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

We have shown previously that Phe(93) in the extracellular domain of the er ythropoietin (EPO) receptor (EPOR) is crucial for binding EPO. Substitution of Phe(93) with alanine resulted in a dramatic decrease in EPO binding to the Escherichia coli-expressed extracellular domain of the EPOR (EPO-bindin g protein or EBP) and no detectable binding to full-length mutant receptor expressed in COS cells. Remarkably, Phe(93) forms extensive contacts with a peptide ligand in the crystal structure of the EBP bound to an EPO-mimetic peptide (EMP1), suggesting that Phe(93) is also important for EMP1 binding . We used alanine substitution of EBP residues that contact EMP1 in the cry stal structure to investigate the function of these residues in both EMP1 a nd EPO binding. The three largest hydrophobic contacts at Phe(93) Met(150), and Phe(205) and a hydrogen bonding interaction at Thr(151) were examined. Our results indicate that Phe(93) and Phe(205) are important for both EPO and EMP1 binding, Met(150) is not important for EPO binding but is critical for EMP1 binding, and Thr(151) is not important for binding either ligand. Thus, Phe(93) and Phe(205) are important binding determinants for both EPO and EMP1, even though these ligands share no sequence or structural homolo gy, suggesting that these residues mag represent a minimum epitope on the E POR for productive ligand binding.