Page 369 - Hematology_ Basic Principles and Practice ( PDFDrive )

Chapter 26 Biology of Erythropoiesis, Erythroid Differentiation, and Maturation 305

superfamily, which includes the receptors for IL-3, GM-CSF, and SIGNAL TRANSDUCTION BY EPOR
IL-5, the EPOR polypeptide contains four conserved cysteine
residues and a WSXWS motif in the extracellular region. Additional Considerable progress has been made in our understanding of
extracytoplasmic sequences of EPOR determine the specificity for EPOR-mediated signal transduction. Early studies demonstrated
EPO binding. The cytoplasmic region of EPOR does not contain a that stimulation of EPOR on primary erythroid cells resulted in
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tyrosine kinase catalytic domain; instead it interacts with cytoplasmic increased calcium ion flux and increased globin mRNA synthesis.
tyrosine kinases. Cross-linking of radiolabeled EPO to cell surface Since the cloning of the EPOR polypeptide and its stable expres-
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EPOR results in formation of at least two major cross-linked protein sion in heterologous cell systems, such as the Ba/F3 cell system,
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complexes of 140 and 120 kDa. The molecular composition of considerable molecular insight has been gained. For instance, it
these complexes remains unsolved but suggests that EPOR contains is now clear that EPO induces homodimerization of the EPOR
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additional subunits or accessory proteins. The extracytoplasmic polypeptide. 248,249 Following receptor dimerization at the cell surface,
region of the EPOR polypeptide contains the EPO binding activity of a series of tyrosine phosphorylation events occurs, resulting in a
the receptor. 221–223 Therefore additional EPOR subunits may provide mitogenic signal and a differentiative signal. 250,251 Initial studies of
other structural and functional elements of the receptor but are not the EPOR signal transduction pathway made use of mutant forms of
required for high-affinity EPO binding. The extracytoplasmic region EPOR stably expressed in the indicator cell line Ba/F3. Ba/F3 cells
of the EPOR polypeptide has been crystalized. 224–226 The crystal are a murine IL-3–dependent pro-B lymphocyte cell line. These cells
structure confirms the dimeric structure of the activated receptor. can be readily transfected with the complementary DNA (cDNA)
Interestingly, small synthetic peptides are capable of inducing EPOR for EPOR, resulting in stable expression of the receptor on the cell
dimerization, suggesting a profitable avenue for EPO-mimetic and surface. Expression of the full-length, wild-type EPOR polypeptide
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EPO-antagonist drug design. EPO-mimetic agents are represented in these cells resulted in EPO-dependent growth and partial EPO-
by polypeptides restricted to the portion of the protein that binds induced erythroid differentiation. 250,251 Expression of truncated
the receptor, by forms of the protein molecularly engineered to forms of the EPOR polypeptide in these cells resulted in variable
increase its glycosylation state and therefore its stability in vivo, or growth responses. For instance, truncation of the membrane proximal
by dimeric forms of proteins obtained by genetic introduction of region of EPOR demonstrated a critical positive regulatory domain
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bridging sites or chemical cross-linking. It is also possible that of EPOR required for mitogenesis. Furthermore, truncation of the
nonpeptide chemicals sharing the same stereo and electric properties carboxy-terminal (C-terminal) 40 amino acids of EPOR resulted in
of the receptor-binding domain of the protein might be identified. As increased EPO-dependent growth, suggesting that the C-terminal
shown for carbamylated EPO, modified isoforms may have biologic region contained a negative regulatory domain normally required for
activity that partially differs from, and is possibly more effective downmodulating EPOR mitogenic signals. 246
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than, the native protein, especially with regard to the activity The biochemical basis for these positive and negative regulatory
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of the growth factor in nonhematopoietic tissues. In addition to domains has been elucidated. The membrane proximal positive regu-
EPO mimetics, erythroid stimulating agents under development are latory region of EPOR binds constitutively to Janus-activated kinase
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represented by modulators of HIF-1α expression, IgA2, an immu- 2 (JAK2), a cytoplasmic tyrosine kinase necessary for erythroid
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noglobin that selectively increases under conditions of anemic stress differentiation, as evidenced by mice lacking the corresponding gene
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and TGF-β superfamily ligand traps. 231 dying at an early embryonic stage. Upon EPO binding to the
EPOR mRNA, originally isolated from murine erythroblast cell receptor, the receptor dimerizes, resulting in activation of prebound
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lines (MEL and HCD57) and from a human erythroid cell line JAK2. The JAK2 next tyrosine phosphorylates multiple signaling
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(OCIM1), has been found in nonerythroid cells as well. EPO proteins in the cell, leading to various mitogenic and differentiative
promotes the differentiation of megakaryocytes at physiologic responses. The negative regulatory domain of EPOR is required for
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concentrations of hormone, suggesting that megakaryocytes have recruiting the phosphatase SHP1 to EPOR. SHP1 binds to an
functional cell surface EPORs. Rat and mouse placenta also have activated tyrosine phosphate on the EPOR polypeptide and rapidly
cell surface EPOR, detected by radiolabeled EPO cross-linking. downregulates JAK2 activity and dephosphorylates the EPOR
EPO promotes a chemotactic effect on endothelial cells, 233,234 sug- polypeptide. Failure to recruit the SHP1 phosphatase can result in
gesting the presence of a cell surface receptor in these cells. Other increased EPOR signaling and a polycythemic state (see Alterations
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studies suggest that EPOR is expressed in neural cells and smooth in EPOR and Its Signaling in Disorders of Erythropoiesis).
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muscle cells. Adverse effects in cancer patients treated with EPO JAK2 is required for appropriate Golgi processing and cell surface
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have been attributed to the effects of EPO on tumor cells. The expression of EPOR. Once activated by EPO/EPOR binding on
functional importance of EPOR expression in nonerythroid cells has the cell surface, JAK2 initiates several events in EPOR-mediated signal
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null
been revealed by rescue experiments in EPOR mice. Because transduction. JAK2 initially activates tyrosine phosphorylation of
null
EPOR mutant mice die of severe anemia between days 13 and 15 several tyrosine residues of the cytoplasmic tail of EPOR. These phos-
of embryonic development, the mutant embryos can be rescued by phorylated tyrosine residues next serve as docking sites for binding of
transgenic expression of EPOR under the control of the hemopoietic- other cytoplasmic effector proteins containing Src homology 2 (SH2)
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specific GATA1 regulatory domain. Under steady-state conditions, domains, such as the p85 subunit of phosphatidylinositol 3-kinase,
the rescued animals are normal, because the gene is expressed only the adaptor protein Shc, 257,258 and STAT5. 259,260 (Examples of signal
in erythroid cells. However, in comparison with normal mice, the transduction proteins expressed in primary human erythroblasts are
increase in plasma EPO concentration in response to induced anemia given in Table 26.2). Once these proteins have docked on EPOR,
was delayed in the rescued animals, suggesting that one of the major they become tyrosine phosphorylated and engage other downstream
functions of EPOR expression in nonerythroid cells is fine-tuning signaling events. In addition, JAK2 activates the Ras/Raf/MAPK
the regulation of the response to stress. 238 (mitogen-activated protein kinase) pathway, further contributing to
The existence of naturally occurring splice variants of the EPOR the EPO-induced mitogenic signal. 261,262 The molecular mechanism
gene encoding EPOR polypeptides of variable length and activity of Ras activation by JAK2 remains unknown but may entail direct
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has been shown. 239–242 The soluble secreted form of EPOR binds binding of the proteins and tyrosine phosphorylation. 262
EPO and thereby competes with the cell surface receptor isoform. Activation of the JAK2/STAT5 signaling pathway has been
The biologic function of alternative forms of the cell surface EPOR, studied in considerable detail. Upon EPOR tyrosine phosphoryla-
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including a truncated form of EPOR found in early progenitors, tion, STAT5 protein binds to a specific phosphorylated tyrosine
remains unknown but may be related either to differential EPO residue of the EPOR. 259,263 Binding is mediated by the SH2 domain
signaling and responses (survival, proliferation, differentiation) at of STAT5. Following EPOR binding, STAT5 itself becomes tyrosine
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different stages in erythroid development or to the establishment phosphorylated at amino acid Y694. Activated STAT5 then dis-
of erythroid-specific versus myeloid-specific niches in the marrow engages from EPOR, undergoes homodimerization, and translocates
microenvironment. to the cell nucleus, where it activates transcription of EPO-inducible