The long term goal of this project is to elucidate mechanisms regulating the differentiation and proliferation of red blood cells. Dysfunctions in red cell development can lead to diseases ranging from anemias to erythroleukemias, and knowledge of the molecular mechanisms regulating erythropoiesis is essential for the design of rational therapies. Erythropoietin is a prime regulator of red blood cell development. This glycoprotein stimulates both the differentiation of erythroblasts from pluripotent progenitor cells and the proliferation of committed erythroid progenitor, cells. With the recent development and acquisition of cell lines that respond to erythropoietin by either proliferating (B6SUt.EP) or differentiating to produce hemoglobin (J2E) plus the acquisition of the recently cloned cDNA for one polypeptide chain of the erythropoietin receptor, biological systems are now in hand to investigate the mechanisms of erythropoietin action at a molecular level. The proposed experiments will examine the mechanisms of erythropoietin action ranging from signal transduction to effects on gene expression. (1) Binding of erythropoietin to its receptor leads to rapid tyrosyl phosphorylation of several membrane proteins including a primary Mr 155,000 substrate, PY155. The nature of PY155 and its role in signal transduction will be investigated first by phosphopeptide mapping of this protein in responsive cells to establish its association with erythropoietin-and possibly IL3- and GMCSF-induced growth, followed by purification of PY155, peptide sequencing and cDNA isolation. (2) The range of cell types capable of supporting an erythropoietin response will be established by expressing the erythropoietin receptor cDNA in a variety of factor-dependent lines and testing for responsiveness to erythropoietin. Effects on other factor-receptor systems, such as IL-3, M-CSF and GMCSF also will be identified. Additionally, functional cytosolic domains of the erythropoietin receptor will be mapped by deleting regions of the cDNA and assaying for function after expression in transfected cells. (3) After establishing which genes are induced by erythropoietin in the cell line J2E, the mechanisms involved in this erythroid induction will be examined by assaying for changes in chromatin structure and in the profile of DNA binding proteins. The role of novel binding proteins will be tested by mutating their cognate sites in globin reporter genes transfected into uninduced and induced J2E cells, and the roles of individual and groups of proteins will be analyzed in cell free systems.
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