The extracytoplasmic domain of the erythropoietin receptor forms a monomeric complex with erythropoietin.
We have generated a truncated form of the erythropoietin receptor (EPO-R), the extracytoplasmic ligand-binding domain, that is secreted from a transfected Chinese hamster ovary (CHO) cell line. The truncated receptor is readily purified from CHO conditioned media as a 33-Kd glycosylated protein, which is converted to a 25-Kd species upon treatment with protein N-glycan glycosidase F. Cross-linking of radioiodinated EPO to the secreted receptor yielded a complex of 72 Kd. Also, the growth of the EPO-dependent cell line, FDCPE, was inhibited in a dose-responsive manner by the truncated receptor. The complex of the secreted receptor and EPO was isolated by gel filtration and shown to be a one-to-one complex of the receptor and growth factor by quantitative amino terminal sequencing. Finally, analysis of the interaction of the receptor and growth factor by gel filtration indicated an apparent dissociation constant of 1.1 nmol/L for the truncated receptor.
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Genetics Institute Inc, Cambridge, MA.
Jones
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D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D006224:Cricetinae; D017077:Culture Media, Conditioned; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017467:Receptors, Erythropoietin / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection
D017077:Culture Media, Conditioned; D009838:Oligodeoxyribonucleotides; D017467:Receptors, Erythropoietin; D011993:Recombinant Fusion Proteins; D004921:Erythropoietin; D004247:DNA