Identification of a critical ligand binding determinant of the human erythropoietin receptor. Evidence for common ligand binding motifs in the cytokine receptor family.
The erythropoietin receptor (EPOR) 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 EPOR to search for amino acid residues involved in erythropoietin (EPO) binding. Mutant proteins were expressed in bacteria as soluble EPO binding proteins (EBP) and characterized for EPO 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 EPO binding in the EBP. More conservative tyrosine or tryptophan substitutions at Phe93 resulted in much less dramatic effects on EPO binding. Biophysical studies indicated that the F93A mutation does not result in gross structural alterations in the EBP. Furthermore, the F93A mutation in full-length EPOR expressed in COS cells abolished detectable EPO 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 EPO binding. These data indicate that Phe93 is a critical EPO binding determinant of the EPOR. 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.
Middleton
S A
SA
R. W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey 08869, USA.
Johnson
D L
DL
Jin
R
R
McMahon
F J
FJ
Collins
A
A
Tullai
J
J
Gruninger
R H
RH
Jolliffe
L K
LK
Mulcahy
L S
LS
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004921:Erythropoietin / Q000378:metabolism; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D017354:Point Mutation; D017433:Protein Structure, Secondary; D018121:Receptors, Cytokine / Q000737:chemistry*; D017467:Receptors, Erythropoietin / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014364:Tryptophan; D014443:Tyrosine
D008024:Ligands; D018121:Receptors, Cytokine; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; D004921:Erythropoietin; D014443:Tyrosine; D010649:Phenylalanine; D014364:Tryptophan; D000409:Alanine