A SEQUENTIAL DIMERIZATION MECHANISM FOR ERYTHROPOIETIN RECEPTOR ACTIVATION

We have probed the interaction of human erythropoietin (EPO) with its receptor (EPO-R) by analyzing a panel of 17 EPO mutants in a variety o f in vitro assays, Mutant proteins were expressed in 293s cells and qu antified by using an N-terminal epitope tag in conjunction with a surf ace plasmon resonance assay, Receptor binding was studied using both a soluble form of the EPO-R extracellular domain in an ELISA-format bin ding competition assay and full-length EPO-R in transfected BaF3 cells , Proliferative activity of the mutants was also determined in the BaF 3-derived cell line and was correlated with the results from binding a ssays. Based on the results of these assays, we identified two distinc t receptor binding sites on the EPO molecule, We propose that one site , containing residues Arg-150 and Lys-152, binds initially to EPO rece ptor on the cell surface. A second site, containing Arg-103 and Ser-10 4 (and possibly Arg-14), is involved in binding a second EPO-R at the cell surface, thus forming a homodimeric receptor complex. Furthermore , we demonstrate that one EPO mutant (R103A), which has previously bee n shown to lack proliferative function, is in fact an EPO antagonist. Taken together, these data support a sequential dimerization mechanism of EPO-R activation.