Erythropoietin receptor-dependent erythroid colony-forming unit development: capacities of Y343 and phosphotyrosine-null receptor forms.
Red cell development depends on the binding of erythropoietin (EPO) to receptors expressed by erythroid colony-forming units (CFUe) and the subsequent activation of receptor-bound Janus kinase (Jak2). Jak2 then mediates the phosphorylation of receptor tyrosine sites and the recruitment of 25 or more Src homology 2 domain-encoding proteins and associated factors. Previous studies have shown that an EPO receptor form containing Jak2-binding domains plus a single phosphotyrosine(343) (PY(343))-STAT5-binding site provides all signals needed for erythroid cell development. However, roles for PY(343) and STAT5 remain controversial, and findings regarding PY-null receptor activities and erythropoiesis in STAT5-deficient mice are disparate. To study activities of a PY-null EPO receptor in primary cells while avoiding compensatory mechanisms, a form retaining domains for Jak2 binding and activation, but lacking all cytoplasmic tyrosine sites, was expressed in transgenic mice from a GATA1 gene-derived vector as a human epidermal growth factor receptor- murine EPO receptor chimera (EE-T-Y343F). The bio-signaling capacities of this receptor form were investigated in CFUe from thiamphenicol-treated mice. Interestingly, this PY-null EPO receptor form supported CFUe development (in the absence of detectable STAT5 activation) at efficiencies within 3-fold of those levels mediated by either an EE-T-Y343 form or the endogenous EPO receptor. However, EE-T-Y343F-dependent Ter119(+) erythroblast maturation was attenuated. In tests of cosignaling with c-Kit, EE-T-Y343F nonetheless retained full capacity to synergize with c-Kit in promoting erythroid progenitor cell proliferation. Thus, EPO receptor PY-dependent events can assist late erythropoiesis but may be nonessential for EPO receptor-c-Kit synergy.
Miller
Chris P
CP
Department of Veterinary Science, Pennsylvania State University, University Park, PA 16802, USA.
Heilman
Destin W
DW
Wojchowski
Don M
DM
D019943:Amino Acid Substitution; D000818:Animals; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D004268:DNA-Binding Proteins / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics; D015672:Erythroid Precursor Cells / Q000166:cytology* / Q000378:metabolism / Q000502:physiology; D004920:Erythropoiesis / Q000187:drug effects / Q000502:physiology; D006133:Growth Substances / Q000494:pharmacology; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D008894:Milk Proteins; D016297:Mutagenesis, Site-Directed; D019000:Phosphotyrosine / Q000502:physiology; D019009:Proto-Oncogene Proteins c-kit / Q000502:physiology; D017467:Receptors, Erythropoietin / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000502:physiology; D050799:STAT5 Transcription Factor; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism
D004268:DNA-Binding Proteins; D006133:Growth Substances; D008894:Milk Proteins; D017467:Receptors, Erythropoietin; D011993:Recombinant Fusion Proteins; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D019000:Phosphotyrosine; D066246:ErbB Receptors; D019009:Proto-Oncogene Proteins c-kit