An elucidation of the erythropoietin ligand-receptor interaction is critical to an understanding of the mechanisms by which erythropoietin controls the production of erythrocytes from bone marrow progenitors. The experiments described in this proposal will investigate the binding site of erythropoietin, the structure of the erythropoietin receptor, and the early cellular events following receptor activation. The relationship between structure and function of erythropoietin will be Investigated by site-directed mutagenesis in an effort to identify the binding site of the hormone. cDNA's which encode for the normal and mutated proteins will be expressed in mammalian cells and analyzed for specific receptor binding and bioactivity. The low abundance of erythropoietin receptors on erythroid progenitors and erythroleukemic cell lines renders the purification of the receptor a difficult task. For this reason, a cDNA library will be synthesized from cells which express the erythropoietin receptor. A newly developed method of screening a cDNA library will utilize erythropoietin as a ligand probe. This expression strategy has recently been successful In the isolation of the cDNA clones for other low abundance growth factor receptors such as the IL-1 and IL-6 receptors. The cDNA which potentially encodes the erythropoietin receptor will be transfected into mammalian cells which lack the receptor. Cells expressing the surface receptor will be identified with labeled erythropoietin. Nucleotide sequence analysis will determine structural similarities with other cloned growth factor receptors. The tissue distribution of the erythropoietin receptor will be examined in mammalian cells by the detection of mRNA transcripts with a radiolabeled cDNA probe and the quantification of surface receptors with anti-receptor antibodies. Transfected cells which express high levels of erythropoietin receptors will provide a model to study receptor activation and subsequent signal transduction. The chromosomal localization of the gene for the erythropoietin receptor will be determined. Further studies will investigate the relationship between retroviruses or oncogenes and the erythropoietin ligand-receptor complex. Site-directed mutagenesis of the cDNA for the erythropoietin receptor will determine the functional sites of the protein. These reagents will be valuable for further study of the effects of erythropoietin on the differentiation and proliferation of erythroid cells.
