Identification of a Critical Ligand Binding Determinant of the Human Erythropoietin Receptor

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.

Mutagenesis Studies of the Human Erythropoietin Receptor

“…The W40A mutation was then expressed as a full-length EPOR in COS cells and failed to bind EPO in the whole cell binding assay (Table I). Further investigation of this mutant full-length receptor utilizing a radiolabeled antibody cell surface detection method (15) revealed no detectable cell surface expression of the W40A full-length receptor (data not shown). These data suggest that W40A is a critical structural determinant of the EPOR.…”

“…Generally, our approach was to screen for mutations resulting in decreased EPO binding activity by testing crude preparations of the mutant EBPs in a high performance-size exclusion chromatography (HP-SEC) assay (16). Although not quantitative, the HP-SEC assay does not require purification of the mutant proteins, is quick and easy to perform, and is capable of identifying mutations resulting in large reductions in EPO binding (15,16). This assay was successful in identifying an EBP mutant with a 1,000-fold increased IC 50 for EPO (15); however, it may not be sensitive enough to detect less dramatic effects on EPO binding, since an EBP mutant (L96A-EBP) with a 200-fold increased IC 50 for EPO still showed EPO binding.…”

“…Although not quantitative, the HP-SEC assay does not require purification of the mutant proteins, is quick and easy to perform, and is capable of identifying mutations resulting in large reductions in EPO binding (15,16). This assay was successful in identifying an EBP mutant with a 1,000-fold increased IC 50 for EPO (15); however, it may not be sensitive enough to detect less dramatic effects on EPO binding, since an EBP mutant (L96A-EBP) with a 200-fold increased IC 50 for EPO still showed EPO binding. Following HP-SEC analysis, selected mutant proteins were purified and analyzed in competition binding assays or expressed and characterized as full-length receptor on the surface of COS cells.…”

“…Detection of EPOR on the surface of COS cells was performed using an anti-EBP monoclonal antibody in a modification of the receptor binding assay described above. This procedure for cell surface detection has been described previously (15).…”

“…Previously, we reported that a single residue, Phe 93 , is a critical ligand binding determinant of the EPOR (15). Here, in a more extensive mutagenesis analysis of the 225-amino acid extracellular domain of the EPOR, we report the effect on EPO binding of single amino acid substitutions created by sitespecific mutagenesis.…”