Ligand binding properties of the human erythropoietin receptor extracellular domain expressed in Escherichia coli.
We developed an assay to directly measure the ligand binding properties of the cloned human erythropoietin receptor (EpoR). The cDNA encoding the extracellular domain of the human EpoR was amplified by polymerase chain reaction and ligated into the prokaryotic expression vector pGEX3X. Synthesis in Escherichia coli was induced and a soluble glutathione S-transferase fusion protein, EREx, was purified by erythropoietin affinity chromatography. Purified EREx was bound to GSH agarose beads and used in a solid phase ligand binding assay. Specific binding of 125I-erythropoietin to EREx beads was demonstrated. A single affinity class (Kd = 1.5 nM) of the binding site was evident on Scatchard analysis. The Kd of this site is quantitatively equivalent to that of the "low" affinity cellular binding site. Kinetic analysis of ligand binding to EREx revealed both the on and off rates to be rapid, with t1/2 of 60 and 40 s, respectively. EREx ligand binding exhibits no obvious metal ion dependence or cross-competition by other hemopoietins. Antibodies to EREx block the binding of erythropoietin to the cellular EpoR. We conclude that the 66-kDa EpoR protein is capable of specific ligand binding and that no covalent modifications or associated molecules are required for this interaction. We speculate that the "high" affinity cellular binding site (Kd less than 0.2 nM) results from the interaction of the EpoR with another molecule, either additional EpoR or associated subunits, that decreases the ligand off rate.
Harris
K W
KW
Department of Medicine, University of Minnesota, Minneapolis 55455.
Mitchell
R A
RA
Winkelmann
J C
JC
D001483:Base Sequence; D002846:Chromatography, Affinity; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D017467:Receptors, Erythropoietin; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D007457:Iodine Radioisotopes; D008024:Ligands; D011956:Receptors, Cell Surface; D017467:Receptors, Erythropoietin; D011993:Recombinant Fusion Proteins; D004921:Erythropoietin; D004247:DNA