Serum form of the erythropoietin receptor identified by a sequence-specific peptide antibody.
The present investigation was undertaken to search for soluble forms of the erythropoietin receptor in human serum using polyclonal antibody against an amino terminal peptide sequence in the extracellular domain. This sequence was located adjacent to the amino terminus at residues 25-38. When this antibody was used for Western blots of solubilized membranes from nucleated bone marrow cells, a protein consistent with native erythropoietin receptor was seen. Purified soluble ectodomain of the erythropoietin receptor displayed appropriate reactivity with this antibody. When sera from normal subjects and patients with a range of hematologic disorders were examined by Western blotting, a protein with a molecular mass of 34 Kd was detected in sera from patients with enhanced erythropoiesis including sickle cell anemia, thalassemia, and megaloblastic anemia. This protein was rarely detected in normal serum but appeared when normal subjects were treated with recombinant erythropoietin and disappeared after full treatment of patients with megaloblastic anemia due to vitamin B12 deficiency. The protein was not detected after myeloablation for bone marrow transplantation but appeared with marrow engraftment. Reactivity of this protein with the peptide antibody was competitively inhibited by the amino terminal peptide sequence. An additional 48 Kd protein was detected that showed minimal variation in intensity with differing degrees of erythropoietic activity. Detection of this protein could not be inhibited by the addition of synthetic peptide. Our findings indicate the presence of a soluble form of the erythropoietin receptor related to the extracellular domain that is highly correlated with enhanced erythropoiesis.
Department of Medicine, Kansas University Medical Center, Kansas City 66160-7402.
R D
RD
Baynes
G K
GK
Reddy
Y J
YJ
Shih
B S
BS
Skikne
J D
JD
Cook
D000595:Amino Acid Sequence; D000906:Antibodies; D015153:Blotting, Western; D001853:Bone Marrow / Q000378:metabolism; D016026:Bone Marrow Transplantation; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000276:immunology; D017467:Receptors, Erythropoietin / Q000032:analysis* / Q000276:immunology
D000906:Antibodies; D009842:Oligopeptides; D017467:Receptors, Erythropoietin