MUTAGENESIS STUDIES OF THE HUMAN ERYTHROPOIETIN RECEPTOR - ESTABLISHMENT OF STRUCTURE-FUNCTION-RELATIONSHIPS

Mutagenesis of the erythropoietin receptor (EPOR) permits analysis of the contribution that individual amino acid residues make to erythropo ietin (EPO) binding. We employed both random and site-specific mutagen esis to determine the function of amino acid residues in the extracell ular domain (referred to as EPO binding protein, EBP) of the EPOR, Res idues were chosen for site specific alanine substitution based on the results of the random mutagenesis or on their homology to residues tha t are conserved or have been reported to be involved in ligand binding in other receptors of the cytokine receptor family, Site-specific mut ants were expressed in Escherichia coli as soluble EBP and analyzed fo r EPO binding in several different assay formats, In addition, selecte d mutant proteins were expressed as full-length EPOR on the surface of COS cells and analyzed for I-125-EPO binding in receptor binding assa ys. Using these methods, we have identified residues that appear to be involved in EPO binding as well as other residues, most of which are conserved in receptors of the cytokine receptor family, that appear to be necessary for the proper folding and/or stability of the EPOR, We present correlations between these mutagenesis data and the recently s olved crystal structure of the EBP with a peptide ligand.