One of the major unanswered questions in the regulation of hematopoiesis is the mechanism by which soluble growth factors interact with specific receptors on hematopoietic cells. This proposal focuses on establishing the structural features of erythropoietin (Epo) that are responsible for its biosynthetic processing and function. Site-directed mutations, both deletions and amino acid replacements, will be introduced into the Epo gene in order to test specific functional properties. Methods will be developed for high level production of normal and mutant Epo's in Cos7, CHO and S. frugiperda cells as well as in E. Coli and in a cell-free translation system. The recombinant Epo products will be purified to homogeneity and characterized biochemically. the following assays will be developed to assess both short-term and long-term effects of normal and mutant products in the Epo-responsive cell line HCD57: equilibrium binding, internalization, Ca2+ influx, intracellular pH, [3H]-thymidine incorporation, and heme synthesis. For the latter 3 measurements, HCD57 cells will be compared to erythroid cells isolated from the spleens of mice treated with phenylhydrazine. We will prepare cassette mutants that correspond to surface epitopes predicted from primary and secondary structure. The properties of these mutants will provide information on the domains of Epo responsible for binding to its receptor as well as for internalization. Particular epitopes of interest will be selected for preparation of additional mutants that will better define these functionally important sites. Site-directed mutants will also be used to investigate the biosynthetic and functional roles of post- translational modifications including N-terminal processing, cleavage of C-terminal Arg, as well as N- and O- linked glycosylation. This cumulative information will be used in developing a computer based prediction of 3-dimensional structure. Concurrently we will attempt to prepare crystals of normal Epo and selected mutants, and if successful, they will be analyzed by x-ray diffraction. From these studies we hope to gain comprehensive information on structure-function relationships of Epo that will contribute to an understanding, at the molecular level, of receptor binding, internalization and signal transduction.
