Mutagenesis Studies of the Human Erythropoietin Receptor: ESTABLISHMENT OF STRUCTURE-FUNCTION RELATIONSHIPS

Francis P Barbone,Steven A Middleton,Dana L Johnson,Frank J Mcmahon,Jennifer Tullai,Robert H Gruninger,Adriane E Schilling,Linda K Jolliffe,Linda S Mulcahy

doi:10.1074/jbc.272.8.4985

Abstract

Mutagenesis of the erythropoietin receptor (EPOR) permits analysis of the contribution that individual amino acid residues make to erythropoietin (EPO) binding. We employed both random and site-specific mutagenesis to determine the function of amino acid residues in the extracellular domain (referred to as EPO binding protein, EBP) of the EPOR. Residues were chosen for site-specific alanine substitution based on the results of the random mutagenesis or on their homology to residues that are conserved or have been reported to be involved in ligand binding in other receptors of the cytokine receptor family. Site-specific mutants were expressed in Escherichia coli as soluble EBP and analyzed for EPO binding in several different assay formats. In addition, selected mutant proteins were expressed as full-length EPOR on the surface of COS cells and analyzed for 125I-EPO binding in receptor binding assays. Using these methods, we have identified residues that appear to be involved in EPO binding as well as other residues, most of which are conserved in receptors of the cytokine receptor family, that appear to be necessary for the proper folding and/or stability of the EPOR. We present correlations between these mutagenesis data and the recently solved crystal structure of the EBP with a peptide ligand.

Highlights

Mutagenesis of the erythropoietin receptor (EPOR) permits analysis of the contribution that individual amino acid residues make to erythropoietin (EPO) binding
Residues were chosen for site-specific alanine substitution based on the results of the random mutagenesis or on their homology to residues that are conserved or have been reported to be involved in ligand binding in other receptors of the cytokine receptor family
We have identified residues that appear to be involved in EPO binding as well as other residues, most of which are conserved in receptors of the cytokine receptor family, that appear to be necessary for the proper folding and/or stability of the EPOR

Summary

Mutagenesis Studies of the Human Erythropoietin Receptor

Residues were chosen for site-specific alanine substitution based on the results of the random mutagenesis or on their homology to residues that are conserved or have been reported to be involved in ligand binding in other receptors of the cytokine receptor family. Selected mutant proteins were expressed as full-length EPOR on the surface of COS cells and analyzed for 125I-EPO binding in receptor binding assays Using these methods, we have identified residues that appear to be involved in EPO binding as well as other residues, most of which are conserved in receptors of the cytokine receptor family, that appear to be necessary for the proper folding and/or stability of the EPOR. Amino acid residues involved in ligand binding have been identified in several receptors of the cytokine receptor family using alanine substitution site-specific mutagenesis.

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION

Receptor binding