Erythropoietin and its Receptors

To watch the companion video to this series, click here.

To read the introduction to this series, click here.


In 1906, French researchers first postulated that a humoral factor regulated the production of red blood cells[1]. Erythropoietin (EPO) was finally isolated from the urine of anemic patients in the 1970’s[2]. EPO is the main and crucial[3] hormonal regulator of red blood cell (erythrocyte) production in the bone marrow and spleen, which is called erythropoiesis. It is a 165-amino acid glycoprotein that belongs to the family of type I cytokines, produced in adulthood by fibroblasts in the renal cortex, which stimulate the survival, proliferation, and differentiation of erythrocytic progenitors[4].

EPO is synthesized most prominently when hypoxic conditions are sensed. When that occurs, hypoxia-inducible factors (HIFs) induce the expression of genes that are normally suppressed by GATA-2 and NF-kB, particularly the EPO gene located on chromosome 7[5]. In the nervous system, it is also synthesized during infection[6], mechanical damage[7], intense neural activity[8], elevated temperature[9], and metabolic stress. It is tempting to speculate that EPO is synthesized during sauna practice, though this has yet to be studied.


EPO is structurally defined by 4 a-helices, named A, B, C, and D. EPO binds to the EPO receptor (EPOR) at two binding sites on its molecule[10], the high-affinity first (site 1; binding affinity of 1 nM) located in helices A, B, D, and a part of the loop connecting A and B, and the low-affinity second (site 2; binding affinity of 1 mM) located in helices A and C[11]. Although the molecules affinity for site 2 is 1000x weaker than its affinity for site 1, binding at both sites appears necessary for full signal transduction, as mutation at one site is sufficient to impair signaling[12][13].

The EPOR has three isoforms that interact with each other and with EPO to dictate the effect of EPO’s eventual agonism of the EPORs. There is a full-length protein, a soluble protein lacking a transmembrane and lacking intracellular domains, and a shortened protein lacking large amounts of intracellular domains[14]. Soluble EPORs have been found in the brain, though in contrast to the full-length EPOR, its expression is downregulated in hypoxic conditions[15]. It is speculated that soluble EPORs function to modulate circulating EPO, dictating the availability of EPO to bind to full-length EPORs. Dopaminergic neurons of the substantia nigra co-express the truncated and full-length isoforms, where it appears that the truncated form may interfere with EPO signaling to the full-length form[16]. Consequently, the levels of co-expression of the truncated form may modulate sensitivity of full-length EPORs to EPO.

Overall, EPORs are expressed on progenitor but not mature cells of skeletal muscle and blood, and they are also expressed in the heart[17], kidney[18], pancreas[19], uterus[20], and brain[21]. In the brain, EPORs isoforms are expressed on neurons, astrocytes, oligodendrocytes, and cerebral endothelial cells[22].


When agonized by EPO at sites 1 and 2, EPOR then activates signaling pathways including the Janus kinase (JAK-2[23]) and the signal transducer and activator of transcription 5A and 5B (STAT-5[24]) pathways[25]. The EPOR contains intracellular domains associated with members of the JAK-2 pathway, without which it would be unable to phosphorylate[26]. Binding at EPOR activates JAK-2 by transphosphorylation, and once activated JAK-2 phosphorylates receptors on tyrosines, which then become docking sites for the STAT-5 transcription factors. STAT-5 transcription factors located at the receptor are then phosphorylated by JAK-5, causing their disassociation from the receptor, translocation to the nucleus of the cell, and transcription of cytokine-responsive genes that regulate cell proliferation, differentiation, and apoptosis[27]. Note that the STAT-5 pathway is also phosphorylated by other cytokines, including growth hormone, interleukin-2, and interleukin-3[28].

Additionally, there is evidence that the EPOR also activate the phosphoinositide-3-kinase (PI3K-AKT) and mitogen-activated protein kinase (MAPK) pathways[29].


There is evidence that EPO exerts some of its biological effects independent of its namesake receptor. The splice variant of EPO that lacks the third exon of the full-length EPO, EV-3, does not activate the EPOR, does not stimulate erythropoiesis, and yet induces neuroprotection in rodents[30]. Moreover, human engineered variants including CEPO, Epobis, and HBSP (discussed in detail in the third article of this series) do not induce erythropoiesis but also produce neuroprotection[31][32][33][34]. While EPO has a greater affinity for the hematopoietic EPOR than it does for non-hematopoietic, tissue-protective receptors, it also appears to require longer exposure to EPORs than it requires for the non-hematopoietic receptors in order to exert an effect[35]. Thus far, candidate receptors for EPO’s non-erythropoietic, neuroprotective effect are the common b chain receptor, the ephrin B4 receptor, and the cytokine receptor-like factor 3.


Also known as CD131, the BCR is a receptor from the cytokine type I receptor family that forms heteromeric receptors with other cytokine receptors involved in hematopoiesis, including those of interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF)[36]. It is co-expressed with the EPOR in a tissue protective heteroreceptor[37] that is developed even in the absence of EPO[38], though it is EPOR is expressed 3x more than BCR in hippocampal regions of rodents[39].

BCR’s activity is upregulated by hypoxic conditions, inflammation, and metabolic stress[40][41]. EPO induces phosphorylation of the BCR[42] and the EPOR/BCR complexes activate JAK similarly to the homomeric EPORs, producing similar downstream responses. The man-made EPO variants CEPO[43] and pHBSP[44] have been shown to produce selective agonism of heteromeric BCR/EPOR units.

Because BCR/EPOR heteromeric units are not expressed in all brain regions that have been observed to receive a neuroprotective effect, and because the EPO splice variant EV-3 is neuroprotective but neither agonizes EPOR homomeric units nor BCR/EPO heteromeric units[45], the neuroprotection offered by EPO is mediated by more than EPOR and BCR.


Ephrins are proteins that characteristically agonize the ephrin receptors (EPH), with mammalian A-ephrins binding to 9 EPH-A receptors and B-ephrins binding to 5 EPH-B receptors. The ephrin signaling to EPH is involved in not only hematopoiesis, angiogenesis, and cancer cell regulation[46], but also neurogenesis[47], axon growth[48], synapse formation and plasticity[49], dendritic morphology[50], and memory development[51].

In studies on cancer cells, it was shown that EPH-B4 receptors are agonized by both ephrin B2 and EPO, and that they are co-expressed with EPORs on heteromeric units, as EPO is co-expressed with BCRs in heteromeric units. Interestingly, EPH-B4 receptors are over 30x less sensitive to EPO than EPORs, indicating that their role likely only predominates under a paucity of EPOR receptors. It was also shown that expression of EPH-B4s in cancer cells was correlated to higher mortality rates, though expression of EPORs was not, and that EPO treatment worsened patient outcomes, likely through agonism of the EPH-B4 receptors. The neurons of rodents also co-express EPH-B4 receptors and EPORs, indicating that some of the neurotrophic and memory-enhancing effects of EPO may be mediated by the EPH-B4 receptors[52].


The CRLF3 is a little-studied cytokine receptor that has a docking site for JAK[53]. An in vitro study on beetle neurons has shown that cell death resulting from hypoxia and serum deprivation can be averted by administration of both EPO and EV-3, which does not agonize the EPO receptor. However, knock-down of the CRLF3 receptor abolishes EV-3’s protective effect on neuronal survival, implying that CRLF3 mediates EV-3’s neuroprotective effect in beetles, although the exact mechanisms are yet unknown[54].

Having discussed endogenous EPO and provided a summary of its action on known receptors, the next article will summarize its effects on health, with a particular focus on cognitive health.

To read the second article in the series, click here.

[1] Carnot, P. (1906). Sur l'activité hémopoiétique du sérum au cours de la régénération du sang. CR Acad Sci., 143, 384-386. [2] Goldwasser, E. (1996). Erythropoietin: a somewhat personal history. Perspectives in biology and medicine, 40(1), 18-32. [3] Wu, H., Liu, X., Jaenisch, R., & Lodish, H. F. (1995). Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor. Cell, 83(1), 59-67. [4] Jelkmann, W. (2011). Regulation of erythropoietin production. The Journal of physiology, 589(6), 1251-1258. [5] La Ferla, K., Reimann, C., Jelkmann, W., & HELLWIG-BÜRGEL, T. H. O. M. A. S. (2002). Inhibition of erythropoietin gene expression signaling involves the transcription factors GATA-2 and NF-κB. The FASEB Journal, 16(13), 1811-1813. [6] Shen, Y., Yu, H. M., Yuan, T. M., Gu, W. Z., & Wu, Y. D. (2009). Erythropoietin attenuates white matter damage, proinflammatory cytokine and chemokine induction in developing rat brain after intra‐uterine infection. Neuropathology, 29(5), 528-535. [7] Campana, W. M., & Myers, R. R. (2003). Exogenous erythropoietin protects against dorsal root ganglion apoptosis and pain following peripheral nerve injury. European Journal of Neuroscience, 18(6), 1497-1506. [8] Morishita, E. M. M. Y. R., Masuda, S., Nagao, M., Yasuda, Y., & Sasaki, R. (1996). Erythropoietin receptor is expressed in rat hippocampal and cerebral cortical neurons, and erythropoietin prevents in vitro glutamate-induced neuronal death. Neuroscience, 76(1), 105-116. [9] Shein, N. A. A., Horowitz, M., Alexandrovich, A. G., Tsenter, J., & Shohami, E. (2005). Heat acclimation increases hypoxia-inducible factor 1α and erythropoietin receptor expression: implication for neuroprotection after closed head injury in mice. Journal of Cerebral Blood Flow & Metabolism, 25(11), 1456-1465. [10] Cheetham, J. C., Smith, D. M., Aoki, K. H., Stevenson, J. L., Hoeffel, T. J., Syed, R. S., ... & Harvey, T. S. (1998). NMR structure of human erythropoietin and a comparison with its receptor bound conformation. Nature structural biology, 5(10), 861-866. [11] Syed, R. S., Reid, S. W., Li, C., Cheetham, J. C., Aoki, K. H., Liu, B., ... & Finer-Moore, J. (1998). Efficiency of signalling through cytokine receptors depends critically on receptor orientation. Nature, 395(6701), 511-516. [12] Matthews, D. J., Topping, R. S., Cass, R. T., & Giebel, L. B. (1996). A sequential dimerization mechanism for erythropoietin receptor activation. Proceedings of the National Academy of Sciences, 93(18), 9471-9476. [13] Zhang, Y. L., Radhakrishnan, M. L., Lu, X., Gross, A. W., Tidor, B., & Lodish, H. F. (2009). Symmetric signaling by an asymmetric 1 erythropoietin: 2 erythropoietin receptor complex. Molecular cell, 33(2), 266-274. [14] Uversky, V. N., & Redwan, E. M. (2017). Erythropoietin and co.: intrinsic structure and functional disorder. Molecular BioSystems, 13(1), 56-72. [15] Soliz, J., Gassmann, M., & Joseph, V. (2007). Soluble erythropoietin receptor is present in the mouse brain and is required for the ventilatory acclimatization to hypoxia. The Journal of physiology, 583(1), 329-336. [16] Marcuzzi, F., Zucchelli, S., Bertuzzi, M., Santoro, C., Tell, G., Carninci, P., & Gustincich, S. (2016). Isoforms of the Erythropoietin receptor in dopaminergic neurons of the Substantia Nigra. Journal of neurochemistry, 139(4), 596-609. [17] Ruifrok, W. P. T., de Boer, R. A., Westenbrink, B. D., van Veldhuisen, D. J., & van Gilst, W. H. (2008). Erythropoietin in cardiac disease: new features of an old drug. European journal of pharmacology, 585(2-3), 270-277. [18] Brines, M., & Cerami, A. (2006). Discovering erythropoietin's extra-hematopoietic functions: biology and clinical promise. Kidney international, 70(2), 246-250. [19] Fenjves, E. S., Ochoa, M. S., Cabrera, O., Mendez, A. J., Kenyon, N. S., Inverardi, L., & Ricordi, C. (2003). Human, nonhuman primate, and rat pancreatic islets express erythropoietin receptors1. Transplantation, 75(8), 1356-1360. [20] Yasuda, Y., Masuda, S., Chikuma, M., Inoue, K., Nagao, M., & Sasaki, R. (1998). Estrogen-dependent production of erythropoietin in uterus and its implication in uterine angiogenesis. Journal of Biological Chemistry, 273(39), 25381-25387. [21] Marti, H. H. (2004). Erythropoietin and the hypoxic brain. Journal of Experimental Biology, 207(18), 3233-3242. [22] Ott, C., Martens, H., Hassouna, I., Oliveira, B., Erck, C., Zafeiriou, M. P., ... & Kolbow, T. (2015). Widespread expression of erythropoietin receptor in brain and its induction by injury. Molecular medicine, 21(1), 803-815. [23] Witthuhn, B. A., Quelle, F. W., Silvennoinen, O., Yi, T., Tang, B., Miura, O., & Ihle, J. N. (1993). JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin. Cell, 74(2), 227-236. [24] Grebien, F., Kerenyi, M. A., Kovacic, B., Kolbe, T., Becker, V., Dolznig, H., ... & Müllner, E. W. (2008). Stat5 activation enables erythropoiesis in the absence of EpoR and Jak2. Blood, The Journal of the American Society of Hematology, 111(9), 4511-4522. [25] Debeljak, N., & Sytkowski, A. J. (2012). Erythropoietin and erythropoiesis stimulating agents. Drug testing and analysis, 4(11), 805-812. [26] Witthuhn, B. A., Quelle, F. W., Silvennoinen, O., Yi, T., Tang, B., Miura, O., & Ihle, J. N. (1993). JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin. Cell, 74(2), 227-236. [27] Morris, R., Kershaw, N. J., & Babon, J. J. (2018). The molecular details of cytokine signaling via the JAK/STAT pathway. Protein Science, 27(12), 1984-2009. [28] Teglund, S., McKay, C., Schuetz, E., Van Deursen, J. M., Stravopodis, D., Wang, D., ... & Ihle, J. N. (1998). Stat5a and Stat5b proteins have essential and nonessential, or redundant, roles in cytokine responses. Cell, 93(5), 841-850. [29] Arcasoy, M. O., & Jiang, X. (2005). Co‐operative signalling mechanisms required for erythroid precursor expansion in response to erythropoietin and stem cell factor. British journal of haematology, 130(1), 121-129. [30] Bonnas, C., Wüstefeld, L., Winkler, D., Kronstein-Wiedemann, R., Dere, E., Specht, K., ... & Sillaber, I. (2017). EV-3, an endogenous human erythropoietin isoform with distinct functional relevance. Scientific reports, 7(1), 1-15. [31] Leist, M., Ghezzi, P., Grasso, G., Bianchi, R., Villa, P., Fratelli, M., ... & Kallunki, P. (2004). Derivatives of erythropoietin that are tissue protective but not erythropoietic. Science, 305(5681), 239-242. [32] Villa, P., Van Beek, J., Larsen, A. K., Gerwien, J., Christensen, S., Cerami, A., ... & Torup, L. (2007). Reduced functional deficits, neuroinflammation, and secondary tissue damage after treatment of stroke by nonerythropoietic erythropoietin derivatives. Journal of Cerebral Blood Flow & Metabolism, 27(3), 552-563. [33] Pankratova, S., Gu, B., Kiryushko, D., Korshunova, I., Køhler, L. B., Rathje, M., ... & Berezin, V. (2012). A new agonist of the erythropoietin receptor, Epobis, induces neurite outgrowth and promotes neuronal survival. Journal of neurochemistry, 121(6), 915-923. [34] Collino, M., Thiemermann, C., Cerami, A., & Brines, M. (2015). Flipping the molecular switch for innate protection and repair of tissues: Long-lasting effects of a non-erythropoietic small peptide engineered from erythropoietin. Pharmacology & therapeutics, 151, 32-40. [35] Brines, M. (2010). The therapeutic potential of erythropoiesis-stimulating agents for tissue protection: a tale of two receptors. Blood purification, 29(2), 86-92. [36] D'Andrea, R. J., & Gonda, T. J. (2000). A model for assembly and activation of the GM-CSF, IL-3 and IL-5 receptors: Insights from activated mutants of the common β subunit. Experimental hematology, 28(3), 231-243. [37] Brines, M., Grasso, G., Fiordaliso, F., Sfacteria, A., Ghezzi, P., Fratelli, M., ... & Pobre, E. (2004). Erythropoietin mediates tissue protection through an erythropoietin and common β-subunit heteroreceptor. Proceedings of the National Academy of Sciences, 101(41), 14907-14912. [38] Jubinsky, P. T., Krijanovski, O. I., Nathan, D. G., Tavernier, J., & Sieff, C. A. (1997). The β chain of the interleukin-3 receptor functionally associates with the erythropoietin receptor. Blood, The Journal of the American Society of Hematology, 90(5), 1867-1873. [39] Tiwari, N. K., Sathyanesan, M., Schweinle, W., & Newton, S. S. (2019). Carbamoylated erythropoietin induces a neurotrophic gene profile in neuronal cells. Progress in Neuro-Psychopharmacology and Biological Psychiatry, 88, 132-141. [40] Bohr, S., Patel, S. J., Vasko, R., Shen, K., Iracheta-Vellve, A., Lee, J., ... & Berthiaume, F. (2015). Modulation of cellular stress response via the erythropoietin/CD131 heteroreceptor complex in mouse mesenchymal-derived cells. Journal of Molecular Medicine, 93(2), 199-210. [41] Brines, M., & Cerami, A. (2012). The receptor that tames the innate immune response. Molecular medicine, 18(3), 486-496. [42] Hanazono, Y., Sasaki, K., Nitta, H., Yazaki, Y., & Hirai, H. (1995). Erythropoietin induces tyrosine phosphorylation of the β chain of the GM-CSF receptor. Biochemical and biophysical research communications, 208(3), 1060-1066. [43] Leist, M., Ghezzi, P., Grasso, G., Bianchi, R., Villa, P., Fratelli, M., ... & Kallunki, P. (2004). Derivatives of erythropoietin that are tissue protective but not erythropoietic. Science, 305(5681), 239-242. [44] Brines, M., Patel, N. S., Villa, P., Brines, C., Mennini, T., De Paola, M., ... & Ghezzi, P. (2008). Nonerythropoietic, tissue-protective peptides derived from the tertiary structure of erythropoietin. Proceedings of the National Academy of Sciences, 105(31), 10925-10930. [45] Bonnas, C., Wüstefeld, L., Winkler, D., Kronstein-Wiedemann, R., Dere, E., Specht, K., ... & Sillaber, I. (2017). EV-3, an endogenous human erythropoietin isoform with distinct functional relevance. Scientific reports, 7(1), 1-15. [46] Suenobu, S., Takakura, N., Inada, T., Yamada, Y., Yuasa, H., Zhang, X. Q., ... & Suda, T. (2002). A role of EphB4 receptor and its ligand, ephrin-B2, in erythropoiesis. Biochemical and biophysical research communications, 293(3), 1124-1131. [47] Ashton, R. S., Conway, A., Pangarkar, C., Bergen, J., Lim, K. I., Shah, P., ... & Schaffer, D. V. (2012). Astrocytes regulate adult hippocampal neurogenesis through ephrin-B signaling. Nature neuroscience, 15(10), 1399. [48] Drescher, U., Bonhoeffer, F., & Müller, B. K. (1997). The Eph family in retinal axon guidance. Current opinion in neurobiology, 7(1), 75-80. [49] Hruska, M., & Dalva, M. B. (2012). Ephrin regulation of synapse formation, function and plasticity. Molecular and Cellular Neuroscience, 50(1), 35-44. [50]Ethell, I. M., Irie, F., Kalo, M. S., Couchman, J. R., Pasquale, E. B., & Yamaguchi, Y. (2001). EphB/syndecan-2 signaling in dendritic spine morphogenesis. Neuron, 31(6), 1001-1013. [51] Dines, M., Grinberg, S., Vassiliev, M., Ram, A., Tamir, T., & Lamprecht, R. (2015). The roles of Eph receptors in contextual fear conditioning memory formation. Neurobiology of learning and memory, 124, 62-70. [52] Pradeep, S., Huang, J., Mora, E. M., Nick, A. M., Cho, M. S., Wu, S. Y., ... & Brock, S. (2015). Erythropoietin stimulates tumor growth via EphB4. Cancer cell, 28(5), 610-622. [53] Boulay, J. L., O'Shea, J. J., & Paul, W. E. (2003). Molecular phylogeny within type I cytokines and their cognate receptors. Immunity, 19(2), 159-163. [54] Hahn, N., Knorr, D. Y., Liebig, J., Wüstefeld, L., Peters, K., Büscher, M., ... & Heinrich, R. (2017). The insect ortholog of the human orphan cytokine receptor CRLF3 is a neuroprotective erythropoietin receptor. Frontiers in molecular neuroscience, 10, 223.