TI  - Shared and unique determinants of the   <prot><prot>erythropoietin</prot> (<prot>EPO</prot>) receptor</prot>   are
      important for binding  <prot>EPO</prot>  and  <prot><prot>EPO</prot> mimetic peptide </prot>.
PG  - 14163-9
AB  - We have shown previously that Phe93 in the extracellular domain of the
      <prot><prot>erythropoietin</prot> (<prot>EPO</prot>) receptor</prot>  (<prot>EPOR</prot>)  is crucial for binding  <prot>EPO</prot> 
      Substitution of Phe93 with alanine resulted in a dramatic decrease in <prot>EPO</prot>
      binding to the Escherichia coli-expressed extracellular domain of the <prot>EPOR</prot>
      (<prot><prot>EPO</prot>-binding protein</prot> or <prot>EBP</prot>) 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  <prot>EBP</prot>  bound
      to an  <prot><prot>EPO</prot>-mimetic peptide</prot> (<prot>EMP1</prot>) , suggesting that Phe93 is also important
      for <prot>EMP1</prot> binding. We used alanine substitution of <prot>EBP</prot> residues that
      contact <prot>EMP1</prot> in the crystal structure to investigate the function of these
      residues in both <prot>EMP1</prot> and <prot>EPO</prot> 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 <prot>EPO</prot> and <prot>EMP1</prot> binding, Met150 is not important for <prot>EPO</prot>
      binding but is critical for <prot>EMP1</prot> binding, and Thr151 is not important for
      binding either ligand. Thus, Phe93 and Phe205 are important binding
      determinants for both <prot>EPO</prot> and <prot>EMP1</prot>, even though these ligands share no
      sequence or structural homology, suggesting that these residues may
      represent a minimum epitope on the <prot>EPOR</prot> for productive ligand binding.
AD  - R. W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey
