Profiling of early gene expression induced by erythropoietin receptor structural variants.

The development of erythroid progenitor cells is triggered via the expression of the erythropoietin receptor (EPOR) and its activation by erythropoietin. The function of the resulting receptor complex depends critically on the presence of activated JAK2, and the complex contains a large number of signaling molecules recruited to eight phosphorylated tyrosine residues. Studies using mutant receptor forms have demonstrated that truncated receptors lacking all tyrosines are able to support red blood cell development with low efficiency, whereas add-back mutants containing either Tyr343 or Tyr479 reconstitute EPOR signaling and erythropoiesis in vivo. To study the contribution of tyrosines to receptor function, we analyzed the activation of essential signaling pathways and early gene induction promoted by different receptor structural variants using human epidermal growth factor receptor/murine EPOR hybrids. In our experiments, receptors lacking all tyrosine residues or the JAK2-binding site did not induce mitogenic and anti-apoptotic signaling, whereas add-back mutant receptors containing single tyrosine residues (Try343 and Tyr479) supported the activation of these functions efficiently. Profiling of early gene expression using cDNA array hybridization revealed that (i) the high redundancy in the activation of signaling pathways is continued at the level of transcription; (ii) the expression of many genes targeted by the wild-type receptor is not supported by add-back mutants; and (iii) a small set of genes are exclusively induced by add-back receptors. We report the identification of several early genes that have not been implicated in the EPOR-dependent response so far.

Identification of an acquired JAK2 mutation in polycythemia vera.

Polycythemia vera (PV) is a human clonal hematological disorder. The molecular etiology of the disease has not been identified. PV hematopoietic progenitor cells exhibit hypersensitivity to growth factors and cytokines, suggesting possible abnormalities in protein-tyrosine kinases and phosphatases. By sequencing the entire coding regions of cDNAs of candidate enzymes, we identified a G:C--> T:A point mutation of the JAK2 tyrosine kinase in 20 of 24 PV blood samples but none in 12 normal samples. The mutation has varying degrees of heterozygosity and is apparently acquired. It changes conserved Val(617) to Phe in the pseudokinase domain of JAK2 that is known to have an inhibitory role. The mutant JAK2 has enhanced kinase activity, and when overexpressed together with the erythropoietin receptor in cells, it caused hyperactivation of erythropoietin-induced cell signaling. This gain-of-function mutation of JAK may explain the hypersensitivity of PV progenitor cells to growth factors and cytokines. Our study thus defines a molecular defect of PV.

Structural requirements of the extracellular to transmembrane domain junction for erythropoietin receptor function.

The erythropoietin receptor (EpoR) is crucial for erythrocyte formation. The x-ray crystal structures of the EpoR extracellular domain lack the juxtamembrane (JM) region and the junction to the transmembrane (TM) domain. Yet the JM-TM regions are important for transmitting the conformational change imposed on the receptor dimer by Epo binding. Cysteine-scanning mutagenesis of the JM-TM regions identified three novel constitutively active mutants, demonstrating close disulfide-bonded juxtapositioning of these residues in the JM (L223C) and N-terminal TM domain (L226C, I227C). Chemical cross-linking defined the interface of the active helical TM dimer and revealed that the JM-TM segment encompassing Leu(226)-Leu(230) is non-helical. Molecular dynamics and NMR studies indicated that the TM-JM junction forms an N-terminal helix cap. This structure is important for EpoR function because replacement of this motif by consecutive leucines rendered the receptor constitutively active.

Identification of the human erythropoietin receptor region required for Stat1 and Stat3 activation.

Signal transducers and activators of transcription (Stat) proteins play important roles in the regulation of hematopoiesis as downstream molecules of cytokine signal transduction. It was previously demonstrated that erythropoietin (EPO), a major regulator of erythropoiesis, activates 3 different Stat members, Stat1, Stat3, and Stat5, in a human EPO-dependent cell line, UT-7/EPO. To clarify the mechanism by which EPO activates Stat1 and Stat3 via the EPO receptor (EPOR), a series of chimeric receptors was constructed bearing the extracellular domain of the granulocyte colony-stimulating factor receptor linked to the transmembrane domain of EPOR and the full length or several mutants of the cytoplasmic domain of EPOR, and these chimeric receptor complementary DNAs were introduced into UT-7/EPO cells. Tyr432 on human EPOR was important for activation of Stat1 and Stat3 and c-myc gene induction. In addition, Jak2 and Fes tyrosine kinases were involved in EPO-induced activation of Stat1 and Stat3. These results indicate that Stat1 and Stat3 are activated by EPO via distinct mechanisms from Stat5.

Identification of tyrosine residues within the intracellular domain of the erythropoietin receptor crucial for STAT5 activation.

FDCP-1 cells are hematopoietic progenitor cells which require interleukin-3 for survival and proliferation. FDCP-1 cells stably transfected with the murine erythropoietin receptor cDNA survive and proliferate in the presence of erythropoietin. Erythropoietin induces the activation of the short forms (80 kDa) of STAT5 in the cells. Erythropoietin-induced activation of STAT5 was strongly reduced in cells expressing mutated variants of the erythropoietin receptors in which tyrosine residues in their intracellular domain have been eliminated. We determined that the erythropoietin receptor tyrosine residues 343 and 401 are independently necessary for STAT5 activation. The amino acid sequences surrounding these two tyrosine residues are very similar. Peptides comprising either phosphorylated Tyr343 or phosphorylated Tyr401, but not their unphosphorylated counterparts, inhibited the STAT5 activation. We propose that these two tyrosine residues of the erythropoietin receptor constitute docking sites for the STAT5 SH2 domain. The growth stimulus mediated by erythropoietin was decreased in cells expressing erythropoietin receptors lacking both Tyr343 and Tyr401. This suggests that STAT5 activation could be involved in the growth control of FDCP-1 cells.

Characterization of three erythropoietin (Epo)-binding proteins in various human Epo-responsive cell lines and in cells transfected with human Epo-receptor cDNA.

Molecular cloning of a cDNA for a mouse erythropoietin (Epo) receptor (EpoR) has facilitated the understanding of the structure of this receptor. However, there is, as yet, no explanation for the discrepancy between the protein recognized by specific antibodies against mouse EpoR and the unexpectedly larger species that can be cross-linked to labeled Epo. It is unclear whether the product of an unidentified gene is included in the EpoR complex. In the present study, we directly compared the cross-linking patterns for human EpoR that were endogenously expressed in three types of Epo-responsive cell, and that was artificially expressed in nonhematopoietic cells after transfection with cDNA for human EpoR. We observed that 85-kD and 105-kD proteins formed ligand-receptor complexes in all the human Epo-responsive cells and, furthermore, that the formation of a complex derived from the 70-kD protein was dependent on the level of expression of the cloned EpoR mRNA in these cells. By contrast, a prominent cross-linked band derived from the 70-kD protein and a weaker band derived from the 80- to 85-kD protein, but no band derived from the 105-kD protein, could be shown in the case of nonhematopoietic cells transfected with the human EpoR cDNA. These observations suggest that the cloned cDNA for human EpoR alone does not allow generation of the complete EpoR in nonhematopoietic cells and that the 105-kD Epo-binding protein may represent the product of an as yet unidentified gene that is expressed in hematopoietic cells.

Activation and inhibition of erythropoietin receptor function: role of receptor dimerization.

Members of the cytokine receptor superfamily have structurally similar extracellular ligand-binding domains yet diverse cytoplasmic regions lacking any obvious catalytic domains. Many of these receptors form ligand-induced oligomers which are likely to participate in transmembrane signaling. A constitutively active (factor-independent) mutant of the erythropoietin receptor (EPO-R), R129C in the exoplasmic domain, forms disulfide-linked homodimers, suggesting that the wild-type EPO-R is activated by ligand-induced homodimerization. Here, we have taken two approaches to probe the role EPO-R dimerization plays in signal transduction. First, on the basis of the crystal structure of the ligand-bound, homodimeric growth hormone receptor (GH-R) and sequence alignment between the GH-R and EPO-R, we identified residues of the EPO-R which may be involved in intersubunit contacts in an EPO-R homodimer. Residue 129 of the EPO-R corresponds to a residue localized to the GH-R dimer interface region. Alanine or cysteine substitutions were introduced at four other residues of the EPO-R predicted to be in the dimer interface region. Substitution of residue E-132 or E-133 with cysteine renders the EPO-R constitutively active. Like the arginine-to-cysteine mutation at position 129 in the exoplasmic domain (R129C), E132C and E133C form disulfide-linked homodimers, suggesting that constitutive activity is due to covalent dimerization. In the second approach, we have coexpressed the wild-type EPO-R with inactive mutants of the receptor missing all or part of the cytosolic domain. These truncated receptors have a dominant inhibitory effect on the proliferative action of the wild-type receptor. Taken together, these results strengthen the hypothesis that an initial step in EPO- and EPO-R-mediated signal transduction is ligand-induced receptor dimerization.