Search

Hematopoietic cytokine receptors, such as the erythropoietin receptor (EpoR), are single membrane-spanning proteins. Signal transduction through EpoR is crucial for the formation of mature erythrocytes. Structural evidence shows that in the unliganded form EpoR exists as a preformed homodimer in an open scissor-like conformation precluding the activation of signaling. In contrast to the extracellular domain of the growth hormone receptor (GHR), the structure of the agonist-bound EpoR extracellular region shows only minimal contacts between the membrane-proximal regions. This evidence suggests that the domains facilitating receptor dimerization may differ between cytokine receptors. We show that the EpoR transmembrane domain (TM) has a strong potential to self interact in a bacterial reporter system. Abolishing self assembly of the EpoR TM by a double point mutation (Leu 240-Leu 241 mutated to Gly-Pro) impairs signal transduction by EpoR in hematopoietic cells and the formation of erythroid colonies upon reconstitution in erythroid progenitor cells from EpoR(-/-) mice. Interestingly, inhibiting TM self assembly in the constitutively active mutant EpoR R129C abrogates formation of disulfide-linked receptor homodimers and consequently results in the loss of ligand-independent signal transduction. Thus, efficient signal transduction through EpoR and possibly other preformed receptor oligomers may be determined by the dynamics of TM self assembly.

Erythropoietin (Epo) is essential for formation of mature red blood cells (RBC). However, the function of Epo receptor (EpoR)-dependent signaling pathways in the regulation of erythropoiesis remains unclear. To determine whether specific Stat signals are required for RBC development, we changed the Stat signaling specificity of the EpoR. The wild-type EpoR activates only Stat5. Thus, we substituted the major Stat5 binding sites (residues 343 and 401) in the EpoR cytoplasmic region with the Stat3 binding/activation motif from gp130. We demonstrated that activated EpoRs containing a single substitution stimulate Stat5 and Stat3, whereas an EpoR with both substitutions stimulates Stat3 but not Stat5. We then determined the ability of these receptors to support fetal liver and adult erythropoiesis. Our results show that erythropoiesis is stimulated by EpoRs that activate Stat5, both Stat5 and Stat3, or Stat3 in place of Stat5. These findings demonstrate that the specificity of EpoR Stat signaling is not essential for RBC development.

J2E cells produce rapid, fatal erythroleukemias in vivo but still respond to erythropoietin (epo) in vitro by differentiating, proliferating and remaining viable in the absence of serum. Mutant epo receptors were introduced into these cells to determine whether they could influence the different biological responses to epo in vitro and the development of erythroleukemias. Three mutant receptors were used as cytoplasmic truncation mutants Delta257 and Delta321 (above box 1 and below box 2 respectively), and the cytoplasmic point mutant W282R (defective for JAK2 activation). Strikingly, the Delta321 mutation produced a hyper-sensitive response in vitro to epo-induced differentiation and viability, but not to proliferation. In contrast with the Delta321 receptor, the Delta257 and W282R mutants inhibited all biological responses to epo due to impaired JAK2 phosphorylation. Significantly, erythroleukemias took almost twice as long to develop with cells containing the W282R mutation, indicating that JAK2 plays an important role in the emergence of these leukemias. These data demonstrate that mutant epo receptors dominantly altered responses of J2E cells to epo in culture and the development of erythroleukemias. Oncogene (2000) 19, 953 - 960.

Inherited mutations in the erythropoietin receptor (EPOR) causing premature termination of the receptor cytoplasmic region are associated with dominant familial erythrocytosis (FE), a benign clinical condition characterized by hypersensitivity of erythroid progenitor cells to EPO and low serum EPO (S-EPO) levels. We describe a Swedish family with dominant FE in which erythrocytosis segregates with a new truncation in the negative control domain of the EPOR. We show that cells engineered to concomitantly express the wild-type (WT) EPOR and mutant EPORs associated with FE (FE EPORs) are hypersensitive to EPO-stimulated proliferation and activation of Jak2 and Stat5. These results demonstrate that FE is caused by hyperresponsiveness of receptor-mediated signaling pathways and that this is dominant with respect to WT EPOR signaling.

The hormone erythropoietin (Epo) is essential for red blood cell development. Epo binds a high affinity receptor on the surface of erythroid progenitor cells, stimulating receptor dimerization and activation of the intracellular signal transduction pathways that support erythroid cell survival, proliferation and differentiation. Biochemical and structural analysis of the erythropoietin receptor (EpoR) is revealing the molecular mechanisms of EpoR function, leading the way to the development of small molecule Epo mimetics. This review focuses on the role EpoR dimerization plays in receptor function.

The cytokine receptor superfamily is characterized by structural motifs in the exoplasmic domain and by the absence of catalytic activity in the cytosolic segment. Activated by ligand-triggered multimerization, these receptors in turn activate a number of cytosolic signal transduction proteins, including protein tyrosine kinases and phosphatases, and affect an array of cellular functions that include proliferation and differentiation. Molecular study of these receptors is revealing the roles they play in the control of normal hematopoiesis and in the development of disease.

We modified BiP, the resident endoplasmic reticulum (ER) heat shock protein 70, to contain an epitopetag sequence close to the C-terminus (BiP-tag); the epitope is derived from an influenza hemagglutinin (HA) subtype and is recognized by the monoclonal antibody 12CA5. This antibody both immunoprecipitates BiP-tag and detects it on Western blots. Using transient expression of cDNAs in COS cells, we studied the interaction of BiP-tag with several membrane proteins. Consistent with previous work on BiP, BiP-tag bound poorly and transiently to newly made wild-type influenza HA glycoprotein and strongly and irreversibly to an HA mutant that misfolds and is retained in the ER. Most newly made erythropoietin receptor (EPO-R) polypeptides are retained in the ER and degraded there; we show here that, in cotransfected COS cells, newly made EPO-R is bound to BiP-tag prior to its degradation. Thus, by several criteria the BiP-tag molecule is fully functional in binding newly made proteins. Because it can be immunoprecipitated by a readily available antibody, it offers several advantages to the study of protein folding in the ER and the role of chaperones in this process.

The erythropoietin receptor (EPO-R) is synthesized in transfected Ba/F3 cells as a major 64-kDa endoglycosidase H (Endo H)-sensitive species, with a single N-linked oligosaccharide, and a minor 62-kDa unglycosylated form. Approximately half of the newly made EPO-R is processed to a mature 66-kDa form with a Golgi-processed Endo H-resistant oligosaccharide, of which only a minor fraction is expressed at the cell surface. Both the Endo H-sensitive and the Endo H-resistant forms of the receptor have a half-life of 45-60 min (Yoshimura, A., D'Andrea, A. D., and Lodish, H. F. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4139-4143). The mature, Endo H-resistant form of the EPO-R appears to be degraded in lysosomes or in other acidic organelles, since receptor degradation is blocked by treatment with NH4Cl, chloroquine, or leupeptin. A fraction of the Endo H-resistant EPO-R molecules is cleaved, generating two fragments of 46 and 39 kDa. The sizes of these fragments and their reactivities with carboxyl-terminal-specific antibodies indicate that the receptor is cleaved at two sites in the exoplasmic domain, 7 kDa apart, and carboxyl-terminal to the N-glycosylation site. Both fragments are membrane anchored and are probably formed in a late or post-Golgi compartment, since their formation is blocked by incubation of cells at 20 degrees C or by incubation with brefeldin A. These membrane-anchored COOH-terminal fragments are probably degraded in lysosomes or in other acidic vesicles as cell fractionation demonstrates that they colocalize with lysosomes, and similar to the intact EPO-R, their degradation is inhibited by NH4Cl. Finally, double labeling immunofluorescence experiments demonstrate that in NH4Cl-treated cells both intact mature EPO-R and the 46- and 39-kDa fragments accumulate in lysosomes and presumably are normally degraded there. The sensitivity of the EPO-R to endoproteolytic cleavages in its exoplasmic domain may relate to its low surface expression and to its extreme metabolic instability.