Shared and unique determinants of the erythropoietin (EPO) receptor are important for binding EPO and EPO mimetic peptide

We have shown previously that Phe⁹³ in the extracellular domain of the erythropoietin (EPO) receptor (EPOR) is crucial for binding EPO. Substitution of Phe⁹³ with alanine resulted in a dramatic decrease in EPO binding to the Escherichia coli-expressed extracellular domain of the EPOR (EPO-binding protein or EBP) and no detectable binding to full-length mutant receptor expressed in COS cells. Remarkably, Phe⁹³ forms extensive contacts with a peptide ligand in the crystal structure of the EBP bound to an EPO-mimetic peptide (EMP1), suggesting that Phe⁹³ is also important for EMP1 binding. We used alanine substitution of EBP residues that contact EMP1 in the crystal structure to investigate the function of these residues in both EMP1 and EPO binding. The three largest hydrophobic contacts at Phe⁹³, Met¹⁵⁰, and Phe²⁰⁵ and a hydrogen bonding interaction at Thr¹⁵¹ were examined. Our results indicate that Phe⁹³ and Phe²⁰⁵ are important for both EPO and EMP1 binding, Met¹⁵⁰ is not important for EPO binding but is critical for EMP1 binding, and Thr¹⁵¹ is not important for binding either ligand. Thus, Phe⁹³ and Phe²⁰⁵ are important binding determinants for both EPO and EMP1, even though these ligands share no sequence or structural homology, suggesting that these residues may represent a minimum epitope on the EPOR for productive ligand binding.