Characterization of the Structure of the Erythropoietin Receptor by Ligand Blotting

• Published in: Blood Vol. 77; no. 12; pp. 2577 - 2582
• Main Authors: Atkins, Harold L., Broudy, Virginia C., Papayannopoulou, Thalia
• Format: Journal Article
• Language: English
• Published: Washington, DC Elsevier Inc 15.06.1991; The Americain Society of Hematology
• Subjects: 550201 - Biochemistry- Tracer Techniques; ANIMAL CELLS; ANIMALS; BASIC BIOLOGICAL SCIENCES; BETA DECAY RADIOISOTOPES; BIOCHEMICAL REACTION KINETICS; Biological and medical sciences; BIOLOGICAL MATERIALS; BLOOD; BLOOD CELLS; BODY FLUIDS; CELL CONSTITUENTS; CELL MEMBRANES; CHEMICAL COMPOSITION; CHEMICAL REACTIONS; CROSS-LINKING; DAYS LIVING RADIOISOTOPES; DISEASES; ELECTRON CAPTURE RADIOISOTOPES; ELECTROPHORESIS; Erythrocyte Membrane - chemistry; ERYTHROCYTES; ERYTHROPOIETIN; Erythropoietin - metabolism; GROWTH FACTORS; Hematologic and hematopoietic diseases; HORMONES; Humans; IMMUNE SYSTEM DISEASES; INTERMEDIATE MASS NUCLEI; INTERNAL CONVERSION RADIOISOTOPES; IODINE 125; IODINE ISOTOPES; Iodine Radioisotopes; ISOTOPE APPLICATIONS; ISOTOPES; KINETICS; LEUKEMIA; Leukemia, Erythroblastic, Acute; LIGANDS; MAMMALS; MATERIALS; Medical sciences; MEMBRANE PROTEINS; MEMBRANES; MICE; MITOGENS; MOLECULAR STRUCTURE; MOLECULAR WEIGHT; NEOPLASMS; NUCLEI; ODD-EVEN NUCLEI; ORGANIC COMPOUNDS; PEPTIDE HORMONES; POLYMERIZATION; PROTEINS; RADIOISOTOPES; REACTION KINETICS; RECEPTORS; Receptors, Cell Surface - chemistry; Receptors, Cell Surface - isolation & purification; Receptors, Cell Surface - metabolism; Receptors, Erythropoietin; Recombinant Proteins - metabolism; RODENTS; TRACER TECHNIQUES; TUMOR CELLS; Tumor Cells, Cultured; VERTEBRATES; Characterization; Human; Binding capacity; Erythropoietin; Pathophysiology; Hemopathy; Molecular biology; Glycoprotein hormone; Hormonal receptor
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V77.12.2577.2577

Erythropoietin (Epo) regulates the growth and differentiation of erythroid cells by binding to a specific receptor. We characterized the native Epo receptor on erythroleukemia cell lines by ligand blotting. Solubilized cell membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose, and probed with125 l-Epo. Specificity was demonstrated by inhibition of125 I-Epo binding by unlabeled excess Epo but not other peptide growth factors and by the cellular distribution of the Epo binding protein. A single membrane protein of 61 Kd ± 4 Kd was sufficient to bind125 l Epo in both human (0CIM2, K562) and murine (GM979, Rauscher, DA-1) cell lines. This finding is consistent with the predicted size of the Epo receptor from the murine cDNA clone. However, chemical crosslinking of125 l-Epo to its receptor has identified two Epo binding proteins of 105 Kd and 85 Kd. This difference may occur because the receptor is size fractionated before Epo binding in the ligand blot, but after Epo binding in crosslinking studies. Ligand blotting demonstrates that the native Epo receptor is composed of a single 61-Kd Epo binding protein, and suggests the presence of additional proteins of 20 to 25 Kd that associate with the receptor after Epo binding.