The Erythropoietin Receptor and Signal Transduction Akihiko Yoshimura and Ken'ichi Arai The Oncologist 1996, 1:337-339.

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THE ERYTHROPOIETIN RECEPTOR AND SIGNAL TRANSDUCTION

ERYTHROPOIETIN (EPO) Erythropoietin (EPO) is a hormone that promotes the proliferation and differentiation of erythroid progenitor cells and regulates the number of erythrocytes in peripheral blood. EPO is produced mainly by the kidneys, and transcription of the EPO gene is promoted by a reduction in the oxygen concentration in the blood. The existence of EPO was suggested near the end of the 19th century by the discovery that hypoxia increases the production of red blood cells. EPO was identified as a serum factor in the 1950s, and in 1970 Miyake and coworkers succeeded in purifying it by using the urine of patients with aplastic anemia as a starting material. The human EPO gene was cloned in 1985 using a partial amino acid sequence from this purified EPO, and it is well known that recombinant EPO is currently used as a drug to treat anemia associated with chronic renal failure and other illnesses. ACTION OF EPO When human bone marrow cells are cultured in a semisolid medium containing EPO, they form small erythroblast colonies in five to seven days, and by day 10 large erythroblast colonies appear that resemble fireworks (burst colonies). The original cells in the former colonies are called colony forming units-erythroid (CFU-E) or late-stage erythroblast progenitor cells and in the latter colonies they are called burst forming units-erythroid (BFU-E) or early-stage erythroblast

progenitor cells. As shown in Figure 1, red blood cells are produced through differentiation from stem cells to BFU-E, CFUE, and erythroblasts. Although EPO acts on both BFU-E and CFU-E cells, CFU-E cells show greater sensitivity to EPO, and other factors such as stem cell factor (SCF), interleukin (IL)-3, IL-4, and granulocyte macrophage colony-stimulating factor (GM-CSF) must be present together with EPO for BFUE cell proliferation. In erythroblasts beyond the CFU-E stage, sensitivity to EPO decreases as the cells mature. THE EPO RECEPTOR AND THE CYTOKINE RECEPTOR FAMILY The EPO receptor gene was cloned by DAndrea and coworkers in 1989 from murine erythroleukemia cells [1]. It became clear that the EPO receptor belongs to the cytokine receptor family that comprises receptors for the various interleukins, GM-CSF, granulocyte colony-stimulating factor (G-CSF), growth hormone and prolactin. The special characteristic of this family of receptors is that they are switched on (i.e., the receptor is activated) and transduce signals to the interior of the cell by the formation of homo- or hetero-oligomers (dimers or trimers). Moreover, hetero-oligomers of these receptors share a common receptor subunit. As shown in Figure 2, the IL-3, IL-5 and GM-CSF receptors have a common subunit, and their ligand specificity is determined by the subunit. In the same manner, the IL-6, LIF and oncostatin M (OSM) receptors all share gp130, which is the subunit of the IL-6 receptor. The IL-2, IL-4 and IL-7 receptors all share the subunit of the IL-2 receptor. All the above receptors are activated by the formation of hetero-oligomers, but the GCSF receptor, EPO receptor, and growth hormone receptor are activated by the formation of homodimers of the same types of molecules [2]. We can see that groups of cytokines such as the interleukins that affect a relatively wide range of cells and have redundant biological activity create this redundancy through the common use of a single receptor subunit. On the other hand, EPO and G-CSF act with high specificity on a relatively limited range of cells, so it was probably unnecessary for their receptors to share one of the subunits.

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Pluripotent hemopoietic stem cells

Hemopoietic progenitor cells

Mature blood cells

Hemopoietic stem cells

BFU-E

CFU-E

Erythroblasts

Erythrocytes

SCF IL-6 IL-3 G-CSF IL-11

SCF IL-3 GM- CSF IL-4

Erythropoietin (EPO)

Figure 1. Production of red blood cells.

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EPO RECEPTOR AND JAK2 KINASE A: Groups with common subunit B: Groups with common gp130 subunit The signal for cellular proliferation and LIF differentiation into erythroblasts is thought to IL-3 IL-5 GM-CSF IL-6 OSM CNTF originate at the EPO receptor. The cytoplasmic domain of the EPO receptor can be divided into two major regions. Roughly half of the cytoplasmic domain, the part lying nearest the plasma membrane, is required for generating the signals for proliferation and differentiation such as the induction of globin synthesis [3, 4]. The remaining half is not required for this signaling, and, conversely, it LIFR LIFR acts to dampen the signals. It is known that a (gp130) (gp130) (gp130) (gp130) tyrosine kinase called JAK2 associates with D: Groups that form homodimers C: Groups with common subunit the region near the plasma membrane, underGrowth goes autophosphorylation, and phosphoryG-CSF EPO hormone IL-2 IL-4 IL-7 lates the EPO receptor, and a transcription factor called a STAT [5]. It is thought that JAK2 plays an important role in promoting cellular proliferation. The STAT is activated by the phosphorylation, and it then translocates to the nucleus, recognizes a specific base sequence in the promoter region of its target gene, and initiates transcription. At present, we know that LIF: Leukemia inhibitory factor the STAT whose activation is mediated by OSM: Oncostatin M the EPO receptor is STAT5, and the target CNTF: Ciliary neurotrophic factor genes are CIS [6], which has an SH2 domain LIFR: LIF receptor (a molecular structure that recognizes a phosphorylated tyrosine) and OSM [7], which is a pleiotropic cytokine. However, Figure 2. Four groups in cytokine receptor family. Black line ( ) is WSXWS motif that is well activation of STAT5 and activation of the conserved in cytokine receptors. target genes are not unique to the EPO kinase-MAP kinase cascade, and ultimately initiates the receptor, and they also occur with the IL-2 and IL-3 transcription of oncogenes such as c-fos and c-jun. receptors. Moreover, the JAK2 substrate that is directly An enzyme called PI3 kinase binds to the tyrosine linked to cellular proliferation is still unknown. At prephosphorylation site of the receptor and a second messensent, studies are under way to determine the transcription ger is born. It is known that this pathway is a requirement factors specific to EPO and their target genes, as well as for DNA synthesis in certain types of fibroblasts. However, the substrates of JAK2. these signal transduction pathways are not unique to the EPO receptor, and they are also activated by most growth RECEPTOR PHOSPHORYLATION AND CESSATION OF THE SIGNAL factor receptors, so they are not necessarily required for On the other hand, tyrosine phosphorylation of the EPO-induced proliferation. Conversely, the tyrosine phosreceptor is necessary at the cytoplasmic tail region far from phatase SH-PTP1 (also called HCP) that has an SH2 the plasma membrane, and the signal transduction pathway domain and is specific to blood cells associates with the that originates with this phosphorylated tyrosine and is tyrosine phosphorylation site of the receptor and promotes mediated by proteins with SH2 domains becomes activated. the dephosphorylation of JAK2. In other words, the role of First, a GTP/GDP exchange factor called SOS, which is SH-PTP1 is to stop generation of the signal [8]. mediated by Shc and Grb2, migrates to the plasma memTherefore, in mutations lacking this cytoplasmic tail brane and converts a ras protein to its GTP form. The actiregion of the receptor far from the plasma membrane, the vated ras protein then activates the Raf-MAP kinase

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Physician Education receptors do not undergo tyrosine phosphorylation, JAK2 activation continues for a longer period of time, and thus the signal is generated more efficiently. In fact, in one patient with a mild case of familial erythrocytosis a mutation was discovered in which the C-terminus of the EPO receptor was missing 70 amino acids [9]. This was a dominant genetic trait, and the patients erythroblasts showed an increased sensitivity to EPO. In this family the impairment was not severe enough to be called an illness, and in fact it is said that this patient was proficient enough athletically to compete for a gold medal at the Olympics. More specifically, the reason that athletes undergo training at high altitudes is to boost EPO production because of the lower oxygen partial pressure, and this brings about the desired effect of sustained athletic capability due to a resultant increase in red blood cells. However, the same effect has occurred naturally in this athlete thanks to accelerated receptor capability. Professor Akihiko Yoshimura Genetic Information Research Department Institute of Life Science Kurume University Kurume, Japan Professor Kenichi Arai Molecular Biology Research Department Institute of Medical Science University of Tokyo Tokyo, Japan

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Erythropoietin receptor and signal transduction.

R EFERENCES
1 DAndrea AD, Lodish HF, Wong GG. Expression cloning of the murine erythropoietin receptor. Cell 1989;57:277-285. 2 Yoshimura A, Longmore G, Lodish HF. Point mutation in the exoplasmic domain of the erythropoietin receptor resulting in hormone-independent activation and tumorigenicity. Nature 1990;348:647-649. 3 Ohashi H, Maruyama K, Liu Y et al. Ligand-induced activation of chimeric receptors between the erythropoietin receptor and receptor tyrosine kinases. Proc Natl Acad Sci USA 1994;91:158-162. 4 Maruyama K, Miyata K, Yoshimura A. Proliferation and erythroid differentiation through the cytoplasmic domain of the erythropoietin receptor. J Biol Chem 1994;269:5976-5980. 5 Witthuhn BA, Quelle FW, Silvennoinen O et al. JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin. Cell 1993;74:227-236. 6 Yoshimura A, Ohkubo T, Kiguchi T et al. A novel cytokineinducible gene CIS encodes an SH2-containing protein that binds to tyrosine-phosphorylated interleukin 3 and erythropoietin receptors. EMBO J 1995;14:2816. 7 Yoshimura A, Ichihara M, Kinjyo I et al. Mouse oncostatin M: an immediate early response gene induced by multiple cytokines through the JAK-STAT5 pathway. EMBO J 1996;15:1055-1063. 8 Klingmuller U, Lorenz U, Cantley LC et al. Specific recruitment of SH-PTP1 to the erythropoietin receptor causes inactivation of JAK2 and termination of proliferative signals. Cell 1995;80:726-738. 9 Chapelle ADL, Traskelin AL, Juvonen E. Truncated erythropoietin receptor causes dominantly inherited benign human erythrocytosis. Proc Natl Acad Sci USA 1993;90:4495-4499.

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