TI  - Identification of a critical ligand binding determinant of the human
      <prot><prot>erythropoietin</prot> receptor</prot>. Evidence for common ligand binding motifs in the
      cytokine receptor family.
PG  - 14045-54
AB  - The <prot><prot>erythropoietin</prot> receptor</prot> (<prot>EPOR</prot>) is a member of a family of cytokine and
      growth factor receptors that share conserved features in their
      extracellular and cytoplasmic domains. We have used site-specific
      mutagenesis within the extracellular domain of the  <prot>EPOR</prot>  to search for
      amino acid residues involved in  <prot>erythropoietin</prot> (<prot>EPO</prot>)  binding. Mutant
      proteins were expressed in bacteria as soluble <prot>EPO</prot> binding proteins (<prot>EBP</prot>)
      and characterized for <prot>EPO</prot> binding activity in a number of different
      assays. Substitution of phenylalanine at position 93 (Phe93) with alanine
      (F93A mutation) resulted in a drastic reduction in  <prot>EPO</prot>  binding in the  <prot>EBP</prot> .
      More conservative tyrosine or tryptophan substitutions at Phe93 resulted
      in much less dramatic effects on <prot>EPO</prot> binding. Biophysical studies
      indicated that the F93A mutation does not result in gross structural
      alterations in the <prot>EBP</prot>. Furthermore, the F93A mutation in full-length  <prot>EPOR</prot> 
      expressed in COS cells abolished detectable  <prot>EPO</prot>  binding. This was not a
      result of processing or transport defects, since mutant receptor was
      present on the surface of the cells. Mutations in the region immediately
      around Phe93 and in residues homologous to other reported ligand binding
      determinants of the cytokine receptor family had small to moderate effects
      on <prot>EPO</prot> binding. These data indicate that Phe93 is a critical  <prot>EPO</prot>  binding
      determinant of the  <prot>EPOR</prot> . Furthermore, since Phe93 aligns with critical
      ligand binding determinants in other receptors of the cytokine receptor
      family, these data suggest that receptors of this family may use common
      structural motifs to bind their cognate ligands.
AD  - R. W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey
