We have shown previously that Phe 93 in the extracellular domain of the erythropoietin (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-binding 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

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... ound 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 crystal structure to investigate the function of these residues in both EMP1 and 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 homology, suggesting that these residues may represent a minimum epitope on the EPOR for productive ligand binding.