Abstract
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.
Highlights
The erythropoietin receptor (EPOR) is a member of a tions as the primary regulator of erythropoiesis by inducing the family of cytokine and growth factor receptors that survival and proliferation of erythroid progenitor cells and share conserved features in their extracellular and cytoplasmic domains
Mutagenesis Strategy—We have previously described a system for the overexpression of the extracellular domain of the human EPOR in E. coli (Ref. 37, see “Experimental Procedures”)
Amino acid residues in the EPO binding proteins (EBP) were selected for substitution with alanine based on their alignment with residues known to be involved in ligand binding in related receptors of the cytokine receptor family
Summary
Mutagenesis and Expression of the EBP—Mutagenesis was performed in a bacterial expression vector designed to produce a soluble EPO receptor (referred to here as an EPO binding protein or EBP) consisting of the 225 amino acid extracellular domain of the human EPOR truncated just prior to the beginning of the transmembrane domain. Transfected COS cells were detached from plates by incubation at 37 °C for 20 to 60 min in 5 ml of PBS containing 0.5 mM EDTA and 0.02% sodium azide, pelleted, and resuspended in binding buffer (RPMI 1640, 25 mM HEPES pH 7.5, 5% bovine serum albumin, 0.02% sodium azide). Detection of EPOR on the surface of COS cells was performed using an anti-EBP monoclonal antibody (mAb) in a modification of the receptor binding assay described above. Duplicate tubes were set up containing 1 g of mAb number 3 and an excess of EBP (10 g), which would be expected to compete with cell surface EPOR for binding to the mAb. After overnight incubation at 4 °C, cells were pelleted and resuspended in 100 l of binding buffer containing 0.1 g (2 Ci) of 125Ilabeled sheep anti-mouse Ig (Amersham). Binding of mAb to the EBP was detected by the addition of horseradish peroxidase-conjugated goat anti-mouse IgG (Jackson Immunoresearch Laboratories, West Grove, PA) and tetramethylbenzidine (Kirkegaard and Perry Laboratories, Gaithersberg, MD) and measuring the absorbance at 450 nm
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