliferation pathways as measured by an increase in phosphorylation of the downstream signaling proteins AKT and ERK. In contrast, no activation by human recombinant erythropoietin was observed in isolated tumor cells. Though tumor samples exhibited a broad range of cell-surface expression of EGFR, c-Met, and IGF-1R, no cell-surface erythropoietin receptor was detected in tumor cells from the 186 tumors examined (by flow cytometry or Western blot). Erythropoiesis-stimulating agents did not act directly upon isolated tumor cells to stimulate pathways known to promote proliferation or survival of human tumor cells isolated from primary and metastatic tumor tissues.

Erythropoietin, a novel versatile player regulating energy metabolism beyond the erythroid system.

Erythropoietin (EPO), the required cytokine for promoting the proliferation and differentiation of erythroid cells to stimulate erythropoiesis, has been reported to act as a pleiotropic cytokine beyond hematopoietic system. The various activities of EPO are determined by the widespread distribution of its cell surface EPO receptor (EpoR) in multiple tissues including endothelial, neural, myoblasts, adipocytes and other cell types. EPO activity has been linked to angiogenesis, neuroprotection, cardioprotection, stress protection, anti-inflammation and especially the energy metabolism regulation that is recently revealed. The investigations of EPO activity in animals and the expression analysis of EpoR provide more insights on the potential of EPO in regulating energy metabolism and homeostasis. The findings of crosstalk between EPO and some important energy sensors and the regulation of EPO in the cellular respiration and mitochondrial function further provide molecular mechanisms for EPO activity in metabolic activity regulation. In this review, we will summarize the roles of EPO in energy metabolism regulation and the activity of EPO in tissues that are tightly associated with energy metabolism. We will also discuss the effects of EPO in regulating oxidative metabolism and mitochondrial function, the interactions between EPO and important energy regulation factors, and the protective role of EPO from stresses that are related to metabolism, providing a brief overview of previously less appreciated EPO biological function in energy metabolism and homeostasis.

The Erythropoiesis Stimulating Agent Omontys/Peginesatide up-Modulates Erythroid Progenitor Formation, and Cell Surface Erythropoietin Receptor Levels.

AbstractAbstract 2089Peginesatide is a peptide-based erythropoietin receptor (EPOR) agonist with recent FDA approval for treating the anemia of chronic kidney disease among adult dialysis patients. Although peginesatide exhibits a 47.9h half-life when given IV in dialysis patients, it is administered once-monthly. Taken together, this predicts that additional functional properties contribute to peginesatide’s durable erythropoiesis stimulating activity. Here we report on three such properties. During ex vivo murine bone marrow erythroid development, compared directly to rHuEPO, peginesatide first enhanced KitposCD71pos progenitor cell expansion (including KitposCD71lowCD36posCD13pos erythromyelo- progenitors). Second, peginesatide exhibited a 1300 minute EPOR residence time vs. 77 minutes for rHuEPO. Third, the culture of EPO-dependent human UT7epo cells in peginesatide led to substantial EPOR up-modulation, as well as an apparent lessening of the processing of mature EPOR’s. Furthermore, in studies that compared peginesatide vs. rHuEPO effects on the erythroid development of human bone marrow-derived CD34pos progenitors, the following differences were observed. At days 6–10 of culture, peginesatide gave rise to increased frequencies (up to 200% as compared to rHuEPO) of KitposCD71pos co-positive erythroid progenitors. At early stages (d2-d4), cell-surface EPOR levels also were elevated among progenitors expanded in peginesatide. When levels of CD13posCD36pos co-positive cells were analyzed, frequencies of these erythromyelo-progenitors also were heightened up to three-fold. Analyses of cultures at later time-points (eg, d10 of culture) indicated that peginesatide and rHuEPO supported the formation of GPAhigh erythroblasts at similarly high frequencies. In addition, cytospin analyses revealed morphological distinctions for multicellular proerythroblast complexes formed in peginesatide. Peginesatide’s persistent erythropoietic activity allowing for once-monthly dosing therefore is likely to involve novel effects on erythromyelo-progenitor recruitment, and increased EPOR cell surface expression including apparent increases in relative levels of full-length EPOR forms. Disclosures:Green:Affymax, Inc.: Employment. Leu:Affymax, Inc.: Employment. Mortensen:Affymax, Inc.: Employment. Young:Affymax, Inc.: Prior employment Other. Schatz:Affymax, Inc.: Employment. Wojchowski:Affymax, Inc.: Membership on an entity's Board of Directors or advisory committees.

ETV6-RUNX1 (TEL-AML1) Deregulates the Erythropoietin Receptor (EPOR) and Promotes Cell Survival.

AbstractAbstract 3641ETV6-RUNX1 (TEL-AML1) fusion is usually an initiating and pre-natal event in childhood acute lymphoblastic leukaemia (ALL). Transformation results in the generation of a persistent pre-leukemic clone, which post-natally converts to ALL following the acquisition of necessary secondary genetic alterations. The erythropoietin receptor gene (EPOR) is consistently highly expressed ectopically in TEL-AML1+ ALL but the presence of a functional receptor on the cell surface and its role, if any, in leukemogenesis driven by TEL-AML1 remains to be confirmed. Similarly, there is much debate as to the role played by the cytokine erythropoietin (EPO) and signaling through EPOR in relation to non-erythroid cell survival. Here, we show by using biotinylated erythropoietin (EPO) and flow analysis that the pre-B ALL TEL-AML1+ cell line (REH) appeared to have higher levels of EPO receptor than other non-TEL-AML1 cell lines. Furthermore, when we “blind screened” CD19+ peripheral blood or bone marrow cells from 10 patients with pre-B ALL, we identified 5 patients that showed high expression of ligand-binding EPO receptor on the cell surface, 4 of which were subsequently shown to be TEL-AML1+. We show that the inducible expression of TEL-AML1 in lymphoid BaF3 cells or its constitutive expression in either a murine transgenic model or in normal human cord blood cells is also sufficient to increase expression of EPOR. In order to further assess the direct regulation of EPOR by TEL-AML1 we next performed EMSA and ChIP experiments to confirm occupancy of AML1 consensus binding sites within the EPOR promoter and luciferase reporter assays to confirm up-regulation of EPOR promoter activity in the presence of TEL-AML1. Given the proposed pro-survival properties of EPO on non-erythroid cells, we next asked if the observed increase in expression of the EPOR in cells expressing TEL-AML1 could correlate with an increased cell survival in the presence of EPO. Cell survival experiments including growth curves, Annexin V staining and analysis of anti-apoptotic gene markers revealed that IL3-dependent cells expressing TEL-AML1 showed a prolonged survival in the presence of EPO alone and the consequent activation of anti-apoptotic pathways. The subsequent removal of TEL-AML1 expression in these cells resulted in cell death even in the presence of EPO, further suggesting that this effect is a consequence solely of fusion gene expression. Moreover, the observed survival in the presence of TEL-AML1 and EPO was enhanced by the co-addition of stem cell factor (SCF). Interestingly, SCF associated intracellular tyrosines have been shown to play an essential role in signaling through EPOR-SCFR interactions. In the presence of EPO, signaling via EPOR was confirmed by phosophorylation of JAK2, STAT5 and AKT and the subsequent up-regulation of BCL-XL. Similarly, EPOR functionality was demonstrated in TEL-AML1 positive patient cells by analysis of JAK2 pathway signaling after EPO induction. We propose that such activation lead to the increase in cell survival also observed in patient cells in the presence of EPO - again suggesting a role for enhanced cell survival through the EPO-EPOR axis. In our final model of pre-leukemia, CD34+ cord blood cells were transduced in vitro with a lentivirus capable of expressing both TEL-AML1 and GFP and ‘primed’ for pre-B lineage commitment. GFP (TEL-AML1) positive cord blood cells also showed increased levels of cell surface EPO receptor when compared to empty vector controls. Collectively, these data support the contention that TEL-AML1 directly activates ectopic expression of a functional EPO receptor and provides cell survival signals that may contribute critically to persistence of the pre-malignant clone in patients. Disclosures:No relevant conflicts of interest to declare.

Recombinant Human Erythropoietin (rHuEPO) In Combination with Chemotherapy Increases Breast Cancer Metastasis In Pre-Clinical Mouse Models

AbstractAbstract 3345 Background:Erythropoietin (EPO) is a key regulator of erythropoiesis, and has been shown to stimulate growth, maintain viability, and promote differentiation of red blood cell precursors. The EPO receptor (EPO-R) is expressed by erythroid cells and by several non-hematopoietic cell types including various neoplastic cells. Erythropoiesis-stimulating agents (ESAs) are used clinically for the treatment of chemotherapy-induced anemia. The results of some recent randomized clinical trials have reported an increased incidence in adverse events and reduced survival in ESA-treated metastatic breast cancer patients receiving chemotherapy, potentially related to EPO-induced cancer progression. These results have raised concerns over ESA treatment in metastatic cancer patients. However, very little pre-clinical data is available regarding the impact of EPO on breast cancer metastasis. The goal of the current study was therefore to determine if EPO can influence the malignant behavior of breast cancer cells and/or influence the metastatic process. Methods:MDA-MB-468, MDA-MB-231, MDA-MB-435, and 4T-1 breast cancer cell lines were treated with recombinant human EPO (rHuEPO; 10 U/ml) or control media and screened for EPO-R mRNA expression levels by RT-PCR, and for EPO-R protein expression by Western blot and flow cytometry. MDA-MB-231 (231) and MDA-MB-435 (435) cell lines were used for functional assays in vitro and in vivo. Untreated or rHuEPO treated cells were grown in 2D and 3D in vitro systems (standard tissue culture plates and 0.6% soft agar, respectively) to determine if rHuEPO influenced growth. In vitro cell survival was also assessed in response to treatment with rHuEPO in the presence or absence of paclitaxel chemotherapy (10mg/ml), radiation (10G), or hypoxic conditions (1% O2). Following mammary fat pad injection, in vivo effects of rHuEPO (300U/kg) alone or in combination with paclitaxel treatment (10mg/kg) were assessed in mouse models of tumorigenicity and spontaneous metastasis. Results:Expression analysis of EPO-R mRNA and protein revealed a large variation in levels across different cell lines. The majority of cell lines did not express cell surface EPO-R by flow cytometry, although two cell lines (231 and 435) did show weak expression of EPO-R mRNA, with only the 231 cell line showing EPO-R expression by Western blot. In vitro, a small protective effect from rHuEPO on radiation-treated 435 cells was seen (p<0.05); however, rHuEPO treatment alone or combined with chemotherapy or hypoxia did not cause a significant increase in cell survival relative to untreated controls cells. In contrast, in vivo studies demonstrated that rHuEPO increased the incidence and burden of lung metastases in immunocompromised mice injected with 231 or 435 cells and treated with paclitaxel relative to mice treated with paclitaxel alone (p<0.05). Conclusions:The lack of an in vitro effect of rHuEPO highlights the importance of in vivo studies to delineate the effects of EPO on the metastatic process. Our novel findings demonstrate that rHuEPO can reduce the efficacy of chemotherapy in the metastatic setting in vivo, and in some cases enhance the inherent metastatic growth potential of human breast cancer cells.This work was supported by funding from the London Regional Cancer Program and Janssen Ortho Canada Disclosures:Xenocostas:Janssen Ortho: Consultancy, Honoraria, Research Funding. Allan:Janssen Ortho: Research Funding.

Analysis of cell surface erythropoietin receptor (EpoR) expression and function in human epithelial tumor tissues

11104 Background: EpoR mRNA and protein expression have been reported at low levels in human tumors implying EpoR may be functional in tumor cells with inferred implications for the use of erythropoiesis stimulating agents in the Oncology setting. However, mRNA studies use bulk tumor tissue which ignores contribution from the stroma and IHC studies have all used Abs that have since been shown to not be specific for EpoR. Therefore this important question remains unresolved. Methods: EpoR expression and function was investigated in viable, non-apoptotic primary human tumor cells (using tumor specific Abs) that were disaggregated from at least 30 patients from each of the following tumor types: breast (incl. metastases), NSC lung, colorectal, and ovarian. Additional tumor types included head and neck, GBM, pancreatic and gastric. Analysis was performed under conditions that were shown to preserve EpoR expression and function in positive control tissues (primary human erythroid progenitors from bone marrow and UT-7 cells). Cell surface EpoR expression was evaluated by flow cytometry using novel EpoR-specific Abs. EpoR function was investigated by analysis of EpoR signaling using phospho-specific Abs specific for STAT5, Erk, Akt etc following ex vivo Epo stimulation (0U- 300U/mL; for 5 and 30 min). Results: No expression of cell surface EpoR was detected in tumor cells in any of the over 130 tumors. In contrast, high levels of expression were observed in positive controls analyzed in parallel. No induction of EpoR signaling was observed in tumor cells at any [Epo], whereas activation was readily detected in tumor cells treated in parallel with a cocktail of known tumor growth factors. Positive controls treated in parallel, showed robust Epo concentration-dependent signaling. No evidence of EpoR expression or function was observed in tumor endothelial cells. Conclusions: These data demonstrate that epithelial tumor cells do not express functional cell surface EpoR and are not responsive to physiological, therapeutic or indeed very high levels of Epo (300U/mL). No significant financial relationships to disclose.

Analysis of Cell Surface Erythropoietin Receptor (EpoR) Expression and Function in Epithelial Human Tumor Tissues Reveals No Detectable Cell Surface Expression or Function

Analysis of Cell Surface Erythropoietin Receptor (EpoR) Expression and Function in Epithelial Human Tumor Tissues Reveals No Detectable Cell Surface Expression or FunctionA number of studies have reported that EpoR mRNA is detectable at low levels in human epithelial tumor tissues and have raised the possibility that this surface EpoR protein expression could render human tumor cells responsive to Epo with inferred implications for the use of erythropoietic stimulating agents in the Oncology setting. To date the only data supporting this hypothesis has been generated using mRNA analysis or protein analysis using antibodies that have subsequently been shown to not be specific for EpoR.To address these possible issues more directly we have analyzed EpoR expression and function in a large panel of human primary tumor tissues isolated by surgical resection from a variety of epithelial tumor cells (including colon, non small cell lung, breast and ovarian tumors). Solid human tumor tissue was disaggregated using a treatment shown to preserve cell surface EpoR density and function in positive controls (below). Flow cytometric analysis of primary tumor cells in the disaggregated tumor population was performed using multiple markers specific for epithelial tumor cells, as well as viability dyes and apoptotic markers to exclude non-viable and or apoptotic cells from the analysis. Using anti-EpoR monoclonal antibodies with high sensitivity and specificity for EpoR, no expression of cell surface EpoR was detected in primary tumor cells in any of the more than 60 human epithelial tumor specimens analyzed. In contrast, high levels of expression were observed in a positive control cell line (UT7/Epo) analysed in parallel, as well as a physiologically relevant primary tissue (differentiated erythroid progenitor cells). Notably, in a fraction of the ovarian and breast tumor tissues cell surface EpoR expression was identified in CD45+ tumor infiltrating leukocytes. No EpoR expression was detected in non tumor cells that were not CD45+ suggesting no contribution from other stromal elements in the tumor. These observations may explain the reported detection of EpoR mRNA in a subset of breast and other solid tumor patients, as those previous analyses involved bulk tumor tissue and did not allow analysis of tumor cell specific expression.To test the possibility that low cell-surface EpoR protein density on tumor cells may be sufficient for function but undetectable by flow cytometry, we evaluated the ability of primary human tumor tissue to support an EpoR-driven signal pathway. EpoR function was analyzed in primary human tumor cells treated with a range of concentrations of recombinant human Epo (rHuEpO; 0.1–300U/mL) under conditions shown to result in productive EpoR dependent signaling in positive control cells/tissues. Analysis of possible EpoR-driven signaling was determined by intracellular flow cytometry using antibodies specific for the phosphorylated forms of STAT5, Akt, Erk1/2, p70S6, STAT3, STAT1, STAT6, JNK, and c-jun. Attribution of any detected signaling to viable tumor cells was performed via the use of a combination of tumor cell specific and viability/apoptosis markers. No evidence of downstream signaling was observed in primary tumor cells in epithelial tumors from over 60 patients at any concentration of rHuEpo, whereas UT7/Epo cells, treated in parallel, showed robust Epo concentration-dependent activation of signaling. Furthermore, activation of these signaling proteins was detected when the same primary human tumor cells were treated in parallel with a cocktail of known human tumor growth factors, confirming that these cells are capable of responding to exogenous stimuli using the same pathways as EpoR and that these signaling events can be readily detected using the platform. Taken together these data support the hypothesis that tumor cells in solid human tumors do not express functional cell surface EpoR and are not responsive to physiological or therapeutically relevant concentrations of Epo or indeed very high levels of Epo (300U/mL).

Protein tyrosine phosphatase 1B participates in the down-regulation of erythropoietin receptor signalling.

Erythropoietin (EPO) is the principal hormone regulating the proliferation of erythroid precursors and their differentiation into erythrocytes. Binding of ligand to the cell-surface EPO-R (EPO receptor) induces dimerization and JAK2 (Janus kinase 2)-mediated tyrosine phosphorylation of the receptor. Less than 1% of the EPO-Rs are displayed on the cell surface; most of the receptor molecules are retained in intracellular compartments, including the ER (endoplasmic reticulum). Using pervanadate (PV) as a potent tool to inhibit cellular PTPs (protein tyrosine phosphatases), we demonstrated previously the accumulation of mature (endoglycosidase H-resistant) tyrosine-phosphorylated EPO-R [Cohen, Altaratz, Zick, Klingmuller and Neumann (1997) Biochem. J. 327, 391-397]. In the present study, we investigated the participation of the ER-associated PTP1B in the dephosphorylation of intracellular EPO-R. We demonstrate tyrosine phosphorylation of EPO-R in BOSC-23T cells co-expressing EPO-R and the 'substrate-trapping' mutant form of PTP1B, PTP1B D181A (referred to as PTP1BD). In vivo interaction between EPO-R and PTP1B suggested that PTP1B dephosphorylates the EPO-R intracellularly. Endoglycosidase H resistance of tyrosine-phosphorylated EPO-R in cells expressing PTP1BD suggested that mature EPO-R is dephosphorylated by PTP1B. Stimulation with EPO of cells co-expressing EPO-R and either PTP1BD or PTP1B resulted in an increase or decrease respectively in phosphotyrosine EPO-R. We thus suggest that PTP1B dephosphorylates EPO-stimulated EPO-R and participates in the down-regulation cascade of EPO-mediated signal transduction.

Ligand-independent oligomerization of cell-surface erythropoietin receptor is mediated by the transmembrane domain.

Binding of erythropoietin (Epo) to the Epo receptor (EpoR) is crucial for production of mature red cells. Although it is well established that the Epo-bound EpoR is a dimer, it is not clear whether, in the absence of ligand, the intact EpoR is a monomer or oligomer. Using antibody-mediated immunofluorescence copatching (oligomerizing) of epitope-tagged receptors at the surface of live cells, we show herein that a major fraction of the full-length murine EpoR exists as preformed dimers/oligomers in BOSC cells, which are human embryo kidney 293T-derived cells. This observed oligomerization is specific because, under the same conditions, epitope-tagged EpoR did not oligomerize with several other tagged receptors (thrombopoietin receptor, transforming growth factor beta receptor type II, or prolactin receptor). Strikingly, the EpoR transmembrane (TM) domain but not the extracellular or intracellular domains enabled the prolactin receptor to copatch with EpoR. Preformed EpoR oligomers are not constitutively active and Epo binding was required to induce signaling. In contrast to tyrosine kinase receptors (e.g., insulin receptor), which cannot signal when their TM domain is replaced by the strongly dimerizing TM domain of glycophorin A, the EpoR could tolerate the replacement of its TM domain with that of glycophorin A and retained signaling. We propose a model in which TM domain-induced dimerization maintains unliganded EpoR in an inactive state that can readily be switched to an active state by physiologic levels of Epo.

New epoetin molecules and novel therapeutic approaches.

Erythropoietin (EPO) is a 34 kDa protein that is the primary regulator of red blood cell production. EPO facilitates its effect by binding to the cell surface EPO receptor which initiates the JAK-STAT signal transduction cascade. The search for small mimetic molecules of EPO has led to the discovery of a family of peptides that demonstrate EPO mimetic activity. A member of this peptide family, EMP1 (EPO mimetic peptide 1), was used to solve the crystal structure of the soluble EPO receptor in complex with this peptide. The structure revealed a 2:2 stoichiometry of receptor to peptide, with each peptide contacting both receptor molecules in a symmetrical fashion. The potency of the EMPs could be improved through the covalent dimerization of two peptide molecules. Further investigations of EMP EPO receptor complex structures revealed the formation of a non-productive receptor dimer using an inactive peptide. An alternative approach towards the identification of an EPO-like mimetic is to target an intracellular signalling molecule such as haematopoietic cell phosphatase (HCP), also known as SHP1. Inhibiting HCP causes responsive cells to be hypersensitive to EPO. The cloned HCP protein has been utilized in screening assays to identify small molecule inhibitors of HCP.

Cell Surface Organization of the Erythropoietin Receptor Complex Differs Depending on its Mode of Activation

During erythroid development erythropoietin (EPO) binds specifically to a receptor primarily present on committed erythroid progenitors, stimulating mitogenic, survival, and differentiative growth response pathways. Other modes of erythropoietin receptor (EPO-R) activation, such as interaction with the env gene Friend virus envelope glycoprotein (F-gp55) of spleen focus-forming virus or specific mutations in the extracellular domain of the EPO-R, give rise to pathological consequences, in vivo and EPO-independent proliferation and differentiation of cultured cells. Activating extracellular receptor mutations result in covalently linked receptor homodimers. These observations and others have led to the proposal that EPO activates the EPO-R by inducing dimer formation on the cell surface. It has been assumed that F-gp55 also induces dimer formation of the EPO-R; however, clear evidence of this is lacking. In addition, EPO and F-gp55 stimulation of the EPO-R elicit different biological responses. To probe whether the cell surface EPO-R is structurally different with these activators, we contrasted the cell surface EPO-R complex formed following receptor activation by EPO, F-gp55, and mutations in the extracellular domain of the receptor. Our results indicate that cell surface forms of activated EPO-R differ, as judged by their differential association with F-gp55 and pattern of associated cell surface proteins. Interestingly, we find that the env gene of an anemic strain of Friend virus, Rauscher virus envelope glycoprotein, does not interact with the EPO-R at the cell surface. Thus, the mode of Rauscher virus envelope glycoprotein-induced erythroblastosis may be distinct from F-gp55-induced erythroblastosis and possibly not involve the EPO-R.

In vitro phosphorylation of the erythropoietin receptor and an associated protein, pp130.

The cytoplasmic domain of the cloned erythropoietin (EPO) receptor (EPOR) contains no protein kinase motif, yet addition of EPO to EPO-responsive cells causes an increase in protein-tyrosine phosphorylation. Here we show that addition of EPO or interleukin-3 (IL-3) to an IL-3-dependent cell line expressing the wild-type EPOR causes a small fraction (less than 5%) of total cellular EPOR to shift in gel mobility from 66 to 72 kDa, due at least in part to phosphorylation. Using biotinylated EPO as an affinity reagent, we show that the 72-kDa species is greatly enriched on the cell surface. To demonstrate that a protein kinase activity associates with cell surface EPOR, cells were incubated with biotinylated EPO and then cross-linked with a thiol-cleavable chemical cross-linker. The avidin-agarose-selected complexes were incubated with [gamma-32P]ATP. After in vitro phosphorylation and denaturation without reducing agent, both antiphosphotyrosine and anti-EPOR antibodies immunoprecipitated labeled 72-kDa EPOR and an unidentified 130-kDa phosphoprotein (pp130), indicating that a protein kinase is associated with cell surface EPOR and that a fraction of the EPOR was phosphorylated on tyrosine residues either in the cells or during the cell-free phosphorylation reaction. Under reducing conditions, the 72-kDa phosphorylated EPOR but not pp130 was immunoprecipitated with an anti-EPOR antibody, suggesting that the pp130 is bound to the EPOR by the thiol-cleavable chemical cross-linker. Previously, we showed that deletion of the 42 carboxy-terminal amino acids of the EPOR allows cells to grow in 1/10 the normal EPO concentration, without affecting receptor number or affinity. Two carboxy-terminal truncated EPO receptors that are hyperresponsive to EPO were poorly phosphorylated during the in vitro reaction, suggesting that the carboxy-terminal region of the EPOR contains a site for phosphorylation or a site for interaction with a protein kinase. Our data suggests that phosphorylation or interaction with a protein kinase in the carboxy-terminal region may down-modulate the proliferative action of the EPOR.

In vitro phosphorylation of the erythropoietin receptor and an associated protein, pp130.

The cytoplasmic domain of the cloned erythropoietin (EPO) receptor (EPOR) contains no protein kinase motif, yet addition of EPO to EPO-responsive cells causes an increase in protein-tyrosine phosphorylation. Here we show that addition of EPO or interleukin-3 (IL-3) to an IL-3-dependent cell line expressing the wild-type EPOR causes a small fraction (less than 5%) of total cellular EPOR to shift in gel mobility from 66 to 72 kDa, due at least in part to phosphorylation. Using biotinylated EPO as an affinity reagent, we show that the 72-kDa species is greatly enriched on the cell surface. To demonstrate that a protein kinase activity associates with cell surface EPOR, cells were incubated with biotinylated EPO and then cross-linked with a thiol-cleavable chemical cross-linker. The avidin-agarose-selected complexes were incubated with [gamma-32P]ATP. After in vitro phosphorylation and denaturation without reducing agent, both antiphosphotyrosine and anti-EPOR antibodies immunoprecipitated labeled 72-kDa EPOR and an unidentified 130-kDa phosphoprotein (pp130), indicating that a protein kinase is associated with cell surface EPOR and that a fraction of the EPOR was phosphorylated on tyrosine residues either in the cells or during the cell-free phosphorylation reaction. Under reducing conditions, the 72-kDa phosphorylated EPOR but not pp130 was immunoprecipitated with an anti-EPOR antibody, suggesting that the pp130 is bound to the EPOR by the thiol-cleavable chemical cross-linker. Previously, we showed that deletion of the 42 carboxy-terminal amino acids of the EPOR allows cells to grow in 1/10 the normal EPO concentration, without affecting receptor number or affinity. Two carboxy-terminal truncated EPO receptors that are hyperresponsive to EPO were poorly phosphorylated during the in vitro reaction, suggesting that the carboxy-terminal region of the EPOR contains a site for phosphorylation or a site for interaction with a protein kinase. Our data suggests that phosphorylation or interaction with a protein kinase in the carboxy-terminal region may down-modulate the proliferative action of the EPOR.